SECTION X OVERVIEW
T his last section of the book draws together principles
from previous chapters to explain some of the rules that
govern the lifestyles of cells. Cells exhibit a remarkable
diversity in their patterns of growth, proliferation, and
death. For example, some human cells (neurons) are
born around the time of birth and live until the person
dies—more than 100 years in a few cases. The fate of
other cells is to live for only a day or two (eg, cells in
the gut lining). Many differentiated cells form by elabo-
rate pathways that employ a carefully choreographed
series of cues from within the cell and from its neigh-
bors. Other cells, such as many in the immune system,
are spawned in excess, followed by selection of the few
with correctly rearranged genes or with productive con-
nections to partner cells. The unlucky majority of their
siblings whose differentiation did not go so well ulti-
mately commit suicide.
Very different strategies maintain populations of cells.
Long-lived cells divide seldom, if at all. In contrast, the
cells that are involved in producing the gut lining grow
and divide at top speed. Most human cells differentiate
Meiosis Ch 45 Mitosis Ch 44
G2 phase and
control of entry
into mitosis Ch 43 Introduction to the
cell cycle Ch 40
S phase and DNA
replication Ch 42
to carry out specific functions and then no longer pro-
liferate. How do cells decide whether to proliferate, to
stop proliferating and differentiate, or to die? This section
answers these and other questions.
Chapter 40 begins the section with an introduction
to the language of the cell cycle. The cell cycle is driven
by changing states of the cytoplasm created by shifting
balances of protein phosphorylation, dephosphoryla-
tion, and degradation machinery. For the cell cycle, the
key kinases are cyclin-dependent kinases (Cdks),
which require an associated cyclin subunit for activity.
Cdks are also regulated by phosphorylation and by
additional protein cofactors that bind and inactivate
them. Cdks are usually stable, but cyclin levels fluctuate,
owing to targeted destruction at particular points in
the cell cycle. In fact, targeted proteolytic destruction
by the proteasome is a key aspect of cell-cycle control.
Each cell-cycle phase is characterized by the activity of
one or more E3 ubiquitin ligases. Each of these targets
particular proteins for destruction by decorating them
with chains of ubiquitin, a protein that was introduced
Programmed cell death Ch 46
Active CDK
Normal cell arrested
at restriction point
STOP
Rb
G1 phase and the
regulation of cell
proliferation Ch 41
Cancer cell passes
restriction point
GO
Rb
Danger
695
in Chapter 23. This sequential destruction of key factors
gives cell-cycle transitions their irreversible character.
The chapters that follow explain how the cell-cycle
machinery controls each step in the proliferation and
differentiation of cells. Chapter 41 begins with newly
born cells in the G1 phase of the cell cycle. These cells
must decide whether to commit themselves to a round
of proliferation or to withdraw from the proliferation rat
race and enter a nondividing differentiated state called
G0. Cells that will proliferate must first pass a control
point known as the restriction point. This is a control
circuit that determines whether internal and external
conditions are suitable for proliferation. Malfunctions of
the restriction point lead to one of the most terrifying
perturbations of the cell cycle: cancer. If DNA damage
is detected during G1 phase, a checkpoint halts cell
cycle progression until either the damage is repaired or
the cell dies. The chapter includes a section on stem
cells and concludes by considering the role of one of
the most famous cell-cycle proteins, p53, in cell-cycle
control.
Cells that decide to proliferate must replicate their
DNA in a timely and accurate manner. Chapter 42
explains the mechanism of DNA replication during the
S phase, including the selection of sites on DNA to initi-
ate replication, the enzymes that copy the DNA, the
regulation of replication by the cell-cycle machinery,
the organization of replicating chromosomes within the
nucleus, and the checkpoints that help the cells to cope
with various problems that they encounter along the
way. Chapter 43 discusses the G2 phase, during which
cells conduct a final “cockpit check” before embarking
on the irreversible process of division. This is also the
last point in the cell cycle at which the genome is
scanned for damage so that it can be repaired before
division. A checkpoint restrains cells from entering into
mitosis if damaged DNA is detected, and the chapter
briefly explains the major pathways of DNA repair.
Chapter 44 describes mitosis, certainly the most
dramatic and complex program in the cell cycle. Mitosis
696
has been studied since the 1800s, but technical advances
have considerably advanced our understanding of how
it is accomplished at the molecular level. Division
requires wholesale reorganization of cellular structures,
including chromosome condensation and the assembly
of the mitotic spindle. In many cells, the nuclear enve-
lope breaks down. Once the chromosomes are all
attached to the microtubules of the mitotic spindle (yet
another important checkpoint here), they are separated
equally and form two daughter nuclei. Finally, cytoki-
nesis separates the two daughter cells.
Chapter 45 considers meiosis, a specialized form of
division that produces the gametes required for sexual
reproduction. In this division, DNA recombination is key
to segregation of the chromosomes. A number of arcane
terms are used to describe the specialized structures
and processes involved. The chapter then explains how
problems with meiosis can lead to genetic diseases and
how studies of chromosome segregation in yeast led to
an understanding of why birth defects become more
prevalent as human mothers age.
Chapter 46 closes the book with a discussion of what
happens when cells commit suicide by apoptosis,
necroptosis, and autophagy. This is not, strictly speak-
ing, a cell-cycle event but instead represents several alter-
native pathways, each with its own machinery and
signaling systems. Apoptosis sometimes results when it
all “runs off the rails” and cells receive insults from which
they cannot recover. But cell death is not always bad:
apoptosis is an essential part of development of meta-
zoan organisms, homeostasis of their organs and tissues,
and can be a last-ditch defense against viral infection.
Malfunctions of apoptotic pathways can lead to cancer.
The concepts that are discussed in this section of the
book build on the ideas in earlier sections. Cells are
wonderfully complex systems whose behavior is driven
by the laws of chemistry and physics. A major challenge
for cell biology in the future is to devise molecular expla-
nations for the complex behaviors exhibited in this
closing section of our book.

Introduction to the Cell Cycle
The cell cycle is the series of events that leads to the
duplication and division of a cell. Research on the molec-
ular events of cell-cycle control revealed that variations
of similar mechanisms operate the cell cycles of all
eukaryotes from yeasts to humans. Furthermore, the
components that regulate cell growth and division also
play key roles in the cessation of cell division that is
required for cells to differentiate. Control of the cell
cycle is of major importance to human health because
cancer is usually caused by perturbations of cell-cycle
regulation. Based on 2010 to 2012 data, 40% of Ameri-
cans will develop cancer during their lifetime.
Although animal cells have a wide variety of special-
ized cell cycles, the cells in the stratified epithelium
that forms skin illustrate the most common types of cell
cycles (Fig. 40.1). The basal layer of the epithelium is
composed of stem cells that divide only occasionally
(see Box 41.2). They can activate the cell cycle on
demand and then return to a nondividing state. Stem cell
populations can replenish themselves by symmetrical
division, but when specific signals induce them to pro-
liferate, usually one daughter cell remains a stem cell and
the other enters a pool of rapidly dividing cells. The
dividing cells populate the upper layers of the epithe-
lium, stop dividing, and gradually differentiate into the
specialized cells that cover the surface.
Like stem cells, fibroblasts of the connective tissue
(see Fig. 28.2) typically are in a nondividing state, but
they can be stimulated to enter the cell cycle following
wounding or other stimuli (see Fig. 32.11). In the most
extreme case, the nervous system contains a few stem
cells and a few dividing glial cells, but most neurons,
once differentiated, can live for more than 100 years
without dividing again.
Principles of Cell-Cycle Regulation
The goal of the cell cycle in most cases is to produce
two daughter cells that are accurate copies of the parent
CHAPTER 40 
(Fig. 40.2). The cell cycle integrates a continuous growth
cycle (an increase in cell mass) with a discontinuous
division or chromosome cycle (the replication and
partitioning of the genome into two daughter cells). The
chromosome cycle is driven by a sequence of enzy-
matic cascades that produce a sequence of discrete
biochemical “states” of the cytoplasm. Progress through
the cell cycle is ratchet-like and irreversible because each
new state arises not only by expression or activation of
a new cohort of activities, but also by destruction or
inactivation of key activities characteristic of the pre-
ceding state. Later sections of this chapter explain
these mechanisms.
Phases of the Cell Cycle
In describing the cell cycle, it is convenient to divide the
process into several phases. Recognition of these phases
began in 1882, when Flemming named the process of
nuclear division mitosis (from the Greek mito, or
“thread”) after he first observed the condensed chromo-
somes. Mitosis was a clear cell cycle landmark, and the
rest of the cell cycle between mitoses was called inter-
phase (Box 40.1).
Once DNA was recognized as the agent of heredity in
the 1940s, it was deduced that it must be duplicated
at some time during interphase so that daughter cells
can each receive a full complement of genetic material.
In 1953, a key experiment identified the relationship
between the timing of DNA synthesis and the mitotic
cycle (Fig. 40.3). This defined the four cell-cycle phases
as they are known today (see Fig. 40.2).
Each cell is born at the completion of the M phase,
which includes mitosis, the partitioning of the chromo-
somes and other cellular components, and cytokinesis,
the division of the cytoplasm. The chromosomal DNA is
replicated during S phase (synthetic phase). The remain-
ing two phases are gaps between mitosis and the S
phase. The G1 phase (first gap phase) is the interval
697
698 SECTION X  n  Cell Cycle

Death Final stage of

differentiation
in skin

Cessation
of cycling
Terminal
differentiation

Rapid
cell cycles
Expansion of
population

Infrequent
cell cycles
Renewal of
Activation stem cell
population

A B Stem cell

FIGURE 40.1  CELL CYCLES IN A STRATIFIED EPITHELIUM. A, Light micrograph of a section of skin, a stratified squamous epithelium,
stained with hematoxylin and eosin (H&E). B, Diagram showing the different types of cell cycles at the various levels of this epithelium.

A. Cell-cycle details Mitosis

B. Cycle phases in cultured cell
(not to scale)
M Cell-cycle phase Length (hours)
G1 10
Check for Check for
damaged or chromosome S 7.5
unduplicated attachment to Cytokinesis
G2 3.5
DNA mitotic spindle
M 1.0
Generation time 22
DNA
ENLARGED VIEW G0
OF CHROMOSOME

C. Time-scaled diagram
G2 (times in hours)
Growth G
1
in mass 22 0
21
Cohesion M
established
in S phase Check for G2
DNA damage
17.5
G1
Check for DNA Restriction point:
S
damage or stalled pass only if environmental
replication forks conditions favorable
S
Chromosome Centrosome
duplication duplication starts 10

FIGURE 40.2  INTRODUCTION TO THE CELL-CYCLE PHASES. A, Diagrams of cellular morphology and chromosome structure across
the cell cycle. B, Length of cell-cycle phases in cultured cells. C, Time scale of cell-cycle phases.
 CHAPTER 40  n  Introduction to the Cell Cycle 699

BOX 40.1  Selected Key Terms

M phase: Cell division, comprising mitosis, when a fully

grown cell segregates the replicated chromosomes to
opposite ends of a molecular scaffold, termed the Nucleus Cell synthesizing DNA
spindle, and cytokinesis, when the cell cleaves between
the separated chromosomes to produce two daughter
cells. In general, each daughter cell receives a comple-
Add 32P, incubate briefly,
ment of genetic material and organelles identical to then wash out free 32P
that of the parent cell.
Interphase: The portion of the cell cycle when cells grow
and replicate their DNA. Interphase has three sections.
The G1 (first gap) phase is the interval between mitosis
and the onset of DNA replication. The S (synthetic)
phase is the time when DNA is replicated. The G2 Photographic
(second gap) phase is the interval between the termina- emulsion
tion of DNA replication and the onset of mitosis. In
multicellular organisms, many differentiated cells no
longer actively divide. These nondividing cells (which
Cell exposes
may physiologically be extremely active) are in the G0 photographic
phase, a branch of the G1 phase. emulsion
Checkpoints: Biochemical circuits that regulate cell-cycle
transitions in response to the physiological condition
of the cell and signals from its environment. Check- VIEW IN
MICROSCOPE
points detect damage to the DNA due to external
agents or problems that arise during DNA replication
and trigger the DNA damage response. Other check-
points detect problems that arise during attachment of
chromosomes to the spindle.

between mitosis and the start of DNA replication. The

G2 phase (second gap phase) is the interval between the
completion of DNA replication and mitosis. All cycling
cells have an M phase and an S phase. The G1 and G2
phases vary in length and are very short in some early
embryos. The following sections describe the stages of
the cell cycle, starting just after the birth of the cell.
G1 Phase
G1 is typically the longest and most variable cell-cycle
phase. When cells are “born” at cytokinesis, they are
roughly half the size they were before mitosis, and
during G1, they grow back toward an optimal size.
During this time, many activities involved in cell-cycle
progression are repressed so that the cell cannot initiate
a new round of proliferation. This repressive control
system is called the restriction point. If the supply of
nutrients is poor or if cells receive an antiproliferative
stimulus such as a signal to embark on terminal differen-
tiation, they delay their progress through the cell cycle
in G1 or exit the cycle to enter G0 (see “G0 and Growth
Control” below). However, if appropriate positive stimuli
are received, cells overcome the restriction point block
and trigger a program of gene expression that commits
them to a new cycle of DNA replication and cell division.
Cancer cells often have defects in restriction point
20% of cells turn the emulsion black
FIGURE 40.3  DISCOVERY OF CELL-CYCLE STAGES. To deter-
mine whether cells synthesize DNA during a defined portion of the cell
cycle or constantly throughout the entire cycle (as is the case in bac-
teria, for example), Howard and Pelc fed a radioactive component of
DNA (32P) to onion root tip cells, spread the cells in a thin layer on a
microscope slide, washed away the 32P that had not become incor-
porated into DNA, and overlayered the slide with photographic emul-
sion. After incubation in the dark, the emulsion was developed like film
and examined with a light microscope. The nuclei of cells active in
replicating their DNA incorporated the radioactive 32P into DNA and
exposed the photographic emulsion above them. Two possible out-
comes were predicted. If cells synthesized DNA constantly during
interphase, then all cells would incorporate the radioactive label. Con-
versely, if each cell synthesized DNA only during a discrete portion of
the cell cycle, then only cells engaged in active replication during the
period of exposure to 32P would expose the photographic emulsion.
When the slides were examined, 20% of the interphase cell nuclei were
labeled, proving that cells synthesize DNA only during a discrete
portion of interphase. Mitotic cells were unlabeled. Assuming that the
cells traverse the cycle at a more or less constant rate, it was possible
to calculate the length of the synthetic phase. Overall, the time between
successive divisions—the generation time—was approximately 30
hours in the root tip cells. If approximately 20% of the cells were
labeled, then approximately 20% of the 30-hour generation time must
be spent in DNA synthesis. Thus, 0.2 × 30, or 6 hours, was spent in
replication. (Data from Pelc HA Sr. Synthesis of DNA in normal and
irradiated cells and its relation to chromosome breakage. Heredity
Suppl. 1953;6:261–273.)
700 SECTION X  n  Cell Cycle
control and continue to grow and attempt to divide even
in the absence of appropriate environmental signals.
G0 and Growth Control
Most cells of multicellular organisms differentiate to
carry out specialized functions and no longer divide.
Such cells are considered to be in the G0 phase. Cells
often enter G0 directly as they exit their last mitosis. G0
cells are not dormant; indeed, they are often actively
engaged in protein synthesis and secretion, and they may
be highly motile. Many G0 cells have a nonmotile primary
cilium, which is an important sensory organelle (see Fig.
38.19). The G0 phase is not necessarily permanent. In
some specialized cases, G0 cells may be recruited to
reenter the cell cycle in response to specific stimuli.
Cell-cycle reentry involves changes in gene expression
and protein stability and disassembly of the primary
cilium, if present. This process must be highly regulated,
as the uncontrolled proliferation of cells in a multicel-
lular organism can lead to cancer.
S Phase
Chromosomes of higher eukaryotes are so large that
replication of the DNA must be initiated at many differ-
ent sites, termed origins of replication. In budding
yeast, the approximately 400 origins are spaced an
average of 30,000 base pairs apart. An average human
chromosome contains about 150 × 106 base pairs of
DNA, approximately 10 times the size of the entire
budding yeast genome, so many more origins are
required. Each region of the chromosome that is repli-
cated from a single origin is referred to as a replicon.
Groups of neighboring replicons cluster in topologically
associating domains (TADs) (see Chapter 8).
Proliferating diploid cells replicate their DNA once,
and only once, each cell cycle. Each origin of replication
is prepared for replication by the formation of a prerep-
lication complex during G1 (a process that is referred to
as licensing). As each origin “fires” during S phase, the
prereplication complex is dismantled and cannot be
reassembled until the next G1 phase. This ensures that
each origin fires only once per cell cycle. The cyclic
nature of origin licensing is driven at least in part by
fluctuations in the activity of cyclin-dependent kinases
and protein destruction machinery (discussed later).
During replication, the duplicated DNA molecules,
called sister chromatids, become linked to each other
by a protein complex called cohesin (see Fig. 8.18). This
pairing of sister chromatids is important for their orderly
segregation later in mitosis (see Fig. 44.16).
G2 Phase
In most cells of metazoans, G2 is a relatively brief period
during which key enzymatic activities that will trigger
the entry into mitosis gradually accumulate and are con-
verted to active forms. When these activities reach a
critical threshold level, the cell enters mitosis. Along the
way, the chromatin and cytoskeleton are prepared for
the dramatic structural changes that will occur during
mitosis. If damaged DNA is detected during G2, a check-
point activates the DNA damage response and delays
entry of the cell into mitosis.
M Phase
During M phase (mitosis and the subsequent cytokine-
sis), chromosomes and cytoplasm are partitioned into
two daughter cells. Mitosis is normally divided into five
discrete phases.
Prophase is defined by the onset of chromosome
condensation and is actually the final part of G2 phase.
TADs disassemble inside the intact nucleus and mitotic
chromosomes begin to form their characteristic array
of loops (see Chapter 8). In the cytoplasm, a dramatic
change in the dynamic properties of the microtubules
decreases their half-lives from approximately 10 minutes
to approximately 30 seconds. The duplicated centro-
somes (centrioles and associated pericentriolar material
in animal cells; see Fig. 34.14) separate and form the two
poles of the mitotic spindle.
Prometaphase begins when the nuclear envelope
breaks down (in higher eukaryotes) and chromosomes
begin to attach randomly to microtubules emanating
from the two poles of the forming mitotic spindle. Other
microtubules originate on chromosomes and within the
mitotic spindle. As both kinetochores on a pair of sister
chromatids attach to microtubules from opposite spindle
poles, the pair of chromatids slowly moves to a point
midway between the poles. When all chromosomes are
properly attached, the cell is said to be in metaphase.
The exit from mitosis begins at anaphase with abrupt
separation of the two sister chromatids from one
another. Most cohesion molecules linking sister chroma-
tids are removed without cleavage during prophase in a
process initiated by mitosis-specific phosphorylation.
Proteolytic cleavage of the remaining cohesin molecules
triggers the metaphase-anaphase transition. During ana-
phase, the separated sister chromatids move to the two
spindle poles (anaphase A), which themselves move
apart (anaphase B). As the chromatids approach the
spindle poles, the nuclear envelope reforms on the
surface of the chromatin. At this point, the cell is said to
be in telophase.
Finally, during telophase, a contractile ring of actin
and myosin assembles as a circumferential belt at the
cortex midway between spindle poles and constricts the
equator of the cell. The separation of the two daughter
cells from one another is called cytokinesis.
Control of Cell-Cycle Progression
Control networks and checkpoints regulate progres-
sion of the cell cycle. Checkpoints are biochemical

circuits that detect external or internal stimuli and send
appropriate signals to the cell-cycle system. The restric-
tion point in G1 phase is a control network that
integrates the physiological state of the cell with its
environment, including input from other cells and
interactions with the surrounding extracellular matrix.
Cells must receive appropriate growth stimuli from
their environment to progress past this point in the
G1 phase; if not they may live on without dying or
commit suicide by apoptosis (see Chapter 46).
DNA damage checkpoints operate throughout
inter­phase. If damage is detected, the DNA damage
response initiates a cascade of events that blocks cell-
cycle progression and can also trigger cell death by apop-
tosis. Problems with DNA replication generally produce
single-stranded DNA and activate the DNA damage
response. This response stabilizes stalled replication
forks so that they can be repaired. During mitosis, the
spindle assembly checkpoint delays the onset of
chromosome segregation until all chromosomes are
attached properly to the mitotic spindle.
The DNA damage response regulates cell-cycle pro-
gression in a three-tier pathway (Fig. 40.4). First sensors
detect DNA damage. These sensors activate transduc-
ers, which include both protein kinases and transcrip-
tional activators. The transducers act on effectors that
ultimately block cell-cycle progression and may also
fulfill other functions. Two key protein kinases, ataxia-
telangiectasia mutated (ATM) and ataxia-telangiectasia
and Rad9 related (ATR), lie at the head of the pathway
and may also act as sensors of DNA damage. They acti-
vate two transducer kinases, Chk1 and Chk2, as well as
a transcription factor called p53 that induces the expres-
sion of a cohort of genes that halt cell-cycle progression
by inhibiting cyclin-dependent kinases as well as genes
Problems at
DNA breaks replication forks
Genotoxic
stress
ATM ATM ATR + cofactors Sensors
dimer monomer bind ssDNA
Chk2 p53 Chk1 Transducers
kinase transcription kinase
factor
Cell cycle proteins Effectors
DNA repair proteins
Apoptosis Cell-cycle DNA repair Responses
arrest
FIGURE 40.4  ELEMENTS OF THE DNA DAMAGE CHECK-
POINT AND RESPONSE SYSTEM. ATM, ataxia-telangiectasia
mutated; ATR, ataxia-telangiectasia and Rad9 related; ssDNA, single-
stranded DNA.
CHAPTER 40  n  Introduction to the Cell Cycle 701
that trigger cell death by apoptosis. Chapters 41 and 43
discuss these proteins in detail.
Biochemical Basis of Cell-Cycle Transitions
Transitions between cell-cycle phases are triggered by a
network of protein kinases and phosphatases that is
linked to the discontinuous events of the chromosome
cycle by the periodic accumulation, modification, and
destruction of several key components. This section pro-
vides a general introduction to the most important com-
ponents of this network.
Cyclin-Dependent Kinases
Genetic analysis of the cell cycle in the fission yeast
Schizosaccharomyces pombe identified a gene called
cell division cycle–2+ (cdc2+) that is essential for cell-
cycle progression during both the G1 → S and G2 → M
transitions (Box 40.2). The product of this gene, a protein
kinase of 34,000 Da originally called p34cdc2, is the pro-
totype for a family of protein kinases that is crucial for
cell-cycle progression in all eukaryotes. This mechanism
of cell-cycle control is so well conserved that a human
homolog of p34cdc2 can replace the yeast protein, restor-
ing a normal cell cycle to a cdc2 mutant yeast. Boxes
40.3 and 40.4 present a number of the key experiments
and experimental systems that led to the identification
of the molecules that drive the cell cycle.
Humans have more than 10 distinct protein kinases
related to p34cdc2, although only a few are involved in
cell-cycle control. To be active, each enzyme must associ-
ate with a regulatory subunit called a cyclin. Thus, they
have been termed cyclin-dependent kinases (Cdks).
p34cdc2, now termed Cdk1, seems to function primarily
in the regulation of the G2 → M transition in animal cells.
Cdk2 (plus Cdk4 and Cdk6 in some cell types) is involved
in passage of the restriction point during G1. Cdk2 also
contributes to the G2 → M transition, although Cdk1 is
the only Cdk absolutely essential for this step (Appendix
40.1). Cdk7 is important for activation of other Cdks,
and also appears to participate in transcribing RNA
and repairing damaged DNA. Other Cdks participate in
diverse processes ranging from transcriptional regulation
to neuronal differentiation. Surprisingly, fibroblasts from
mice that lack Cdk2, Cdk4, or Cdk6 are viable; other
Cdks can drive the cell cycle if necessary. The mice suffer
developmental difficulties because those genes are
needed for the differentiation of particular cell types.
Cyclins
Cdks require cyclin binding for catalytic activity (Fig.
40.13). Cyclins were discovered in rapidly dividing inver-
tebrate embryos as proteins that accumulate gradually
during interphase and are abruptly destroyed during
mitosis (see Fig. 40.11). This process of cyclic accumula-
tion and destruction inspired their name.
702 SECTION X  n  Cell Cycle
BOX 40.2  Use of Genetics to Study the Cell Cycle
Studies of the distantly related budding and fission yeasts
Saccharomyces cerevisiae and Schizosaccharomyces pombe
(see Fig. 2.8) were important for understanding the cell
cycle for several reasons. First, the proteins that control the
cell cycle are remarkably conserved between yeasts and
mammals. Second, both yeast genomes are small, simplifying
the discovery of important gene products. Third, genetic
analysis is straightforward, as both yeasts can grow as hap-
loids, and both efficiently incorporate cloned DNA into their
chromosomes by homologous recombination.
These two yeasts evolved very different strategies for cell
division. Budding yeasts divide by assembling a single bud
on the surface of the cell every cell cycle. Fission yeasts
divide by fission across the center of an elongated cell. A
useful feature of using yeast to study the cell cycle is that
the stage of the cell cycle is revealed by the cellular morphol-
ogy in the light microscope. For budding yeast, unbudded
cells are in G1, cells with buds smaller than the mother cell
are in S phase, and cells whose buds are similar in size to
the mother cell are in G2 or M. For fission yeast, cell length
provides a yardstick for estimating cell-cycle position.
The cell cycles of both yeasts differ from those of animal
cells. In budding yeast, much of the 90-minute cell cycle is
spent in G1. Thus, the networks controlling the G1 → S transi-
tion are particularly amenable to study. In contrast, a fission
yeast spends most of its 2-hour cell cycle in G2. S phase
follows separation of sister chromatids and occurs prior to
cytokinesis. Thus, the control of the G2 → M transition is
readily studied in fission yeast. During mitosis, the nuclear
envelopes of both yeasts remain intact, so chromosomes
segregate on a spindle inside the nucleus.
Genetic studies revealed that the yeast cell cycle is a
dependent pathway whereby events in the cycle occur nor-
mally only after earlier processes are completed. The cell
cycle can be modeled as a line of dominoes, each domino
corresponding to the action of a gene product that is essen-
tial for cell-cycle progression (Fig. 40.5) and the nth domino
Model of the cell cycle as a
simple dependent pathway
Wild type
CDC mutant
FIGURE 40.5  THE CELL CYCLE MAY BE MODELED AS A
SIMPLE DEPENDENT PATHWAY. A cell division cycle (CDC)
mutation can block further progression along the pathway at a
characteristic point in the cell cycle.
falling only when knocked down by the (n-1)th domino.
According to the model, mutations in genes that are essential
for cell-cycle progression cause an entire culture of yeast to
accumulate at a single point in the cell cycle (the point at
which the defective gene product first becomes essential).
This is referred to as the arrest point. Fig. 40.5 shows this
by including a “mutant” domino that does not fall over when
struck by the upstream domino. Mutants that meet this cri-
terion are called cell division cycle mutants or CDC
mutants. Genetic screens for CDC mutants have identified
many important genes involved in cell-cycle control.
Because CDC genes are essential for cell-cycle progres-
sion, it is impossible to propagate strains of yeast carrying
CDC mutants unless the mutants have a conditional lethal
phenotype. The most commonly used conditional lethal
mutations are temperature sensitive (ts). Many yeast
temperature-sensitive mutants are viable at 23°C (the per-
missive temperature), but cease dividing at 36°C (the
restrictive temperature). Temperature-sensitive proteins
often have an altered amino acid sequence, but occasionally,
the lack of a gene product can cause a ts phenotype. More
recently, the use of auxin-inducible degrons (see Chapters 6
and 23) has enabled experimenters to study the conse-
quences of depleting an essential protein from yeast in a
matter of minutes.
Fission yeasts with CDC mutants affecting the entry into
mitosis have distinctive morphologies. Cells mutant in Wee1
(a kinase that keeps cyclin-dependent kinase–1 [Cdk1] inac-
tive prior to mitosis) enter mitosis prematurely and are
shorter than normal (Fig. 40.6B). In contrast, cells lacking
Cdc25 (a phosphatase that counteracts Wee1 and activates
Cdk1) are unable to undergo mitosis but continue their
growth cycle, therefore becoming greatly elongated (Fig.
40.6C). This simple morphologic assay allowed straightfor-
ward classification of yeast CDC genes into those that stimu-
late progression through mitosis and those that retard entry
into mitosis.
A. Wild type B. Wee1 mutant C. Cdc25 mutant
FIGURE 40.6  FLUORESCENCE MICROGRAPHS OF
FISSION YEAST CELLS ILLUSTRATING PHENOTYPES OF
CELL-CYCLE MUTATIONS. Cell walls and nuclei are stained.
A, Wild-type cells. B, A wee1 mutation that accelerates entry into
mitosis at the restrictive temperature. C, A cdc25 mutation that
delays entry into mitosis at the restrictive temperature. (Courtesy H.
Ohkura, Wellcome Trust Institute for Cell Biology, University of Edin-
burgh, United Kingdom.)

BOX 40.3  Studies of the Cell Cycle in Vitro
Amphibian oocytes and eggs are storehouses of most compo-
nents needed for cell-cycle progression. Oocytes are arrested
in G2 until a surge of the hormone progesterone causes them
to “mature” into eggs, which are then naturally arrested in
metaphase of the second meiotic division (see Chapter 45).
After fertilization, the embryo of the South African clawed
frog (Xenopus laevis) undergoes a rapid burst of cell divi-
sions. An initial cell cycle 90 minutes long is followed by a
rapid succession of 11 cleavages spaced only 30 minutes
apart to produce an embryo of 4096 cells (Fig. 40.7).
Thirty minutes per cycle is insufficient to transcribe and
translate all the genes needed to make the daughter cells that
are produced at each division. The frog solves this problem
1 Cleavage 11 Cleavages
90 minutes 30 minutes
apart
A B C they are readily accessible to biochemical manipulation. For
Somatic cell enlarged ×10
FIGURE 40.7  CLEAVAGES SUBDIVIDE THE EGG DURING
XENOPUS EARLY DEVELOPMENT. A, Fertilized egg. B, Two-cell
stage. C, Multicellular embryo. Compare size of somatic cell and
egg.
A. Tightly B. Eggs crushed C. Added sperm
packed by centrifugation nucleus with
eggs membrane
removed
Lipid
Centrifuge
hard
Extract
Xenopus
eggs
Pellet
FIGURE 40.8  USE OF XENOPUS EGG EXTRACTS TO STUDY THE CELL CYCLE. A–B, Making a Xenopus egg extract that is
competent to carry out cell-cycle oscillations in vitro. C–F, A cycling Xenopus extract undergoes alternating S and M phases. G1 and G2
phases are minimal (as they are during early development of the frog).
Humans have at least 16 different cyclin proteins that
range in size from 35 to 130 kD. The highly conserved
cyclin box domain, which docks with the Cdk partners,
is the defining structural feature of these proteins. Only
a handful of cyclin isoforms are involved in cell-cycle
control. Of those that are, some function during G1
phase, others during G2 phase, and still others during
M phase.
Positive Regulation of Cyclin-Dependent Kinase
Structure and Function
Cdks monomers are intrinsically inactive, so they depend
on activation by cyclins and are regulated by positive and
CHAPTER 40  n  Introduction to the Cell Cycle 703
by making oocytes extremely large (~500,000 times the
volume of a typical somatic cell) and storing within them
vast stockpiles of the structural components needed to make
cells. As a result, only DNA and a very few proteins need be
synthesized during early embryonic divisions. In addition to
structural components, many factors that regulate normal
cell-cycle progression are also stockpiled in oocytes. These
features make Xenopus oocytes an excellent source of mate-
rial for biochemical analysis of the cell cycle.
Remarkably, it is possible to make cell-free extracts from
Xenopus eggs that progress through the cell cycle in vitro
(Fig. 40.8). Nuclei from G1 cells or haploid sperm nuclei,
when added to these extracts, efficiently replicate their DNA
and proceed through the cell cycle into mitosis, complete
with chromosome condensation, nuclear envelope break-
down, chromosome alignment on a spindle, and anaphase
segregation of sister chromatids without any additions to
the tube. Because these events occur in a cell-free milieu,
example, antibodies and other proteins can be added to the
extracts, and their effect on the cell cycle can readily be
determined. Thus, the Xenopus extract system offers a pow-
erful tool for testing the role of various proteins in the cell
cycle in higher eukaryotes.
D. Reassembly E. DNA replication F. Mitosis
of nucleus
negative controls. Like other eukaryotic protein kinases
(see Fig. 25.3), Cdks have a bilobed structure with the
active site in a deep cleft between a small N-terminal and
larger C-terminal domain. Monomeric Cdks have a flex-
ible T loop that blocks the mouth of the catalytic pocket.
In addition, a short α-helix is oriented such that a glu-
tamic acid required for adenosine triphosphate (ATP)
hydrolysis points away from the catalytic cleft. As a
result, ATP bound by the monomeric kinase cannot
transfer its α-phosphate to protein substrates (see
Fig. 40.13A).
Several different mechanisms regulate Cdk activity
(Fig. 40.14). On one hand, cyclin binding and
704 SECTION X  n  Cell Cycle

BOX 40.4  Discovery of Factors Essential for Cell-Cycle Progression

The best early evidence for the existence of positive induc-
ers of cell-cycle transitions in mammals was obtained in cell
fusion experiments. When cultured cells in S phase were
fused with cells in G1, the G1 nuclei initiated DNA replication
shortly thereafter. In contrast, if S phase cells were fused
with G2 cells, the G2 nuclei did not rereplicate their DNA
until after passing through mitosis. The most dramatic results
were obtained when mitotic cells were fused with inter-
phase cells. This caused the interphase cells to enter into
mitosis abruptly (as judged by nuclear envelope breakdown
and chromosome condensation). The phenomenon was
termed premature chromosome condensation (PCC).
The mitotic inducer could work in any cell-cycle phase (Fig.
40.9). If mitotic cells were fused with cells in G1 phase,
interphase chromosomes condensed into long, single fila-
ments. If the interphase cell was in G2 phase, the duplicated
chromosomes appeared as double filaments. If the inter-
phase cell was in the S phase, the partially replicated chro-
mosomes condensed into a complex pattern of single and
double condensed regions separated by regions of decon-
densed chromatin corresponding to sites where DNA was
actively replicating at the time of fusion.
Working independently, developmental biologists who
were interested in the control of cell division during early
development in frogs also discovered an activity that could
cause interphase cells to enter the M phase. They used a
micropipette to extract a tiny bit of cytoplasm from a mature
egg that was arrested in metaphase of meiosis II and
inject it into oocytes (which are in G2 phase). The oocytes
rapidly entered M phase, with concomitant chromosome
condensation and nuclear envelope disassembly (Fig. 40.10).
This stimulation to enter M phase is called maturation,
and the unknown factor present in the egg cytoplasm
that induced oocyte maturation was termed MPF, or
maturation-promoting factor (now often referred to as
M phase–promoting factor). It was realized early on that
MPF might be related to the inducer of mitosis detected in
the PCC experiments. In fact, extracts from mitotic tissue
culture cells could induce meiotic maturation when injected
into oocytes. Similar extracts from cells in other phases
of the cell cycle did not cause the G2/M phase transition
in oocytes.
Other cell biologists studying protein synthesis in starfish
and sea urchin embryos noticed a curious protein that
seemed to accumulate across the cell cycle but was then
destroyed during mitosis. They were well aware of the work
on MPF, and immediately suspected that their protein,
which they called cyclin, might be somehow involved in
MPF activity (Fig. 40.11).
In a third line of investigation, geneticists working on
yeasts realized that the cell cycle could be dissected through
the isolation of cell division cycle (CDC) mutants (see Box
40.2). The analysis of the cell cycle with these mutants
dominated cell-cycle research to such an extent that many
human genes that are important in cell-cycle control bear
the CDC name if they are related to well-characterized yeast
genes. The best-known genes to emerge from this analysis
were Cdc2 (Cdk1) and Cdc25, both of which were deter-
mined genetically to encode proteins that actively promote
the G2/M transition. Other genes, such as Wee1, were found
to encode activities that act as antagonists that inhibit the
G2/M transition.
When eventually purified from Xenopus eggs (Fig. 40.12),
active MPF consisted primarily of two polypeptides of 32,000
and 45,000 Da. The smaller component of MPF is the
Xenopus equivalent of the fission yeast Cdc2 gene product
(now known as Cdk1). The larger component of Xenopus
MPF is a B-type cyclin. Only 15 years later was it recognized
that fully functional MPF also requires Greatwall kinase
and its small substrates to inhibit protein phosphatase PP2A-
B55δ and give Cdk activity a chance to take off and trigger
mitotic entry.

A. M–G1 fusion B. M–S fusion C. M–G2 fusion

5 µm

FIGURE 40.9  FUSION WITH MITOTIC CELLS CAUSES INTERPHASE CELLS TO ENTER MITOSIS PREMATURELY, NO MATTER
WHERE THEY ARE IN THE CELL CYCLE. The resulting prematurely condensed chromosomes are single threads if the interphase cell
was in G1 phase (A), double threads if the cell was in G2 phase (C) and a complex mixture of both interspersed with uncondensed regions
if the cell was in S phase (B). M, mitosis. (From Hanks SK, Gollin SM, Rao PN, et al. Cell cycle-specific changes in the ultrastructural orga-
nization of prematurely condensed chromosomes. Chromosoma. 1983;88:333–342.)
 CHAPTER 40  n  Introduction to the Cell Cycle 705

BOX 40.4  Discovery of Factors Essential for Cell-Cycle Progression—cont’d

A B C D

Meiotic Nucleus
spindle Nuclear disassembly Meiotic
and chromosome spindle
condensation

Suck out Inject it Oocyte enters

cytoplasm into oocyte M phase
Egg Fully grown oocyte Egg
FIGURE 40.10  THE EXPERIMENT THAT IDENTIFIED MATURATION-PROMOTING FACTOR. A, The box shows the meiotic spindle
in a Xenopus egg arrested in metaphase II of meiosis. B, The box shows the interphase nucleus in a mature oocyte. Following injection of
MPF, the nucleus disassembles, mitotic chromosomes form (C), and the cell assembles a meiotic spindle (D). Disassembly of the oocyte
nucleus and entry into M phase is called maturation, and the factor triggering this event was named maturation-promoting factor (MPF).

A. SDS gel of
proteins in
A purified MPF
75
B 97

Molecular weight (kDa)

67
B
Intensity of bands A and B

50

43
Cyclin B
A

25

30
Cdk 1

Cleavage Induction of M phase (%) 0 0 0 0 50 85 25 0 0 0

index
0 B. H1 kinase
0 1 2
activity
Time (hours)
Fraction number 6 9 12 15
FIGURE 40.11  EXPERIMENTAL IDENTIFICATION OF A
CYCLIN. Newly synthesized proteins (labeled with 35S-methionine) FIGURE 40.12  PURIFICATION OF MATURATION-PROMOT-
in fertilized sea urchin eggs were separated by sodium dodecylsul- ING FACTOR (MPF). A, Sodium dodecylsulfate (SDS) polyacryl-
fate (SDS) polyacrylamide gel electrophoresis. It was noted that the amide gel electrophoresis of fractions from the final step of the
protein labeled A (which was named cyclin) first accumulated, was purification. The numbers at the bottom show the percentage of
greatly reduced at the metaphase/anaphase transition, and then oocytes that entered M phase when a portion of each column frac-
began to accumulate again. Protein B, which is not involved in  tion was injected (the classical MPF assay). The roughly 32-kD band
cell-cycle regulation, accumulated progressively over this time. is Cdk1 (p34cdc2). The roughly 45-kD band is cyclin B. B, Assay of
“Cleavage index” refers to the percentage of dividing cells observed the ability of the column fractions to phosphorylate histone Hl. This
in the microscope at varying times after fertilization. (From Evans T, is a standard assay for active Cdk enzymes. (Modified from Lohka
Rosenthal ET, Youngblom J, et al. Cyclin: a protein specified by M, Hayes MK, Maller JL. Purification of maturation-promoting factor,
maternal mRNA in sea urchin eggs that is destroyed at each cleav- an intracellular regulator of early mitotic events. Proc Natl Acad Sci
age division. Cell. 1983;33:389–396.) U S A. 1988;85:3009–3013.)

phosphorylation of the T loop stimulate enzyme activity.
On the other, Cdks are inhibited by phosphorylation of
residues adjacent to the ATP-binding site and binding of
inhibitory proteins.
Cyclin binding causes the T loop to retract away from
the mouth of the catalytic pocket (see Fig. 40.13B). In
addition, the secondary structure of the N-terminal
domain is altered, allowing the bound ATP to assume a
conformation suitable for reaction with substrates.
Despite these changes, the Cdk–cyclin complex has
low catalytic activity. Full activation of most Cdks
requires a kinase called Cdk-activating kinase (CAK),
which phosphorylates a threonine in the T loop of the
Cdk (this threonine gives the loop its name). In
706 SECTION X  n  Cell Cycle

A. Cdk2 B. Cdk2–cyclin A C. Active Cdk2–cyclin A

N lobe Active site

PSTAIR ATP
helix

T loop

C lobe Cyclin A Phosphorylation

site

FIGURE 40.13  ATOMIC STRUCTURES OF CYCLIN-DEPENDENT KINASES. A, Cdk2. The PSTAIR helix, found in most Cdks, is named
after a sequence of six amino acids that binds to cyclins (one letter code). (For reference, see Protein Data Bank [PDB; www.rcsb.org] file 1DM2.)
B, Cdk2–cyclin A (kinase at basal activity level). (PDB file 1FIN.) C, Cdk2–cyclin (kinase fully active following phosphorylation of threonine160). (PDB
file 1JST.) ATP, adenosine triphosphate.

vertebrates, CAK is composed of Cdk7-cyclin H. The A. Kinase activation B. Inactive forms

phosphorylated threonine fits into a charged pocket on
the surface of the enzyme, flattening the T loop back
even farther from the mouth of the catalytic pocket (see INK4
Cyclin
Figs. 40.13C and 40.14A). This stimulates the catalytic
activity up to 300-fold, in part because the flattened Cdk
T loop forms part of the substrate-binding surface. Threo-
nine phosphorylation also stabilizes the association of Cyclin
Inactive kinase cannot
Cdk2 with cyclin A. bind
In addition to their cyclin partner, Cdk1 and Cdk2 Cyclin
bind an additional small Cdc kinase subunit (Cks) protein
to their C-terminal domain, away from the active site.
Bound Cks enables the kinase to better hold onto its INK4
substrates and increases the efficiency with which Cdks
can phosphorylate substrates at multiple sites (a hall-
mark of Cdk target phosphorylation). Interference
p27 with ATP use

Negative Regulation of Cyclin-Dependent Kinase

Structure and Function
At least two mechanisms slow or stop the cell cycle by
inactivating Cdks (see Fig. 40.14). During G2 phase, the CAK
phosphorylation
protein kinases Myt1 and Wee1 hold Cdk1 in check by
phosphorylating threonine14 and tyrosine15 in the roof of ATP
cannot
the ATP-binding site. These phosphates interfere with bind
ATP binding and hydrolysis. Threonine14 and tyrosine15 Wee1 and Myt1
phosphorylation
are accessible to the regulatory kinases only following
cyclin binding, so this phosphorylation of Cdks depends,
Cdc25
at least in part, on the availability of cyclins. phosphatase
In mammals, three Cdc25 phosphatases (see Fig. dephosphorylation
25.5) reverse these inhibitory phosphorylations. Cdc25A Interference
regulates both the G1 → S and G2 → M transitions and Active kinase with ATP use
is essential for life of the cell. Ccd25B is dispensable for FIGURE 40.14  POSITIVE AND NEGATIVE REGULATION OF
mitosis, but it is essential for the production of gametes CYCLIN-DEPENDENT KINASES. A, Pathway of activation by cyclin
in meiosis. Cdc25C is a target of the G2 DNA damage binding and phosphorylation. B, Pathways of inactivation by inhibitor
binding and phosphorylation. When INK4 binds, twisting of the Cdk
checkpoint that prevents cells from undergoing mitosis
upper lobe blocks cyclin binding or interferes with ATP hydrolysis.
with damaged DNA (see Fig. 43.11), but cells can survive When p27 binds, a loop insinuates into the upper lobe of the Cdk and
without it. blocks ATP binding. (For reference, see PDB files 1B17 [Cdk2-INK4],
A parallel mechanism for inactivating Cdks involves 1FIN and 1BI7 [Cdk2-INK4-cyclin], and 1JSU [Cdk2-p27-cyclin A].)
binding of subunits from two families of small inhibitory CAK, Cdk-activating kinase.

proteins called the cyclin-dependent kinase inhibitors
(CKIs) and inhibitors of Cdk4 (INK4) (for their names,
see Appendix 40.1). When activated in the DNA damage
response pathway, p53 turns on transcription of the CKI
p21, which inhibits Cdk-cyclin A. CKI p27Kip1 inactivates
complexes of Cdk2 and cyclin A by having a protein loop
invade the N-terminal domain of the Cdk, disrupting its
structure and competing with ATP for binding to the
active site (see Fig. 40.14B).
Members of the INK4 family preferentially inactivate
Cdk4 and Cdk6 in two ways (see Fig. 40.14B). First,
binding to monomeric Cdk distorts the orientation of
the N- and C-terminal lobes, so cyclin D does not bind.
INK4 family inhibitors also inhibit preformed Cdk4/6–
cyclin D complexes by binding the Cdk and distorting
the ATP-binding site so that the kinase uses ATP much
less efficiently.
Cdk inhibitors are important for growth regulation
during the G1 and G0 phases of the cell cycle (see Chapter
41). They also play a critical role in the cell-cycle arrest
that occurs in response to DNA damage and to antipro-
liferative signals. Mutations in the INK4 locus are strongly
linked to cancer.
Role of Phosphatases in
Counter-Balancing Cdk Activity
Two important phosphatases counter-balance Cdk activ-
ity in mitosis. Just as Cdks exhibit cyclic behavior and
are activated in mitosis, these counteracting phospha-
tases must also be cyclic—but they are inhibited in
mitosis. Protein phosphatase 1 (PP1), associates with
numerous targets on chromosomes and the mitotic appa-
ratus, and is highly active during mitotic exit. Many
target proteins have a simple loop with the sequence
RVS/TF that inserts into a groove on the enzyme. During
mitosis, Cdk phosphorylation of adjacent sites often
blocks this interaction with substrates. Cdk1-cyclin B
also phosphorylates the PP1 catalytic subunit during
mitosis, thereby inactivating the enzyme.
PP2A is more directly involved in Cdk regulation. It
is a trimeric enzyme with catalytic, scaffolding, and
regulatory subunits. The latter include B55α-δ and
B56α-ε (see Appendix 40.1). PP2A-B55δ is largely
responsible for removing phosphates added by Cdks.
Consequently PP2A-B55δ must be inhibited to allow
Cdks to drive the cell into mitosis. The Greatwall (Gwl)
kinase regulates PP2A. Gwl is unusual, because 500
amino acids are inserted into its large lobe roughly adja-
cent to the T loop (see Fig. 40.13). Phosphorylation
by Gwl allows two small proteins, Arpp19 (cyclic
adenosine monophosphate [cAMP]-regulated phospho-
protein 19) and ENSA (α-endosulfine), to bind and
inhibit PP2A-B55δ. Thus, Gwl confers the necessary
cyclic behavior on PP2A. Understanding how Gwl is
turned on and off is important for developing a full
CHAPTER 40  n  Introduction to the Cell Cycle 707
picture of mitotic regulation, and this is a subject of
active study.
Role of Protein Destruction in
Cell-Cycle Control
During mitosis active Cdk1–cyclin B–Cks phosphorylates
key substrates leading to dramatic reorganization of the
cell and, ultimately, to separation of sister chromatids on
the mitotic spindle. Once chromatids are separated, the
cell must return to a state with low levels of Cdk activity
so that nuclear envelope reassembly, spindle disassem-
bly, and cytokinesis can occur.
Exit from mitosis requires Cdk inactivation by the
ubiquitin-directed proteolytic machinery. The destruc-
tion of A- and B-type cyclins inactivates Cdk1 and Cdk2.
This allows PP1, PP2A-B55δ, and other phosphatases to
reverse the action of Cdks and bring mitosis to a close.
Ubiquitylation also results in proteolysis of a protein
called securin, which regulates the onset of sister chro-
matid separation at anaphase.
Ubiquitin-mediated destruction of cyclins involves a
cascade of three enzymes described in Chapter 23 (see
Fig. 23.3). First, an E1 enzyme (ubiquitin-activating
enzyme) activates the small protein ubiquitin by
forming a thioester bond between the ubiquitin
C-terminus and a cysteine on the enzyme. Activated ubiq-
uitin is next transferred to another thioester bond on an
E2 enzyme (ubiquitin-conjugating enzyme). The E2
often cooperates with an E3, which is important for
imparting substrate specificity, to transfer ubiquitin to
the ε-amino group of a lysine on a target protein. The
resulting polyubiquitinated proteins are usually targets
for destruction by the cylindrical 26S proteasome (see
Fig. 23.8). This large multienzyme complex functions
like a cytoplasmic garbage disposal, grinding target pro-
teins down to short peptides and spitting out intact
ubiquitin monomers for reuse in further rounds of
protein degradation. Its role was originally thought to be
the removal of damaged proteins from the cytoplasm;
however, it is now recognized as a central factor in cell-
cycle control.
The key E3 ligase regulating cyclin proteolysis is a large
(15-subunit) complex called the anaphase-promoting
complex/cyclosome (APC/C) (Fig. 40.15). The APC/C
is inactive during the S and G2 phases of the cell cycle.
Phosphorylation by Cdk1-cyclin B-Cks1 and binding of
the protein coactivator Cdc20 activate the APC/C in
early mitosis. APC/CCdc20 then triggers the metaphase–
anaphase transition.
An important checkpoint, the spindle assembly
checkpoint, regulates APC/CCdc20 during mitosis,
keeping it inactive until all kinetochores are produc-
tively attached to spindle microtubules. The checkpoint
effector is the mitotic checkpoint complex whose for-
mation is triggered by unattached kinetochores. This
708 SECTION X  n  Cell Cycle

SCFSkp2, SCFβ–TrCP
APC/CCdc20 APC/CCdh1 APC
(active) (active)
Cdc20 Cdh1
E2 Cdh1 Degron
SCFSkp2

Cdk
cyclin

P M A T
P M G1 S G2 P M A T
P M G1 APC/CCdh1 surface and backbone

FIGURE 40.15  TWO FORMS OF THE ANAPHASE-PROMOTING COMPLEX/CYCLOSOME (APC/C) CONTROL THE CELL CYCLE.
At the metaphase-anaphase transition, the APC/C with associated Cdc20 triggers the onset of anaphase by signaling the degradation of securin
and cyclin B. During mitosis Cdh1 phosphorylated by Cdk1–cyclin B is unable to bind the APC/C, so APC/CCdh1 activity is low. As Cdk1–cyclin
B activity declines in anaphase, Cdh1 binds the APC/C and APC/CCdh1 drives the exit from mitosis into G1. APC/CCdh1 remains active throughout
G1 but is inactivated following synthesis of the specific inhibitor Emi1. After the onset of S phase, SCF (shown here adding a ubiquitin chain to
a docked substrate) directs the degradation of cell-cycle substrates such as p27Kip1, following their phosphorylation by protein kinases.

complex inhibits APC/CCdc20 by acting as a competitive A. Structure of SCFSkp2-E2 complex

substrate. As a result, cyclin B and securin are stable until Skp 2
the checkpoint is satisfied. A few substrates, including ~50 Å E2
cyclin A, bind directly to the APC/C without requiring
Cdc20, so they are marked for degradation even when
the checkpoint is active. Skp 1 RbX 1
Exiting from mitosis and allowing the G1 cell to
prepare chromatin for DNA replication (see Chapter 42)
requires low Cdk activity and destruction of Cdc20. This
is accomplished late in mitosis by Cdh1, a different Cullin 1
co-activator of the APC/C. Phosphorylation of Cdh1 by
Cdks blocks its binding to the APC/C early in mitosis.
Thus, APC/CCdh1 forms only after cyclin levels (and there- B. Some F-box proteins C. Their target substrates
fore Cdk activity) decline late in mitosis. As cells pass Skp 2
F E2F-1: Cell-cycle regulator
from G1 into S phase, a newly synthesized inhibitory
protein, Emi1, binds to and inactivates APC/CCdh1. This F box
p27Kip1: Cdk2 inhibitor
allows cyclins to accumulate during S and G2. Remark-
Skp 1
ably, APC/CCdh1 is also involved with regulating the
activity of synapses in nondividing neurons. β-TrCP1 Cdc25A: Cdk1 activator
After the G1 → S transition and throughout the remain- F Wee1: Cdk1 inhibitor
der of interphase members of a different family of E3
activities called SCF regulate the levels of proteins that Emi1: APC/CCdh1 inhibitor
control Cdk and other cell cycle factors. SCF is named β-Catenin: Cell-proliferation
Skp 1 regulator
after three of its four subunits: Skp1, cullin, and F-box
protein (Fig. 40.16). SCF is a molecular toolbox built on
FIGURE 40.16  STRUCTURE AND FUNCTION OF SCF.
a bow-shaped scaffold formed by the cullin subunit. The A, Structure of SCFSkp2. Left, SCF recognizes target proteins through
fourth subunit, Rbx1, binds near the C-terminus of cullin its F-box subunit (Skp2 in this case). Right, Ubiquitin is then transferred
and uses a protein motif called a RING finger to dock to from an E2 enzyme. The whole is assembled on a rigid bow-like scaf-
a ubiquitin-linked E2 enzyme. Skp1 binds to the other fold composed of the cullin subunit. Structures of F-box proteins Skp2
end of the cullin, where it provides a docking site for an and β-Trcp (B) and a list of several of their known target proteins (C).
F-box protein that recognizes and binds the substrate.
(The F-box got its name because it was first discovered
in cyclin F.) Humans have 78 F-box proteins, giving SCF SCF is fundamentally different from the APC/C,
enormous versatility. Two examples shown in Fig. 40.16 because it is constitutively active. However, it ubiquity-
are Skp2, which targets the Cdk inhibitor p27 helping to lates substrates only after they have been phosphory-
drive the G1 → S transition, and β-Trcp, which targets lated, often by Cdks. This feature links SCF activity to
Cdc25A, Wee1, and Emi1. the cell cycle.
 CHAPTER 40  n  Introduction to the Cell Cycle 709

1 2 3 4 5 1 2 3 4

Increasing Cdk activity

Cdk1-cyclin B
Cdk2-cyclin A
Cdk2-cyclin E

P M A T P M A T
P M G1 S G2 P M G1
Increasing APC activity

APC/CCdh1 APC/CCdc20

Emi1
SCFSkp2, SCFβ–TrCP
PP2AB55δ, PP1

FIGURE 40.17  DIAGRAM SHOWING THE CHANGING STATES OF THE CYTOPLASM AS CELLS TRAVERSE THE CELL CYCLE.
Between G2 and G1 are shown the various stages of mitosis: P, prophase; PM, prometaphase; M, metaphase; A, anaphase; T, telophase. The
states of the cytoplasm discussed in the text are shown as green arrows across the top. Increased Cdk activity is shown as peaks upwards from
the central bar. Increasing anaphase-promoting complex/cyclosome (APC/C) activity is shown as a mirror image, with peaks going down from
the central bar. APC, anaphase-promoting complex; PP1, protein phosphatase 1; PP2A, protein phosphatase 2A.

Changing States of the Cytoplasm During
the Cell Cycle
The cell cycle is characterized by five discrete physiolog-
ical states of the cytoplasm (Fig. 40.17). Changing levels
of Cdk activity drive transitions between these states,
sometimes counteracted and sometimes reinforced by
targeted proteolysis.
1. Early mitosis. Cdk2-cyclin A peaks in prophase,
followed by Cdk1-cyclin B. When the nuclear enve-
lope breaks down, the APC/CCdc20 starts degrading
cyclin A, but the spindle checkpoint inhibits destruc­
tion of other substrates. When the last chromo-
some has attached correctly to spindle microtubules
(metaphase), the checkpoint is satisfied, and APC/
CCdc20 starts to degrade cyclin B and securin, an inhibi-
tor of a key protease called separase. Their degrada-
tion continues throughout metaphase.
2. Anaphase and mitotic exit. When securin levels fall
below a critical threshold, active separase cleaves a key
component of the cohesin ring (see Fig. 8.18). This
triggers sister chromatid separation. Cyclin destruction
continues throughout anaphase and telophase, and
falling Cdk1 activity allows the formation of APC/CCdh1,
which marks Cdc20 for destruction along with the
remaining B-type cyclins. SCFβ-Trcp destruction of Emi1
allows APC/CCdh1 to be active when it forms.
3. G1 phase. APC/CCdh1 and Cdk inhibitors of the CKI
and Ink4 families cooperate to inhibit Cdk activity.
Low Cdk activity is required for cytokinesis, spindle
disassembly, chromosome decondensation, nuclear
envelope reassembly, reactivation of transcription,
reassembly of the Golgi apparatus, and assembly of
prereplication complexes on the chromosomes.
4. G1–S phase transition. Growth signals from the envi-
ronment promote the transcription of Cyclin E. If
the levels pass a critical threshold, a burst of Cyclin
E-Cdk2 activity allows the cell to pass the restriction
point, leading to synthesis of proteins required for
DNA replication and cell-cycle progression. Cdk phos-
phorylation targets the CKI peptides for destruction
by SCF allowing Cdk2 to become activated. In addi-
tion, APC/CCdh1 is inactivated by newly synthesized
Emi1.
5. S–G2 phase. Cdk activity remains high throughout the
remainder of the cell cycle, and SCF continues to
degrade selected proteins tagged by Cdk phosphoryla-
tion. SCFβ-Trcp destruction of Cdc25A keeps Cdk1 inac-
tive, preventing a premature entry into mitosis. The
APC/C remains inactive, allowing mitotic cyclins to
accumulate. It is not known what ultimately triggers
entry into mitosis, but an important factor may be a
switch in the specificity of SCFβ-Trcp, which spares
Cdc25A and instead degrades the Cdk-inhibitory
kinase Wee1.
Although this sounds complicated, the underlying
principles are actually quite straightforward. The follow-
ing chapters discuss the cell-cycle transitions in greater
710 SECTION X  n  Cell Cycle

detail and show how the process is modulated in
response to a changing environment.
ACKNOWLEDGMENTS
We thank Tim Hunt, David Morgan, and Jonathon Pines
for their suggestions on revisions to this chapter.
SELECTED READINGS
Bartek J, Lukas J. DNA damage checkpoints: from initiation to recovery
or adaptation. Curr Opin Cell Biol. 2007;19:238-245.
Brown JS, Jackson SP. Ubiquitylation, neddylation and the DNA damage
response. Open Biol. 2015;5:150018.
Craney A, Rape M. Dynamic regulation of ubiquitin-dependent cell
cycle control. Curr Opin Cell Biol. 2013;25:704-710.
Hartwell LH, Weinert TA. Checkpoints: Controls that ensure the order
of cell cycle events. Science. 1989;246:629-634.
Hunt T. On the regulation of protein phosphatase 2A and its role in
controlling entry into and exit from mitosis. Adv Biol Regul. 2013;
53:173-178.
Lorca T, Castro A. The Greatwall kinase: a new pathway in the control
of the cell cycle. Oncogene. 2013;32:537-543.
Morgan DO. The Cell Cycle: Principles of Control. London: New
Science Press; 2007: 297p.
Nasmyth K. A prize for proliferation. Cell. 2001;107:689-701.
Nurse P. A long twentieth century of the cell cycle and beyond. Cell.
2000;100:71-78.
Primorac I, Musacchio A. Panta rhei: the APC/C at steady state. J Cell
Biol. 2013;201:177-189.
Qian J, Winkler C, Bollen M. 4D-networking by mitotic phosphatases.
Curr Opin Cell Biol. 2013;25:697-703.
Stukenberg PT, Burke DJ. Connecting the microtubule attachment
status of each kinetochore to cell cycle arrest through the spindle
assembly checkpoint. Chromosoma. 2015;124:463-480.
Wieser S, Pines J. The biochemistry of mitosis. Cold Spring Harb Per-
spect Biol. 2015;7:a015776.

APPENDIX 40.1

Inventory of the Enzymes of the Cell-Cycle Engine

Cyclin-Dependent Kinases and Their Cyclin Partners
Kinase Cyclin (+ Other) Partner Function
Cdk1 (p34cdc2) A Mammals: triggers G2 → M transition. Yeasts: triggers G1 → S and G2 →
B1, B2 (Xenopus has 5 B-type M transitions. Cyclin A is synthesized in S and destroyed starting at
cyclins) prometaphase. Cyclins B are synthesized in S/G2 and destroyed
Cdk1–cyclin B binds Cks1 following the completion of chromosome attachment to the spindle.
(Cdc kinase subunit) Cyclins A1, B3 function preferentially in meiosis.
Cdk2 A, E Triggers G1 → S transition. Can be replaced by other Cdks in mouse.
Cdk4, Cdk6 D1–D3 Phosphorylation of the retinoblastoma susceptibility protein (pRb) in G1.
Triggers passage of the restriction point and cyclin E synthesis in some
cell types. Extracellular growth factors control synthesis of D cyclins.
Can be replaced by other Cdks in mouse.
Cdk5 CDK5R1 or CDK5R2 Neuronal differentiation, sensory pathways.
Cdk7 (CAK) H; also binds assembly factor Cdk activation by phosphorylation of the T loop. Also in TFIIH, important
MAT1 for regulation of RNA polymerase II transcription and DNA repair.
Cdk8 C Regulation of RNA polymerase II transcription.
Cdk9 T Regulation of RNA polymerase II transcription.
Cyclin Inhibitors
Inhibitor Cdk Substrates Function
CKI: p21Cip1/Waf1 most Most Cdk-cyclin complexes Induced by p53 tumor suppresser. Cell-cycle arrest after DNA damage.
Cdk-cyclin complexes Binds PCNA (proliferating cell nuclear antigen; see Chapter 42) and
inhibits DNA synthesis. Promotes cell cycle arrest in senescence and
terminal differentiation. At low levels, may help assemble active
Cdk-cyclin complexes.
CKI: p27Kip1 Most Cdk-cyclin complexes Cell cycle arrest in response to growth suppressers like TGF-β and in
contact inhibition and differentiation.
CKI: p57Kip2 Most Cdk-cyclin complexes Important in development of the palate.
INK4: p15Ink4b Cdk4, Cdk6 Cell-cycle arrest in response to transforming growth factor (TGF)-β.
Altered in many cancers.
INK4: p16Ink4a Cdk4, Cdk6 Cooperates with the retinoblastoma susceptibility protein (pRb) in
growth regulation. Cell-cycle arrest in senescence. Altered in a high
percentage of human cancers. This gene overlaps the gene for p19ARF,
an important regulator of the p53 tumor-suppresser protein.
INK4: p18Ink4c Cdk4, Cdk6 Cell-cycle arrest in response to growth suppressers.
INK4: p19Ink4d Cdk4, Cdk6 Cell-cycle arrest in response to growth suppressers.
 CHAPTER 40  n  Introduction to the Cell Cycle 711

APPENDIX 40.1

Inventory of the Enzymes of the Cell-Cycle Engine­—cont’d

Other Components
Enzyme Substrates Functions
Wee1 kinase Cdk1 Y15 Nuclear kinase. Inhibits Cdk1-cyclin B in G2.
Myt1 kinase Cdk1 T14 + Y15 Cytoplasmic kinase. Inhibits Cdk1-cyclin B in G2.
Greatwall (Gwl) kinase Arpp19, ENSA (α-endosulfine) Phosphorylated Arpp19 and ENSA inhibit PP2a, allowing active Cdk1-
(MASTL in humans) cyclin B to accumulate and trigger mitotic entry
Cdc25A phosphatase Cdk1 T14, Y15 Promotes G1 → S transition and G2 → M transition. Essential for life of
the cell.
Cdc25B phosphatase Cdk1 T14, Y15 Promotes G2 → M transition. Essential in meiosis.
Cdc25C phosphatase Cdk1 T14, Y15 Promotes G2 → M transition. Dephosphorylates Cdk1 complexed to
cyclins A, B at T14 and Y15. Not essential for life.
PP2A phosphatase Many proteins phosphorylated Regulated by ENSA/Greatwall. With its targeting subunits B55α-δ and
by Cdk1-cyclin B B56α-ε it regulates many activities during mitotic exit and cytokinesis.
PP1 phosphatase Many targets Associates with many “targeting subunits,” which can be, for example,
regulatory proteins, such as RepoMan or can be structural subunits
of the kinetochore (see Chapter 8). It is inactivated by Cdk
phosphorylation during mitosis, but has a key role in mitotic exit.
APC/CCDC20 Cyclin B, securin many others E3 ubiquitin ligase active during M. Requires high Cdk activity to
function. Destruction of cyclins and other substrates essential for exit
from mitosis. Contains 15 subunits plus the specificity factor Cdc20.
APC/CCdh1 Cyclins A, B, many others E3 ubiquitin ligase active during G1. Requires low Cdk activity to
function. Keeps Cdk activity low in G1 through cyclin proteolysis.
Contains 15 subunits plus the specificity factor Cdh1.
SCF Cyclin E, many others Class of E3 ubiquitin ligases containing Skp1 + cullin + Rbx1 + an F-box
protein. Humans have 78 F-box proteins (Caenorhabditis elegans
has more than 300), acting as specificity factors for substrates
phosphorylated at specific sites, including cyclin E and Cdk inhibitors.
This page intentionally left blank

G1 Phase and Regulation of
Cell Proliferation
D uring the G1 phase of the cell cycle, each cell makes
a key decision: whether to continue through another
cycle and divide or to remain in a nondividing state
either temporarily or permanently. During development
of metazoans, cells exit the cell cycle as the first step
toward forming differentiated tissues. In adults, strict
regulation of the timing and location of cell proliferation
is critical to avoid cancer.
Cells enter G1 phase at the end of a proliferation cycle,
after completing mitosis. To be free to decide whether
to proliferate or differentiate, the cell must inactivate the
remnants of the proliferation machinery from the pre-
ceding cell cycle. This is initiated in late M-phase by
inactivating cyclin-dependent kinases (Cdks [see Chapter
40]) via proteolytic destruction of their cyclin subunits.
This continues in G1 phase and is accompanied by syn-
thesis and stabilization of Cdk-inhibitory proteins. The
absence of Cdk activity activates a regulatory network
that represses the transcription of many genes that
promote cell-cycle progression. While this repressive
network is active, the cell cannot proceed through
the cell cycle. The repression can be switched off if the
cell is stimulated by specific signals from the surround-
ing medium, extracellular matrix, and other cells (see
Chapters 27 and 30). If these signals are diffusible sub-
stances, they are known as mitogens. Mitogens can
trigger another round of DNA replication and mitosis,
but first, the cell must pass a major decision point in G1
called the restriction point (Fig. 41.1).
In metazoans, many cells cease cycling, either tempo-
rarily or permanently, exiting the cell cycle into a state
known as G0 (Fig. 41.1). This frequently accompanies
their acquisition of specialized, differentiated character-
istics. Occasionally, it is desirable in tissues for cells in
G0 to reenter the cell cycle to replace lost cells. Special-
ized cells called stem cells fulfill this role in tissue
maintenance.
This chapter describes how cells decide whether to
exit from the cell cycle into the G0 phase, how they return
CHAPTER 41 
to the cycle from G0, and how they regulate their progress
through the G1 phase. It also considers some of the points
at which defects in cell cycle control lead to cancer.
G0 Phase and Growth Control
Cells stop cycling in three ways. First, they may receive
external signals instructing them to withdraw from the
cell cycle, enter into G0, and differentiate, as discussed
later. Second, cells may find themselves in an environment
with insufficient mitogens to drive proliferation. Under
these conditions many cell types enter a transient nondi-
viding state known as quiescence, while they wait for
conditions to improve. Third, cells that have suffered DNA
damage because of a loss of cell-cycle control undergo
senescence, or may, in some cases, commit suicide by
apoptosis (see Chapter 46). Senescence is a permanent
nondividing state that is physiologically distinct from G0
and from which cells normally cannot exit. One physiolog-
ical signal that can lead to senescence is a critical shorten-
ing of the telomere regions of the chromosomes in cells
Lack of mitogens
Antiproliferation signals
G2 M (eg, contact inhibition,
TGF-β telomere damage)
G0
Restriction point:
Regulates G1 progression Proliferation signals
to S phase Nutrients
S Blocks cell-cycle progression G1
unless nutrients & mitogens
are continuously present
FIGURE 41.1  CELL CYCLE SHOWING MAJOR LANDMARKS
IN THE G1 PHASE. TGF-β, transforming growth factor–β.
713
714 SECTION X  n  Cell Cycle
TUMOR
Blood vessels
Normal epithelial cells
NORMAL
FIGURE 41.2  DISRUPTION OF NORMAL TISSUE ARCHITEC-
TURE BY UNCONTROLLED PROLIFERATION OF CANCER
CELLS. Lower right, Normal thyroid tissue. Upper left, A thyroid tumor
with loss of the normal gland structure. (Courtesy Clara Sambade,
IPATIMUP, Porto, Portugal.)
that have divided more than a critical number of times.
Overexpression of telomerase (see Fig. 7.14) can, when
combined with suitable mitogenic stimuli, prevent cells
from undergoing senescence in tissue culture.
Most cells in multicellular organisms are differentiated
(adapted to carry out specialized functions) and no
longer divide. They typically form specialized tissues,
each with a distinctive structural organization that is
important for its function. Unscheduled cell division can
severely disrupt the organization of such tissues (Fig.
41.2). Accordingly, tissues strictly regulate both the loca-
tion and the frequency of cell division. In most tissues,
divisions normally occur at a low rate, producing new
cells in numbers just sufficient to replace those that die.
Under special circumstances, however, such as in
response to wounding (see Fig. 32.11), the rate of cell
division may increase dramatically. This highlights an
important constraint on cell-cycle control in multicellular
organisms: To make organized tissues, cells must exit
from the cell cycle, but some cells must also retain the
ability to reenter the active cell cycle when needed to
repair injuries or replace worn-out cells.
Cells that stop cycling to differentiate are said to have
left the cycle and entered a nonproliferating state called
G0 (Fig. 41.1). G0 may last hours or days, or even for the
life of the organism, as it does for most neurons. It is
important to note that nondividing cells are not
dormant: G0 cells can be biochemically very active and
continue to expend large amounts of energy for many
Contact
inhibition
Cdk2–cyclin E–p21
Cdk2–cyclin E–p27
(inactive)
Differentiation p27
signals Cdk2–cyclin E
p21
p16 p27
Senescence Cdk4-p15
p15
Cdk4–cyclin D–p27 Cdk4-p16
R (inactive)
TGF-β Cyclin D
(degraded)
Various signals Cdks inactive
stimulate synthesis Hypophosphorylated Rb
of Cdk inhibitors binds E2F and inactivates genes
required for cell-cycle progression
FIGURE 41.3  HOW EXTERNAL STIMULI ACT ON CDK INHIBI-
TORS TO CAUSE CELLS TO ENTER A NONDIVIDING G0 STATE.
TGF-β, transforming growth factor–β.
BOX 41.1  Cdk Inhibitor Scorecard
The regulation of G1 progression requires the action of
two families of Cdk inhibitory proteins (see Chapter 40).
Cyclin-dependent kinase inhibitors (CKIs), which include
p21Cip1 and p27Kip1, usually inhibit all Cdks and block cell-
cycle progression, but under special circumstances, they
can actually activate Cdk4/6-cyclin D and promote cell
proliferation during G1. Cell-cycle blocks imposed by CKIs
tend to be temporary. INK inhibitors, which include
p15Ink4B and p16Ink4a, are specialized at inhibiting Cdk4/6-
cyclin D. These can promote a profound and often perma-
nent cell-cycle arrest by activating the Rb pathway of gene
repression.
ongoing processes. Because of turnover, all cells must
continuously synthesize housekeeping proteins. They
must also expend energy to maintain intracellular pH
and ionic composition and to power intracellular motil-
ity. In addition, many specialized G0 cells consume large
amounts of energy to synthesize and secrete protein
products and generate action potentials. Energy metabo-
lism is particularly dramatic in muscle cells that are
responsible for all body movements. Thus, most G0 cells
should be regarded as active cells that just happen no
longer to be engaged in cell division.
Transforming growth factor–β (TGF-β) is an example
of an external signal that arrests progress through the
cycle and regulates differentiation and tissue morpho-
genesis (Fig. 41.3). TGF-β stimulates a receptor serine/
threonine kinase that activates SMAD transcription
factors (see Fig. 27.10). SMADs suppress Cdk-4 synthesis
and increase expression of CKI (cyclin-dependent kinase
inhibitor) and Ink4 class Cdk inhibitors (Box 41.1).
p15Ink4B preferentially inactivates Cdk4–cyclin D and
Cdk6-cyclin D complexes. It also displaces CKI class
 CHAPTER 41  n  G1 Phase and Regulation of Cell Proliferation 715
inhibitors from the Cdk4–cyclin D, permitting them to
transfer to Cdk2–cyclin E complexes in the nucleus. This
further inhibits cell-cycle progression.
The CKI p27Kip1 helps arrest the cell cycle of normal
cells when they become crowded by neighboring cells
(contact inhibition; see ahead, Fig. 41.11) or if their
environment lacks mitogens. Genetic analysis in mice
revealed that p27Kip1 also regulates cell-cycle progression
during development. Mice lacking p27Kip1 are 30% larger
than their normal littermates by several weeks of age.
This is at least partly because cells in many organs
undergo extra rounds of cell division.
An analogous mechanism limits proliferation during
the differentiation of muscle. The transcription factor
MyoD drives expression of the CKI inhibitor p21Cip1,
which helps arrest proliferation and start muscle differ-
entiation (Fig. 41.3). p21Cip1 stops cell-cycle progression
in at least two ways. First, it binds Cdk–cyclin complexes
and stops them from promoting cell-cycle progression.
It also blocks DNA replication by inhibiting the DNA
replication factor proliferating cell nuclear antigen
(PCNA [see Chapter 42]) that is required for DNA poly-
merase δ activity.
BOX 41.2  Stem Cells in Mammals
The defining feature of stem cells is their capacity to self-
renew while producing daughter cells with the capacity to
differentiate into more specialized cells under the control of
intrinsic and environmental cues. Stem cells play a key role
in the development of multicellular organisms in addition
to providing cells for the renewal and regeneration of
adult tissues.
Each multicellular organism begins as a single cell (a fertil-
ized egg) with a genome encoding the information required
to produce an adult. The divisions prior to implantation in
the uterine wall produce a small group of epiblast cells that
go on to form the embryo. The other cells that are produced
at this stage are specialized to support the embryo. Epiblast
cells are termed pluripotent because their progeny can
form all the specialized cells of the adult. Although the plu-
ripotent cells disappear as the embryo develops, epiblast
cells can be propagated and maintained permanently in
culture without losing their pluripotency if optimal condi-
tions are provided. These cell lines are called embryonic
stem cells (ES cells).
Most adult tissues set aside a few tissue stem cells that
have the capacity to renew themselves and to produce
daughter cells that differentiate into a limited range of spe-
cialized cells (see Figs. 28.1, 28.4, and 40.1). Adult stem cells
have diverse patterns of cell-cycle regulation. Some tissue
stem cells continue the cell cycle throughout life. For
example, epithelial stem cells give rise to mature cells that
continuously replace the skin and the lining of the gastroin-
testinal tract. Hematopoietic stem cells in bone marrow
give rise to both short-lived and long-lived differentiated
blood cells. In other organs, such as liver and skeletal muscle,
Both p21Cip1 and p16Ink4a contribute to the permanent
cell-cycle arrest of senescent cells. p16Ink4a activates
inhibitory proteins of the retinoblastoma protein (Rb)
and E2F families (see later) that bind to promoters and
recruit histone methyltransferases, forming heterochro-
matin that permanently inactivates genes required for
proliferation (see Fig. 8.7). Once cells exit the cycle,
multiple redundant pathways block reentry by reinforc-
ing the primary inhibition of Cdk activity. In addition, a
specialized histone variant H1o replaces histone H1 in
G0 cells, resulting in more condensed chromatin. This
represses transcription generally. However, not all gene
expression is suppressed in differentiated cells, many of
which synthesize large amounts of specific proteins (eg,
digestive enzymes secreted by the pancreas).
Moving Into and Out of G0: Stem Cells
Stem cells are professionals at moving back and forth
between G0 and proliferative cell cycles. One of their
roles is to replace worn-out parts of tissues as differenti-
ated cells age or die as a result of various misadventures.
Box 41.2 provides a brief introduction to stem cells.
tissue stem cells are held in reserve unless the tissue is
damaged, when they produce daughter cells to repair the
damage. Stem cells are present even in organs that have a
limited capacity for renewal and regeneration, such as the
nervous system. The potential for regeneration from stem
cells has stimulated research to find ways of using embryonic
or tissue stem cells to repair damaged or diseased organs in
human patients. Stem cells have also been useful for produc-
tion of transgenic animals for scientific research (eg, knock-
out mice).
Discovery and Defining Features of
Stem Cells
Pioneering work on blood cell development (see Fig. 28.4)
established the existence of stem cells and defined many of
the concepts that apply to all types of stem cells. The key
experiment was to inject bone marrow cells from a normal
mouse into a mouse that had been irradiated to kill all the
cells that produce blood cells. Transplantation of bone
marrow cells rescued the irradiated mice from death from
anemia, bleeding, and infections. The transplanted bone
marrow contained precursor cells that formed colonies of
proliferating cells that regenerated the full range of blood
cells. The blood-forming colonies in the spleen, each of
which formed from a single stem cell, contained either one
or, infrequently, several types of differentiating blood cells.
This experimental system first revealed the existence of
several different types of progenitor cells in bone marrow
with the dual capacity to proliferate and to give rise to more
differentiated cells (see Fig. 28.4). These committed pro-
genitor cells have a limited proliferation capacity and can
Continued
716 SECTION X  n  Cell Cycle
BOX 41.2  Stem Cells in Mammals—cont’d
give rise to only to specific subsets of blood cells, such as
red blood cells, platelets, granulocytes, or lymphocytes. In
contrast, there are a very few multipotent hematopoietic
stem cells in bone marrow. They replenish the pool of com-
mitted progenitor cells, ultimately acting as a source of all
types of blood cells, while maintaining themselves through-
out the life of the individual. Antibodies for surface markers
can now be used to distinguish and purify the various types
of hematopoietic progenitors as well as stem cells from mice
and humans. Once separated from the far more numerous
mature and differentiating cells in bone marrow, stem cells
can be used for transplantation into patients with bone
marrow defects.
Most multipotent hematopoietic stem cells are in the G0
phase of the cell cycle. A low level of metabolic activity is
thought to contribute to their longevity, which can poten-
tially exceed the life span of the individual. When stimulated
by demand for more blood cells, growth factors drive multi-
potent stem cells into a cell cycle that culminates in an
asymmetrical division. One daughter cell is another multipo-
tent stem cell. The second daughter cell enters the proliferat-
ing pool of blood cell precursors as a committed progenitor
cell. Committed progenitor cells and their progeny prolifer-
ate vigorously and differentiate into mature blood cells. An
adult human produces more than one million blood cells
every second.
Cytokines and other growth factors regulate proliferation
and differentiation at every stage of blood cell production.
The later stages are best understood. For example, the cyto-
kine erythropoietin acts through a kinase-coupled receptor
to activate a cytoplasmic transcription factor that stimulates
the proliferation and differentiation of the red blood cell
lineage. Other cytokines guide the differentiation of granu-
locytes and monocytes. Hematopoietic stem cells respond to
the same families of growth factors that control other aspects
of development, including Wnts (see Fig. 30.7), Notch (see
Chapter 24), fibroblast growth factor (see Fig. 24.4), and
insulin-like growth factor (see Fig. 24.4). However, too little
is known about these regulatory mechanisms to grow hema-
topoietic stem cells in the laboratory.
Properties of Adult Stem Cells
Years of detailed analysis in the laboratory and clinic estab-
lished hematopoietic stem cells as a model for stem cells in
other tissues. General features include the capacity for self-
renewal and the production of daughters that proliferate and
differentiate. This dichotomy can be achieved by asymmetri-
cal cell divisions guided by the same types of internal cues
that control unequal divisions of cells in early embryos (Fig.
41.4). Symmetrical divisions yielding two daughter stem
cells can also expand the numbers of stem cells during
growth to maturity and during regeneration of damaged
tissues.
Stem cells depend on local environmental cues to main-
tain their status as stem cells. These special environments,
called stem cell niches, are created by tissue cells and the
extracellular matrix. Niche cells anchor stem cells with adhe-
rens junctions and provide cell surface and secreted proteins
that activate the signaling pathways that regulate the cell
Niche cell
Adherens
junction
Stem cell Asymmetrical Stem cell
division +
Committed
cell
Symmetrical Differentiated
division 2 stem cells progeny
FIGURE 41.4  TWO PATTERNS OF STEM CELL DIVISION.
Asymmetrical divisions create two daughter cells: a stem cell that
remains associated with its niche cell to maintain the pool of stem
cells and one that is committed to multiply and produce differenti-
ated progeny. Symmetrical divisions produce two stem cells to
expand the pool of stem cells.
cycle of the stem cell. Some of these factors stimulate divi-
sion; others inhibit differentiation. The niches occupied by
germ cells and neural stem cells from invertebrates are par-
ticularly well characterized. During asymmetrical divisions
of these stem cells, the renewed stem cell stays behind in
the niche, while the daughter that is destined to differentiate
into an egg, sperm, or neuron is released. In bone marrow,
osteoblasts (see Fig. 32.5) and endothelial cells (see Fig.
30.13) provide niches for hematopoietic stem cells.
Epidermal Stem Cells
Skin is an example of a continuously renewing organ with a
considerable capacity for regeneration (see Fig. 40.1). Mul-
tipotential and committed stem cells contribute to both
renewal and regeneration. Committed stem cells reside in
the basal layer of the epidermis. Asymmetrical cell divisions
oriented at right angles to the basal lamina produce two
daughter cells. The daughter touching the basal lamina
carries on as the stem cell. The apical daughter cell divides
multiple times and differentiates into an ascending column
of cells, forming the superficial layers of the epidermis (see
Fig. 35.6). Multipotent stem cells associated with hair folli-
cles give rise to all the cells of the hair follicle and also serve
as a reserve for the committed epidermal stem cells in the
event of injury (Fig. 41.5).
Skeletal Muscle Stem Cells
Small numbers of stem cells reside in a niche sandwiched
between the basal lamina and the giant multinucleated
muscle cells. If the muscle is damaged, these quiescent “satel-
lite cells” multiply and produce muscle cells that regenerate
the tissue. Positive signals for proliferation and differentiation
come through receptor tyrosine kinases and the mitogen-
activated protein (MAP) kinase pathway (see Fig. 27.6) and
other pathways. Restraining signals are provided by myo-
statin, a member of the transforming growth factor (TGF)-β
family (see Fig. 27.10). Inactivation of the myostatin pathway
results in massive enlargement of muscles in mice and
humans. Muscles are capable of regenerating multiple times,
 CHAPTER 41  n  G1 Phase and Regulation of Cell Proliferation 717

BOX 41.2  Stem Cells in Mammals—cont’d

Epidermis

t
haf
ir s
r
Ba
sa

ye
Ha
l la
Sebaceous
gland
Dermis

Bulge

Hair
bulb
A B C
FIGURE 41.5  STEM CELLS FROM SKIN. Multipotent stem cells of the skin reside in the hair follicle bulge (green cells in A, diagram
in C). They move up and repair the epidermis during wound healing, and they move down and generate new hair growth during the hair
cycle. B, Depicts a Nude mouse grafted with the cultured cell progeny of a single “bulge” stem cell and displaying a large tuft of hair, all
derived from that single stem cell. (A and C, From Fuchs E, Tumbar T, Guasch G. Socializing with the neighbors: stem cells and their niche.
Cell. 2004;116:769–778. B, From Blanpain C, Lowry WE, Geoghegan A, et al. Self-renewal, multipotency, and the existence of two cell
populations within an epithelial stem cell niche. Cell. 2004;118:635–648.)

so the stem cell population renews itself during regeneration
or is augmented by stem cells that migrate through the blood
from bone marrow or other tissues.
Cancer Stem Cells
Stem cells may play a role in cancer, acting as a source for
proliferating cells that make up the bulk of the tumor. If true,
this concept helps explain why it is relatively easy to reduce
the size of tumors by targeting dividing cells but difficult to
completely eliminate residual tumor stem cells, which may
divide less frequently. It is thought that the spread of cancer
from the primary tumor to other tissues (metastasis)
requires circulating cancer cells to find locations (niches)
where they can establish themselves as stem cells.
Meristematic Stem Cells in Plants
The growth of plants depends on carefully orchestrated pro-
liferation and differentiation of cells derived from stem cells
called meristems. Through asymmetrical divisions, these
relatively inactive cells give rise to daughters that proliferate
at the tips of shoots and roots. The proliferating cells dif-
ferentiate into specialized tissues such as flowers, while the
stem cells maintain a pool of slowly replicating cells in a
special niche.
Use of Stem Cells to Make
Transgenic Animals
Because embryonic stem cells grow in culture, they can be
manipulated experimentally. They can be transfected with
DNA, and if the proper sequences are present, this DNA can
replace a region of the endogenous chromosome by homolo-
gous recombination. If the modified embryonic stem cells
are subsequently injected into developing embryos at the
blastocyst stage, they are, with low frequency, able to colo-
nize the cell population that will produce germ cells. When
such chimeric embryos grow to adulthood, a proportion of
their gametes will carry a chromosome with the modification
engineered in the embryonic stem cells. Furthermore, this
chromosome will now be inherited by all progeny of that
embryo, giving rise to a line of transgenic animals. This
method is widely used in research to knock out genes by
designing the original DNA construct so that when it enters
the chromosome by homologous chromosome, a critical
region of a target gene is deleted or disrupted. The use of
knockout mice has revolutionized the study of developmen-
tal biology by allowing investigators to determine the func-
tion of specific genes in living animals.
Note the distinction between transgenic animals and
reproductive cloning. “Cloned” animals are produced by
introducing a somatic cell nucleus into an enucleated egg.
Experiments first in frogs and later in mammals, such as
Dolly the sheep, established that egg cytoplasm can repro-
gram gene expression of differentiated cell nuclei, and
enable the development of a cloned animal. Transfer of
nuclei from lymphocytes and olfactory neurons has been
used to derive healthy adult mice. This approach involves
the reversal of epigenetic changes in the nucleus that drove
the differentiation of the adult cell. The molecular mecha-
nisms underlying reprogramming are not well understood
and the success rate at obtaining healthy animals through
nuclear cloning is very low. This cloning does not involve
the use of stem cells, but embryonic stem cells can be
derived from the blastocyst stage of the cloned embryos.
Therapeutic Applications of Stem Cells
Where committed stem cells can be isolated from an adult
organ, it is now possible to regenerate damaged tissues by
transplanting these stem cells from patients themselves or
from donors. The best example is transplantation of bone
marrow stem cells to treat patients whose bone marrow has
been damaged by cancer, chemotherapy, or other disease.
Adverse immunologic reactions are a challenge for trans-
plants from donors other than an identical twin. On one
hand, the immune system of the recipient can reject the
transplanted cells. On the other hand, lymphocytes contami-
nating the donor stem cells can mount an immunologic
attack on the recipient. Using purified hematopoietic stem
cells (ideally, the patient’s own stem cells) rather than mixed
bone marrow cells avoids this problem. Knowing how to
expand hematopoietic stem cells in vitro would be helpful.
This approach is already used for treating burns with epider-
mal stem cells. Normal skin is used as a source of committed

Continued
718 SECTION X  n  Cell Cycle
BOX 41.2  Stem Cells in Mammals—cont’d
skin stem cells, which are multiplied in culture and used to
regenerate all the layers of the skin.
Stem cells might be used to regenerate other damaged
tissues, including the insulin-producing cells that are lost in
Type I diabetes, but appropriate stem cells are not available
for many organs, including the pancreas, brain, and heart.
Indeed, even with appropriate stem cells in hand, much
remains to be learned about how to grow them and then
direct them to differentiate into mature tissues.
Embryonic stem cells can potentially supply all cells nec-
essary to replace any damaged tissue, but sources of human
embryonic stem cells are limited, and acquiring them from
early embryos discarded by fertility clinics is unacceptable
to some people. Patient-specific (autologous) embryonic
stem cells can be derived via somatic cell nuclear transfer
(cloning) followed by expansion of epiblast cells from the
embryo. However, the production of such “artificial” human
embryos is also highly controversial.
Adult stem cells are an alternative to embryonic stem
cells. This approach has the advantage that stem cells can
be isolated from bone marrow, blood, and skin by using
antibodies that recognize specific surface protein “markers”;
however, these specialized stem cells normally do not
produce differentiated cells for regeneration of other tissues.
An alternative method to generate pluripotent stem cells
is based on the transient reintroduction into a differentiated
cell of a group of specific transcription factors commonly
expressed in stem cells. These include Sox2, Oct4, Klf4, and
Reentry Into the Cell Cycle
Cells in the G0 phase may reenter the growth cycle
in response to specific stimulation by mitogens, often
induced by injury or normal cell turnover. Cultured fibro-
blasts are favored for laboratory studies of this process,
as they readily enter a quiescent state mimicking G0
phase when deprived of serum (ie, mitogens and growth
factors) and rapidly reenter the cell cycle when serum
is restored. This response reproduces that found in
wounded tissues. When a living tissue is wounded (see
Fig. 32.11), fibroblasts are exposed to serum that has
leaked from damaged blood vessels. In response, they
divide and colonize the wound, where they lay down
new extracellular matrix to repair the damage.
Serum stimulates three waves of gene expression in
cultured quiescent fibroblasts (Fig. 41.6). The first
includes more than 100 “immediate early” genes.
These include transcription factors of the Jun, fos, myc,
and zinc finger families (see Chapter 10) that activate
numerous downstream genes required for cell growth
and division. Other immediate early genes encode tissue
remodeling factors, cytokines (growth factors), extracel-
lular matrix components (fibronectin), plasma mem-
brane adhesion proteins (integrins), and cytoskeletal
proteins (actin, tropomyosin, vimentin), as well as
Myc. When these factors are ectopically expressed in dif-
ferentiated cells, they can induce the expression of proteins
required for pluripotency, as well as factors required to
change the epigenetic landscape of the cell. This results in
a stable perpetuation of pluripotency. These induced plu-
ripotent stem (iPS) cells, offer the promise that they can
be differentiated into any desired specific cell type for
medical applications. This procedure potentially eliminates
the problem of the availability of autologous stem cells as
many cell types can be reprogrammed to iPS cells. This
technology is rapidly developing, as researchers attempt to
improve the generation of functional cell types from iPS cells
When injected into immunodeficient mice, iPS cells make
tumors containing all cell types, called teratocarcinomas.
This clearly shows that the iPS cells are pluripotent. They
can also generate all cell types in tissue culture when appro-
priate growth factors necessary for self-renewal are removed
from culture media. However, this differentiation is difficult
to control. We cannot yet generate specific fully functional
cell types necessary for therapies with high purity from
pluripotent stem cells, except for a few cell types such as
retinal pigment epithelial (RPE) cells. iPS cell-derived RPE
cells were transplanted for the treatment of macular degen-
eration in 2014, and appeared to be successful in blocking
the degeneration. However, as of 2016, this was the only
clinical trial performed with iPS cell-derived cells. Investiga-
tion on how to control iPS cell differentiation is one of the
main obstacles to be overcome for regenerative medicine.
Immediate early Delayed
transcription factors early genes
Immediate early
Levels of expression
structural proteins
Immediate early
tissue repair
proteins
0 1 2 3 4 5 6
Time (hours)
Cdk inhibitors
Addition
of serum or
growth factor
FIGURE 41.6  PATTERNS OF EXPRESSION OF IMMEDIATE
AND DELAYED EARLY GENES DURING THE RETURN OF
GROWTH-ARRESTED FIBROBLASTS FROM G0 TO ACTIVE
PROLIFERATION AND THE CELL CYCLE.
activities involved in angiogenesis (blood vessel forma-
tion), inflammation, and coagulation. These proteins
facilitate the movement of fibroblasts into wounds and
initiate the repair of tissue damage.
Expression of a second wave of “delayed early”
genes encoding a variety of proteins that are required for
cell growth and proliferation, including cyclin D, pre-
cedes the onset of the S phase. Genes activated after the
onset of the S phase are referred to as “late” genes. Both

delayed early and late gene transcription require synthe-
sis of the transcription factors encoded by immediate
early genes.
These waves of transcription in response to mitogens
enable the G0 cells to pass through a “gate” and reenter
the active cell cycle. This gate is analogous to the restric-
tion point, a critical aspect of G1 control that regulates
the proliferation of all normal cells.
The Restriction Point: A Critical G1
Decision Point
All eukaryotes have a mechanism that operates during
the G1 phase to ensure that cells duplicate their genome
only when the environment is supportive and the chro-
mosomes are undamaged. Healthy yeast cells do not
embark on a round of DNA replication and division until
they reach an appropriate minimum size (actually, they
probably measure their ribosome content and ongoing
rate of protein synthesis). This is important because after
cell division, the daughter cell (bud) is smaller than the
mother and needs more time to grow before it divides
if the population is to maintain a constant cell size.
Whether dividing mammalian cells also monitor their
size is not yet settled.
The influence of cell size on the division cycle was
first demonstrated in an elegant microsurgery experi-
ment (Fig. 41.7). Two Amoeba proteus cells were grown
under identical conditions in parallel cultures. Each day,
a portion of the cytoplasm was amputated from one
amoeba, and the other was left untouched as a control.
Under those circumstances, the cell that suffered the
amputations did not divide for 20 days. During this time,
the control amoeba divided 11 times. When the
Amoeba
Nucleus
B. Experiment
FIGURE 41.7  A MICROSURGERY EXPERIMENT DEMONSTRATES THAT AMOEBAE WILL NOT DIVIDE IF THEY ARE PREVENTED
FROM ATTAINING A SUFFICIENT SIZE. A, Control cell continues to divide. B, Experimental cell does not divide. (For reference, see Prescott
DM. Relation between cell growth and cell division. II: The effect of cell size on cell growth rate and generation time in Amoeba proteus. Exp Cell
Res. 1956;11:86–98.)
CHAPTER 41  n  G1 Phase and Regulation of Cell Proliferation 719
amputations were stopped, the amoeba that had been
operated on divided within 38 hours. The interpretation
of this experiment was that the repeated amputations
prevented the experimental amoeba from ever attaining
a size sufficient to turn on the division program. Evi-
dence suggests that some types of human cells have a
similar size control while others do not.
An essential aspect of growth control during the G1
phase involves monitoring the external environment
for nutrient availability and for signals to proliferate
(mitogenic signals) coming from other cells and from
the extracellular matrix. In a classic experiment, when
three flasks containing populations of cultured cells were
starved by deprivation of amino acids, serum, or phos-
phate respectively, they all stopped cycling in G1. When
the missing ingredients were restored, cells in all three
flasks resumed the cell cycle and entered the S phase at
about the same time. This was surprising because amino
acids are needed to make protein, serum provides growth
factors and mitogens, and phosphate is needed for syn-
thesis of DNA and phospholipids (needed to make mem-
branes). This experiment was interpreted as evidence
that all three types of starvation caused cells to arrest at
a single point in the G1 phase, termed the restriction
point. The restriction point is now defined as the point
after which the cell cycle will proceed even if mitogenic
factors are withdrawn (Fig. 41.8). This supremely impor-
tant aspect of cell-cycle control prevents cells from divid-
ing at inappropriate times and in inappropriate places.
Defects in restriction point control are among the most
common causes of cancer.
Genetic analysis also revealed a point in the G1 phase
after which budding yeast cells appear to be committed
to completion of the cycle. Cells that are starved for
A. Control
720 SECTION X  n  Cell Cycle
Restriction point
M G1
Cell continues to cycle only if Cell committed
extracellular signals are received to cycle
FIGURE 41.8  RESTRICTION POINT. During late G1, cells assess
external and internal stimuli and decide whether to commit to a further
round of DNA replication and division.
nutrients arrest at, or just prior to, this point, termed
START. The mammalian restriction point resembles
yeast START in a number of aspects, but they are not
exactly equivalent, owing to differences between animal
and yeast cell cycles.
Regulation of Cell Proliferation by
the Restriction Point
The restriction point is a molecular “gate” that regu-
lates the expression of genes required for cell-cycle pro-
gression. The gate is based on proteins that are related
to the Rb susceptibility protein and a family of essential
transcription factors known as E2F.
In brief, E2F is a master transcriptional regulator that
activates many of the genes whose products drive DNA
replication and cell-cycle progression. Rb regulates the
cell cycle by binding to E2F and converting it into a
repressor of those same cell-cycle genes. When Rb is
bound to E2F, the cell is said to be arrested at the restric-
tion point. Escape from this arrest involves Cdk activa-
tion and subsequent Rb phosphorylation. This releases
E2F, which then drives cell-cycle progression. An alterna-
tive way to pass the restriction point gate depends on
a potent regulator called Myc, which is discussed
separately later.
Mammals have three Rb-related proteins (pRb, p107,
and p130) and approximately eight E2F family members.
These together constitute a complex multifunctional
network. This chapter refers to the families generically
as Rb and E2F. Some E2F proteins form a heterodimer
with one of three DP (differentiation-regulated transcrip-
tion factor-1 protein) family members. This dimer associ-
ates with the promoter region of E2F target cell-cycle
genes (Fig. 41.9A). Rb binding converts E2F/DP from a
transcriptional activator to the Rb/E2F/DP repressor. Rb
also recruits histone deacetylases, enzymes that remove
acetyl groups from a wide range of proteins, including
the aminoterminal tails of histones (see Fig. 8.7). This
causes compaction of chromatin structure and represses
genes required for cell-cycle progression.
Nonproliferating cells have low Cdk activity in G1
before the restriction point. First, cyclin D messenger
RNA (mRNA) levels are low, so little protein is made.
Second, any cyclin D that is made is retained in the cyto-
plasm, where it is phosphorylated by glycogen synthase
kinase (GSK)-β (see Fig. 30.7) and marked by SCF (Skp,
Cullin, F-box containing complex) for degradation (see
S
Fig. 40.16). Rb/E2F/DP represses the expression of the
genes for cyclins E and A required for Cdk2 activation.
Furthermore, high levels of the CKI class Cdk inhibitor
p27Kip1 inhibit any Cdk2–cyclin E or Cdk2–cyclin A that
happens to be present (see Fig. 40.14).
Signals from mitogens and the extracellular matrix
open the restriction point gate. Stimulation of receptor
tyrosine kinases (see Chapters 25 and 27) or integrins
(see Chapter 30) activates Ras and the mitogen-activated
protein (MAP) kinase/extracellular signal–regulated
kinase (ERK) cascade (see Fig. 27.6). The output of this
cascade stimulates transcription of D-type cyclins (Figs.
41.9 and 41.10) and also inactivates GSK. This allows
cyclin D to accumulate in nuclei. Nuclear cyclin D binds
to and activates Cdk4 and Cdk6 (referred to hereafter as
Cdk4/6–cyclin D), producing an initial pulse of Cdk
activity that is later amplified by Cdk2–cyclin E and
Cdk2–cyclin A. Cdk activity in early G1 is regulated by
adjusting the relative levels of the three D-type cyclins
as well as the levels of Ink4 and CKI inhibitors. This
regulation of cyclin D levels and Cdk inhibitors provides
the crucial link between extracellular mitogens and the
cell cycle.
Mitogens also stimulate transcription of the CKI class
Cdk inhibitor p27Kip1. This protein actually activates
Cdk4/6–cyclin D complexes in two ways. First, the
receptor associated tyrosine kinases Jak or Src phos-
phorylate p27, inducing a structural change within the
Cdk4/6–cyclin D-p27Kip1 complex that activates the
kinase. This links mitogen signaling to Cdk-4/6 activa-
tion. It also promotes the nuclear import of Cdk4/6–
cyclin D. This both leads to full activation of Cdks by
Cdk-activating kinase, a nuclear enzyme (see Chapter
40), and increases the stability of cyclin D. All of this
depends on the continuous presence of mitogenic
signals; if these cease, then cyclin D stability rapidly
declines again, since following dephosphorylation, p27
and p21 again act as Cdk4/6–cyclin D inhibitors.
Cdks push the cell past the restriction point by phos-
phorylating Rb, causing it to dissociate from E2F (Fig.
41.9B; see also Chapter 40 and Appendix 40.1). The E2F/
DP heterodimer remains bound to promoter regions and
now potently activates, rather than represses, the tran-
scription of genes that stimulate cell proliferation. The
proteins produced synthesize DNA (DNA polymerase α,
accessory factors, and enzymes that synthesize nucleo-
tide precursors; see Chapter 42), promote cell-cycle pro-
gression (cyclins E and A, Cdk1, and Cdc25), and regulate
cell-cycle progression (pRb, p107, Emi1).
The chain of events as mitogens break the blockade
on cell-cycle progression imposed by Rb involves a posi-
tive feedback loop as follows. Cdk4/6–cyclin D com-
plexes begin to phosphorylate Rb. This releases some
E2F and permits the initial expression of genes that
 CHAPTER 41  n  G1 Phase and Regulation of Cell Proliferation 721

A. Absence of mitogens
Tyrosine External
kinase Seven-helix signals
receptor receptors
Cyclin D
Ras Cyclin D
Transcription (stable)
Restriction
point
+ Cdk 4/6
M G1 S Kinase phosphorylates p27 p27-P
tyrosine on p27
Cdk 4/6–cyclin D–p27-P
E2F/DP Rb Gene off (active kinase)

Nucleosome Histone Histone Enters

deacetylase N-terminal tails nucleus
Phosphorylates Rb
Histone deacetylation results in chromatin
compaction and repression of transcription
Passage of
restriction point
B. Mitogens present
G1 S G2 M
Tyrosine
kinase Seven-helix FIGURE 41.10  HOW GROWTH FACTORS REGULATE CDK4/6
receptor receptors
Steroid ACTIVITY: THE ROLE OF D-TYPE CYCLINS AND P21. Mitogen-
Ras receptors cAMP induced tyrosine phosphorylation of p27 determines if it assembles
Raf PKA active or inactive CDK4/6–cyclin D complexes–linking assembly of
MEK Synthesis and stability of active kinase to mitogens.
ERK cyclin D, etc

M G1
Histone
4/6 D Rb deacetylase
Cdk 4/6 Cell-cycle genes
cyclin D RNA (cyclins A, E, Cdk1)
E2F/DP pol II DNA replication genes
AC AC AC AC
AC AC AC AC AC
Acetylated "open" chromatin favors transcription
FIGURE 41.9  REGULATION OF CELL-CYCLE PROGRESSION
BY THE E2F/DP/RB COMPLEX. A, The Rb/E2F/DP complex
recruits histone deacetylases (see Chapter 8) and represses specific
genes that are required for cell-cycle progression. This blocks cell-
cycle progression at the restriction point. B, Phosphorylation of Rb by
Cdks alleviates this block and permits passage of the restriction point.
cAMP, cyclic adenosine monophosphate; MEK, mitogen-activated
protein kinase kinase; PKA, protein kinase A.
encode cyclin E, cyclin A, and CDC25A. Active Cdk2-
cyclin E can initiate p27Kip1 degradation, permitting the
rapid accumulation of active Cdks. The restriction point
probably is passed here, and from this point on Cdks
remain active until cyclin destruction in late mitosis.
Cdk2–cyclin E participates in a second wave of Rb
phosphorylation on many sites, leading to the wholesale
liberation of E2F/DP and a surge in transcription of genes
that trigger the onset of DNA replication (S phase entry)
and promote progression through the cell cycle. As the
cell cycle proceeds, Rb phosphorylation is maintained
first by Cdk2–cyclin A and then later by Cdk1–cyclin B
S
until the exit from mitosis. Rb is dephosphorylated at the
mitosis-G0 or G1 transition. This enables it once again to
bind E2F and close the restriction point gate to exit from
the next G1.
The transcriptional regulator Myc drives an alterna-
tive pathway for G1 exit that is also stabilized by
mitogenic signals. Association of myc with one partner
AC activates the transcription of cyclins E and D2. Associa-
tion with a different partner downregulates the tran-
scription of Cdk inhibitors of both the CKI and INK
classes. These activities partly explain why Myc can act
as an oncogene—a protein that helps transform normal
cells into cancer cells (explained further later).
Restriction Point and Cancer
Cancer is a complex class of diseases in which genetic
changes within clones of cells lead to production of cell
populations whose uncontrolled growth can disrupt
tissue function and ultimately kill the individual. Two in
five Americans will be affected by cancer during their
lifetimes. This sounds very high, but considering the
number of cell cycles that are required to produce a
human composed of approximately 1014 cells, and con-
sidering the over 1 million cell divisions that occur per
second in a healthy adult, the disease is actually remark-
ably rare on a per cell basis. This is at least partly because
multiple genetic alterations are required to transform a
normal cell into a cancer cell. Furthermore, the cell cycle
is highly regulated by a web of negative feedback path-
ways that hold in check activities driving cellular
722 SECTION X  n  Cell Cycle
Cells traversing the cell cycle
Normal Cancer
Contact inhibition:
Signaling from cadherin-based
adherens junctions stops cells Transformed cells
from cycling. They arrest in G1. continue to cycle
FIGURE 41.11  LOSS OF GROWTH CONTROL IN TRANS-
FORMED CELLS.
proliferation. In fact, many cancer-causing mutations
that disturb growth control pathways are actually delete-
rious in normal cells and cause them to undergo senes-
cence or commit suicide by apoptosis (see Chapter 46).
Dysregulation of cell proliferation in the G1 phase
causes most types of cancer. This is readily seen in the
laboratory when cells are grown on plastic tissue culture
dishes. Most normal cells proliferate until they cover the
surface completely, forming a monolayer. When the
monolayer is confluent (ie, when cells are touched by
other cells on all sides), signaling initiated by cadherin
proteins (see Fig. 30.7) causes cells to express p27Kip1
and to arrest their cell-cycle progression in G1. This is
called contact inhibition of growth (see Chapter 30,
in the section titled “Cadherin Family of Adhesion Recep-
tors”). Cancer cells lack this control, so they keep pro-
liferating and piling up on top of one another as long as
nutrient and mitogen supplies last (Fig. 41.11). Cells that
lose this aspect of growth regulation are said to be
transformed.
Malfunction of the restriction point is an extremely
common contributor to transformation. Indeed, one or
more components of the p16/cyclin D/Cdk-4/Rb system
are mutated in most human cancers. In addition, several
cancer-causing viruses, such as simian virus 40 (SV40),
papillomaviruses, and adenovirus, make proteins that
facilitate the G1 → S transition by binding Rb and
liberating E2F.
Most cancer cells have abnormalities in the activities
of two classes of genes. Oncogenes are genes whose
inappropriate activation can cause oncogenic (cancer-
ous) transformation of cells. The protein products of
most oncogenes regulate cellular growth and prolifera-
tion. They are typically components of signal trans­
duction pathways that are controlled by feedback
mechanisms. Tumor suppressors are genes whose
inactivation can lead to cancerous transformation. Their
protein products typically inhibit products of oncogenes
or negatively regulate cell proliferation. Several genes
that are involved in restriction point control can act as
either oncogenes or tumor suppressors.
More than 100 oncogenes have been identified thus
far. Most normally function in signal transduction path-
ways that lie downstream of mitogens that stimulate
cell-cycle progression. Their inappropriate activation
can mimic the effects of persistent mitogenic stimula-
tion, thereby uncoupling cells from normal environmen-
tal controls and leading to uncontrolled proliferation and
cancer. For example, Ras activates the MAP/ERK kinase
cascade and accumulation of cyclin D (Fig. 41.9). Ras
genes are mutated in approximately 15% of human
cancers. Inappropriate activation of Ras tricks the cell
into thinking that it is receiving mitogenic signals,
leading it to express cyclin D, phosphorylate Rb, and
proliferate.
Fortunately, in most cells, mutations that result in
uncontrolled proliferation usually lead to DNA damage
(oncogenic stress) and activate a protective pathway
leading to senescence. Other proteins involved in restric-
tion point control can also act as oncogenes if hyperac-
tivated. These include E2F1, cyclin D (overexpressed in
50% of breast cancers), and Cdk4. In each case, activa-
tion of the protein causes inappropriate transcription of
genes promoting cell-cycle progression, bypassing the
restriction point, and leading to uncontrolled cell cycles
and cancerous transformation (Fig. 41.12).
Rb is one of the best-characterized tumor-suppressor
genes. As discussed earlier, a primary function of Rb is
to block cell-cycle progression until sustained mitogenic
stimulation results in its inactivation. It is therefore not
surprising that loss of Rb can lead to inappropriate cell-
cycle progression and cancer. Rare individuals who
inherit one defective Rb gene tend to develop retinoblas-
tomas as children and osteosarcomas as adults. The
cancer arises when the “good” allele is inactivated in a
proliferating cell (this is called a somatic mutation). Such
cancers are rare and occur only later in life in individuals
who inherit two good Rb genes, as two independent
somatic mutations (two “hits”) are required in the same
proliferating cell. Homozygous loss of Rb is lethal during
embryogenesis. This is partly because under some cir-
cumstances, the unleashed E2F can act as a potent
inducer of apoptotic cell death (see Chapter 46).
P16Ink4a is another important tumor suppressor
involved in G1 growth control. Normally, it suppresses
Cdk4/6 activity in nondividing cells (see next section;
also see Chapter 40), thereby reinforcing the ability of
Rb to maintain the growth arrest of G1 cells (Fig. 41.12).
Mutations in the p16Ink4a gene are very common in
cancer, but this is partly because this gene is fascinat-
ingly complex (Fig. 41.16). Mutations in other INK4 Cdk
inhibitors and the CKI protein p27Kip1 are also found in
cancer, although less frequently.
 CHAPTER 41  n  G1 Phase and Regulation of Cell Proliferation 723

KEY Transcription
=

Increasing Cdk activity

prevented Normal cell arrested Nutrients
Transcription of cyclins A, E, = +1 at restriction point Mitogens
Cdk 1 DNA replication genes
STOP Cdk2–cyclin E
No external signals
Rb
Cdk4/6–cyclin D

Normal cell passes M G1 S

Cdk 4/6–cyclin D restriction point

Increasing ubiquitin ligase activity

External signals (active) Restriction point
Rb GO

+1

APC/CCdh1 Emi1
Rb mutant cell passes SCFSkp2
restriction point SCFβ-TrCP

No external signals GO
Degraded proteins: Cyclins Cdc25A p27Kip1
Rb Danger

+1
FIGURE 41.13  PROTEOLYTIC ACTIVITIES IN G1 PHASE.

No external signals Cell with active oncogene Phosphorylated p27Kip1 is recognized by a specific E3
passes restriction point
Active oncogene Cdk 4/6–cyclin D ubiquitin ligase called SCFSkp2 (see Figs. 40.15 and 40.16).
mimics external (active)
signals Rb GO The resulting destruction of p27Kip1 permits a burst of
Danger
Cdk2-cyclin E activation in a positive feedback loop that
+1 rapidly amplifies Cdk activity at the initiation of the S
phase. Later in the S phase, phosphorylation of the DP
Cdk 4/6–p16Ink4a Differentiated cell expressing
subunit of E2F causes its dissociation from DNA, recogni-
(inactive) p16 does not cycle tion by SCF, and destruction. This is essential to com-
External signals plete S phase. SCF also targets cyclins D1 and E for
may be present
STOP destruction, the former when mitogens are limiting and
Rb the latter following autophosphorylation during progres-
sion through the S phase.
Progression throughout G1 requires the activity of
*p16Ink4a
a second E3 ubiquitin ligase known as the anaphase-
Differentiated cell mutant for promoting complex or cyclosome (APC/C). A specific
Cdk 4/6 p16 passes restriction point
cofactor, Cdc20, activates the APC/C to trigger the
metaphase-to-anaphase transition (see Chapter 40). As
External signals Cdk 4/6–cyclin D the cell leaves mitosis, Cdh1 replaces Cdc20 and targets
may be present (active)
Rb GO many cell-cycle regulatory proteins for degradation,
Danger
including Cdc20, A- and B-type cyclins, and factors
+1 involved in DNA replication. Destruction of these target
proteins during mitotic exit and G1 (Fig. 41.13) likely
contributes to the requirement for transcription and de
FIGURE 41.12  HOW ACTIVATED ONCOGENES OR MUTA-
TIONS IN THE RB OR P16 TUMOR SUPPRESSOR PROTEINS novo synthesis of these proteins at the moment cells
CAN LEAD TO ABNORMAL PASSAGE OF THE RESTRICTION decide whether to enter or not a new cycle of genome
POINT AND CANCER. replication.

Proteolysis and G1 Cell-Cycle Progression Integrity of Cellular DNA Monitored by

Just as controlled destruction of proteins is key to the
transition of cells from mitosis to the G1 phase (see
Chapter 40), proteolysis also fulfills several key roles
during progression from G1 into the S phase (Fig. 41.13).
For example, when Cdk2–cyclin E is activated following
cyclin D synthesis, it phosphorylates its inhibitor p27Kip1.
a G1/S Checkpoint
The S phase is a point of no return in the history of any
dividing cell. Given the semiconservative mechanism of
DNA replication, whereby existing DNA strands serve as
templates for newly synthesized strands, any DNA defect
that passes unnoticed through the S phase becomes
724 SECTION X  n  Cell Cycle
Undamaged DNA dsDNA breaks
(ATM)2 ATR not (ATM)2
inactive signaling
E2F drives p53 present E2F
expression of in low amounts active
cell cycle genes in cytoplasm
Normal cell-cycle progression
FIGURE 41.14  THE G1/S CHECKPOINT AND DNA DAMAGE RESPONSE.
perpetuated as a mutation that is transmitted to all future
progeny of the cell. Furthermore, any single-stranded
nick in DNA may become a full-fledged chromosome
break if present during replication. To avoid these prob-
lems, cells have a quality control mechanism to block
entry into the S phase if damaged DNA is detected.
This quality control mechanism involves a check-
point that operates throughout the G1 and S phases
(Fig. 41.14). Checkpoints are biochemical circuits super-
imposed on the normal cell cycle. When activated, the
G1/S checkpoint triggers a DNA damage response
that blocks cell-cycle progression. The block may be
temporary but, in some cases, checkpoint activation
leads to senescence or cell death by apoptosis. Sensor
proteins detect DNA damage and activate this check-
point. In the subsequent DNA damage response, the
sensor proteins activate protein kinases and a key tran-
scriptional regulator that block cell-cycle progression
(see Fig. 40.4).
The DNA damage response has fast and slow compo-
nents: The former is analogous to applying the brakes in
a car; the latter is analogous to removing the wheels and
putting it up on blocks. Both components start with
the protein kinases, ATM and ATR (see Fig. 40.4). ATM
and ATR are related to the lipid kinase phosphatidylino-
sitol 3-kinase (see Fig. 26.7), but their only known
substrates are proteins (see Chapter 40). People lacking
ATM have the disease ataxia-telangiectasia, which is
characterized by immunodeficiency, photosensitivity,
cerebellar degeneration, and an elevated incidence of
leukemias and lymphomas. Loss of ATR is fatal.
DNA damage that disrupts ongoing DNA replication
and produces single-stranded DNA activates ATR, which
phosphorylates and activates a downstream kinase called
Chk1. Chk1 targets the essential phosphatase CDC25A
(Fig. 41.14), marking it for destruction. Because CDC25A
is required to remove inhibitory phosphate groups from
inactive Cdks, its destruction applies a rapid brake to
cell-cycle progression.
ssDNA
ATM ATR active
active localized to
damage site
Mdm2 p53 Chk1
inactive active kinase
in nucleus
Cdc25A
p21 Degraded
Apoptosis Stable cell- Rapid cell-
cycle arrest cycle arrest
DNA double-strand breaks vigorously activate ATM,
which directly and indirectly stabilizes and activates a
critical tumor suppressor, p53. This transcription factor
has been called the “guardian of the genome,” because
in response to DNA damage it also applies a rapid brake
to cell cycle progression. In some cases this arrest leads
to senescence, a permanent cessation of the cell cycle
(putting the car up on blocks).
p53 is very powerful medicine for the cell cycle and
must be carefully regulated by a ubiquitin ligase (E3)
called Mdm2 (mouse double-minute 2; the human ortho-
log is Hdm2) that keeps p53 levels low when the cell
cycle is running normally (Fig. 41.15A). Both p53 and
Mdm2 protein shuttle in and out of the nucleus (see
Chapter 9). When the two proteins associate in the cyto-
plasm, Mdm2 promotes rapid degradation of p53
by the ubiquitin/proteasome system (see Chapter 23).
Because p53 directly stimulates expression of Mdm2, a
feedback loop keeps levels of p53 low. Loss of the Mdm2
gene in mice is lethal unless the p53 gene is also lost.
Both p53 and Mdm2 are phosphorylated following
DNA damage (Fig. 41.15B). These phosphorylations
prevent Mdm2 from binding, so p53 is stabilized, and its
concentration in the nucleus increases dramatically. The
phosphorylations also make p53 a more potent transcrip-
tional activator. The result is a burst of transcription of
p53-regulated genes.
p53 is rapidly activated in response to DNA damage
resulting from hyperproliferation of cells following loss
of restriction point control (oncogenic stress). It also
responds to DNA damage induced by the environment.
If the damage is rapidly repaired, cells continue to cycle,
but if the damage is too severe, p53 induces senescence
or apoptotic cell death (see next paragraph).
In the case of oncogenic stress following loss of
restriction point control (eg, by Ras mutations) E2F stim-
ulates the expression of the tumor suppressor protein
p19Arf (alternate reading frame). This binds and seques-
ters Mdm2 in the nucleolus (Fig. 41.15C), allowing p53
 CHAPTER 41  n  G1 Phase and Regulation of Cell Proliferation 725

A. Healthy cell B. Irradiation C. Oncogene activation

Mdm2 ubiquitin ligase ATM phosphorylates and activates First the irradiation pathway is
directs destruction of p53, which blocks Mdm2 binding activated, then phosphorylation
p53 in cytoplasm of E2F promotes transcription of
p19Arf, which sequesters Mdm2
in nucleoli, releasing active p53

DNA
damage E2F p19Arf
Nucleolus Mdm2
ATM p19Arf
p53 Mdm2 Mdm2
p53-Mdm2 activated cannot p53
bind activated

p53-Mdm2 p53
Ub p53
Ub Active nuclear p53 drives expression Active nuclear p53 drives expression
Ub of proteins that arrest the cell cycle of proteins that arrest the cell cycle
p53-Mdm2 and promote cell death (apoptosis) and promote cell death (apoptosis)

5 3 + Mdm2 p53
p

activated Active p53 also drives expression of the ubiquitin

p53 levels low ligase Mdm2, providing negative feedback on p53
throughout cell Mdm2

FIGURE 41.15  P53 REGULATION AND THE DNA DAMAGE CHECKPOINT IN G1. A, Healthy cell. B, After irradiation, Mdm2 (mouse
double-minute 2) can no longer bind p53, which accumulates in active form in the nucleus. C, After oncogene activation, Mdm2 is sequestered
in the nucleolus, and active p53 accumulates in the nucleus. Activated p53 can induce either cell-cycle arrest or cell death.

to accumulate in the nucleoplasm. There, it activates A. One gene, two promoters

transcription of the CKI p21, stopping the cell cycle. If
this arrest is prolonged, p16INK4A is induced, activating
Rb and leading to permanent cell cycle arrest (senes-
cence). p53 activation can also induce apoptosis by acti-
vating transcription of genes for proapoptotic proteins,
including Bax, BH3-domain proteins, Puma, CD95 (Fas/
Apo1), and Apaf-1 (Fig. 41.15; also discussed in Chapter
46). The decision whether to induce senescence or
death is very complex and may be regulated by different
posttranslational modifications of p53. One way or the
other, p53 serves its function as guardian, as the outcome
is that aberrantly proliferating cells are either perma-
nently silenced or removed, and the body is protected.
p53 is mutated or deleted in about half of all human
cancers. Families that carry a mutated p53 allele have
Li-Fraumeni syndrome with an elevated risk of cancers.
Mice lacking p53 are viable but a defective G1 DNA
damage checkpoint results in cancers while young. This
illustrates a common theme that in many cases check-
point components are not essential for life as long as
nothing untoward occurs. Checkpoints exist primarily
to deal with problems that arise during cell-cycle pro-
gression. However, the elevated cancer rates in
Li-Fraumeni syndrome patients indicate that although
p53 is not essential for the passage of every cell cycle, it
is essential for long-term genetic stability and for main-
taining a proper balance among cell proliferation, dif-
ferentiation, and death during the lifetime of a mammal.
The p19Arf protein (in humans, the protein is smaller
and so is called p14Arf) is extremely unusual, as it is
encoded in a common gene with the Cdk inhibitor
1β 1α 2 3
Ink4a
Arf
B. Two key proteins Ink4a/Arf
p16Ink4a p19Arf
Cdk4-cyclin D1 Rb Mdm2 p53
FIGURE 41.16  DUAL CONTROL OF G1 PROGRESSION BY
THE P16INK4A/P19ARF GENE. This gene encodes two completely dif-
ferent proteins that are key to avoiding cancer. A, The intron–exon
structure of the p16Ink4A/p19Arf gene. B, p16Ink4A and p19Arf negatively
regulate the restriction point via Rb and the DNA damage checkpoint
via p53, respectively.
p16Ink4a (Fig. 41.16). Despite having different promoters
(that respond to different stimuli), the two genes not
only overlap but also share a common exon. Neverthe-
less, the two proteins have no common amino acid
sequences because the shared exons are read in different
frames in the mature mRNAs (mRNAs) for the two pro-
teins. Thus, the p16Ink4a/p14Arf locus encodes two vital
protective factors with different jobs. It is not surprising
that mutations in this key locus are found in between
25% and 70% of human cancers.
G1 Regulation: A Matter of Life and Death
To commit to a new cycle of proliferation, cells must
pass through the restriction point gate. The key to this
726 SECTION X  n  Cell Cycle
gate is the phosphorylation of Rb by Cdks, so signals
such as mitogens that activate Cdks set up a feedback
loop that promotes passage of the gate. Of course, in the
real world, accidents happen, and the G1/S checkpoint
and DNA damage response provide a way to block
cell-cycle progression even in the presence of growth
factors and mitogens. The complex G1 regulatory net-
works have a potential impact on all of us. If they are
disrupted by mutations or damage, the result is cancer.
In fact, very few cancers have intact restriction point
control networks.
ACKNOWLEDGMENTS
We thank Jiri Bartek, Ludger Hengst, Keisuke Kaji, and
Marcos Malumbres for their suggestions on revisions to
this chapter.
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 CHAPTER 42 

S Phase and DNA Replication

A ccurate replication of DNA, which is crucial for cel- protein complex associated with the fork that is actively
lular propagation and survival, occurs during the S phase replicating the DNA is known as the replisome. Accumu-
(DNA synthesis phase) of the cell cycle. This chapter lating evidence suggests that the replisome is stationary at
begins with a brief primer on the events of replication the fork as it “reels in” replicating DNA rather than moving
and then discusses its regulation. Next, the chapter along the DNA like a train on a track.
covers the proteins at origins of replication that ensure
that each region of DNA is replicated once and only once Replicating DNA
per cell cycle. It closes by discussing how the structure
of the nucleus influences replication.
Mechanism of
chain elongation
DNA Replication: A Primer
O O
One of the most exciting byproducts of the Watson- P
O O–
Crick model for the structure of DNA was a predicted H2C O Base 1
mechanism for DNA replication. Because DNA strand Growing DNA chain
pairing is determined by complementary base pairing, H H H H

it was logical to propose the existence of “DNA poly- H

O O
merases,” enzymes that would move along a single strand P
O O–
of DNA, recognize each base in turn, and insert the H2C O Base 2 Free 3'-hydroxyl end
proper complementary base at the end of the growing
chain. Thus, one might have surmised that only a H H H H

single enzyme was required for DNA synthesis. In fact, H

O O
DNA replication in cells involves a complex macromo- P
O O–
lecular machine. H2C O Base 3
In the chemical reaction of DNA replication, the 3′
hydroxyl at the end of the growing DNA strand makes a H H H H

nucleophilic attack on the α-phosphate of the incoming OH H O O–

P
nucleoside triphosphate to form a phosphodiester bond. Nucleophilic O O O–
This incorporates the nucleotide into the growing chain attack P
O O O–
and releases pyrophosphate (Fig. 42.1). This reaction P
requires the presence of a template strand of DNA that O O–
specifies, via base pairing, which of the four nucleoside H2C O Base 4 Entering dNTP
triphosphates is added to the growing molecule. H H H H
The exact site on the chromosomal DNA where replica- OH H
tion begins is termed the origin of bidirectional repli-
FIGURE 42.1  MECHANISM OF DNA POLYMERIZATION. A 3′
cation. As the term bidirectional implies, two sets of
OH group at the end of a growing DNA chain makes a nucleophilic
DNA replication machinery head off in opposite directions attack on the α-phosphate of a triphosphate precursor in the active
from the origin. At the replication fork, one parental site of polymerase (enzyme not shown here). dNTP, deoxynucleoside
DNA molecule splits into two daughters (Fig. 42.2). The triphosphate.

727
728 SECTION X  n  Cell Cycle

Lagging strand
polymerase complex

Lagging strand Polymerase/

5' primase
3'
RNA RPA CMG
Nascent DNA primer Replication Helicase
(Okazaki fragment) fork 3'
Origin of
5'
bidirectional Replisome
replication
5' Nascent DNA
Leading strand
3' Leading strand polymerase complex

FIGURE 42.2  KEY COMPONENTS AND EVENTS AT THE REPLICATION FORK. All enzymes are closely associated in the replisome
complex, but are shown separate here for clarity.

The bidirectional nature of DNA replication causes a A. E. coli chromosome

fundamental problem, as the chemical reaction of DNA
synthesis invariably proceeds in a 5′ to 3′ direction.
Replication of the so-called leading strand, in which
DNA polymerase ε moves in a 3′ to 5′ direction along
the template (laying down nascent DNA in a 5′ to 3′
direction) poses no problems; the polymerase simply
chases behind the replication fork (Fig. 42.2). However,
the other template strand faces in the opposite direction,
apparently requiring a DNA polymerase to synthesize
DNA in the wrong direction as the replication fork
progresses away from the origin (ie, adding nucleotides
in a 3′ to 5′ direction). No DNA polymerase with this
polarity has been found. Instead, this lagging strand
replicates in a series of short segments. Every time
the DNA strands have been peeled apart (unwound)
by 200 nucleotides or so, probably corresponding to
the unwinding of a single nucleosome, a polymerase/
primase complex (Figs. 42.2 and 42.12) initiates DNA
synthesis on the lagging strand, and DNA polymerase
δ runs away from the fork, back toward the replication
origin, again synthesizing nascent DNA in a 5′ to 3′
direction. Thus, lagging strand synthesis proceeds in
bursts in a direction opposite to the overall direction
of fork movement. Synthesis of each lagging strand
fragment stops when DNA polymerase runs into the 5′
end of the previous fragment, displacing the RNA primer
with which it was initiated (see later). Thus, the lagging
strand is copied in a highly discontinuous fashion into
short fragments known as Okazaki fragments (named
after their discoverer [Fig. 42.2]). Fig. 42.12 describes
the enzymes and events at the replication fork in
greater detail.
Origins of Replication
Bacteria such as Escherichia coli replicate their circular
chromosomes using two replication forks starting from
a single origin of replication (Fig. 42.3A), but eukary-
otes must use multiple origins of replication to duplicate
Origin
oriC
500,000 bp
B. Portion of eukaryotic chromosome
Active Active
Dormant Dormant
origin origin
origins origins
130,000 bp
FIGURE 42.3  A, The Escherichia coli chromosome is a simple
replicon with a single origin of replication. In cells, this chromosome
has a complex, highly supercoiled structure. B, Eukaryotic chromo-
somes have multiple origins of replication, most of which remain
dormant unless needed.
their large genomes during the S phase, which can be as
short as a few minutes in some early embryos. These
numerous origins are distributed along the chromosome:
up to 600 to 700 in budding yeast and more than 100,000
in human cells. The origins are distributed so that all the
DNA is replicated in the available time, and many more
origins are prepared than are actually needed.
How is the “firing” of all these origins orchestrated so
that each is used no more than once per S phase? Cells
manage this problem by a mechanism termed licensing,
which ensures that each segment of DNA is replicated
just once per cell cycle. Replication of the origin removes
the license, which cannot normally be renewed until the
cell has completely traversed the cycle and has passed
through mitosis.
The portion of chromosomal DNA replicated by the
two bidirectional forks initiated at a single origin is

termed a replicon. The classic replicon is the E. coli
chromosome (which is 4 × 106 base pairs [bp] in size)
with a single genetically defined replicator site called
oriC (Fig. 42.3). An initiator protein (product of the E.
coli DnaA gene [Fig. 42.13]) binds to this origin and
either directly or indirectly promotes melting of the DNA
duplex, giving the replication machinery access to two
single strands of DNA. Other factors unwind the DNA,
leading to the full assembly of the replisome, which
powers a wave of DNA replication proceeding outward
in both directions along the DNA (a replication “bubble”)
at approximately 750 to 1250 bases per second.
An average human chromosome contains approxi-
mately 150 × 106 bp of DNA. Because the replication
machinery in mammals moves only approximately 20 to
40 bases per second (partly reflecting the fact that the
DNA is packaged into chromatin and partly reflecting the
slower speed of the eukaryotic replisome), it would take
up to 2000 hours to replicate this length of DNA from a
single origin. In most human cells the S phase takes
approximately 10 hours. This means that at least 25
to 125 origins of replication are required to replicate
an average chromosome in the allotted time. In fact,
origins of replication are much more closely spaced
than this. It is estimated that 30,000 to 50,000 origins of
replication “fire” during each cell cycle to replicate
the entire human genome, but many more dormant
origins are licensed for use if necessary. These dormant
origins are essential for resolving replication stress
(see later).
To explain the events at origins of replication, the
budding yeast Saccharomyces cerevisiae serves as a
good example. Its DNA replication is better understood
than that of any other eukaryote and although its origins
of replication are specialized, the proteins that act on
them are conserved across metazoa.
Replication Origins in Saccharomyces cerevisiae
Approximately 400 origins of replication participate in
replicating the budding yeast genome. A major break-
through in understanding DNA replication in S. cerevi-
siae was the identification of short (100 to 150 bp)
segments of DNA that act as replication origins in vivo
when cloned into a yeast plasmid (circular DNA mole-
cule). These autonomously replicating sequences
(or ARS elements) allow yeast plasmids to replicate in
parallel with the cellular chromosomes (Fig. 42.4). ARS
elements are often, although not always, bona fide rep-
lication origins in their native chromosomal context.
Replication always initiates within ARS elements, but not
all ARS elements act as origins of DNA replication in
every cell cycle.
Yeast replication origins are spaced approximately
every 30,000 bp, with a maximum separation of approxi-
mately 130,000 bp. Even this longest interval should
replicate easily within the 30 minutes available during
CHAPTER 42  n  S Phase and DNA Replication 729
A
Plasmid
Grow cells
Selectable
marker gene Introduce into under selective
yeast cells conditions
B
ARS
Plasmid
Grow cells
Selectable
marker gene Introduce into under selective
yeast cells conditions
FIGURE 42.4  PLASMID ASSAY FOR IDENTIFICATION OF AN
AUTONOMOUSLY REPLICATING SEQUENCE (ARS) ELEMENT
(ORIGIN OF DNA REPLICATION) IN BUDDING YEAST. The
plasmid has a selectable marker gene (eg, a gene required for the
synthesis of an essential amino acid) plus (in panel B) an ARS element.
This plasmid is transferred into growing yeast cells that are defective
in the marker gene carried by the plasmid; these cells are then plated
out on agar medium that lacks the essential amino acid. A, A plasmid
lacking an ARS fails to replicate and is lost from the cells. These cells
cannot grow into colonies on plates that lack the essential amino acid.
B, If the plasmid contains an ARS element, it replicates along with the
chromosomal DNA and is maintained in the population. These cells
grow into colonies in the absence of the essential amino acid.
the S phase. Because the number of origins exceeds the
number required to replicate the genome within the
allotted time, some origins need not “fire” every cell
cycle. The probability that any given origin will be used
in a given cell cycle ranges from less than 0.2 to greater
than 0.9. It is important to note that replication by a
passing fork coming from an adjacent origin inactivates
dormant origins. This prevents re-replication of genomic
regions during the cell cycle.
The ARS element does two things to establish an
origin of replication. First, its conserved sequences act
as binding sites for a protein complex that marks it as
a potential origin. Second, it has nearby sequences that
are readily induced to unwind by separating the base-
paired strands.
Budding yeast ARS elements share a common DNA
sequence motif called the ARS core consensus
sequence: 5′-(A/T)TTTAT(A/G)TTT(A/T)-3′ (Fig. 42.5).
Single base mutations at several locations within this
sequence completely inactivate ARS activity. Other, less
well-conserved DNA sequences also contribute to the
activity of the ARS as a replication origin. One of these,
termed B1, together with the ARS core, forms the bind­
ing site for a complex of six proteins termed the origin
730 SECTION X  n  Cell Cycle
ORC
complex
ABF-1 OBR
(origin of
bidirectional 90°
replication) ATP ATP
B3 B2 DNA B1 ARS
unwinding core
A A A
element T T T T A TG T T TT
FIGURE 42.5  ORGANIZATION OF THE AUTONOMOUSLY
REPLICATING SEQUENCE (ARS)-1 ELEMENT. The ORC (origin
recognition complex) binds to the ARS core sequence plus element
B1. B2 is a sequence that can readily be induced to unwind. The OBR
(origin of bidirectional replication) is the site where DNA synthesis
actually begins. B3 is a binding site for an auxiliary factor called ABF-1
that is both a transcriptional activator and an activator of the ARS
element. ATP, adenosine triphosphate. (For reference, see Protein Data
Bank [PDB; www.rcsb.org] file 4X6C.)
recognition complex (ORC [see later section]). The
DNA unwinding element is another short sequence (B2)
located a bit further along the DNA. DNA synthesis
begins at an origin of bidirectional replication midway
between the ORC binding site and the DNA unwinding
element.
ORC was identified by its ability to bind the 11-bp ARS
core sequence (Fig. 42.5). This binding has two notewor-
thy features. First, the subunits of the ORC complex are
AAA adenosine triphosphatases (ATPases; see Box 36.1)
and adenosine triphosphate (ATP) hydrolysis is required
for ORC to bind ARS DNA. Second, in yeast, the ORC
complex remains bound to the origins of replication
across the entire cell cycle. Thus, something other than
the presence of ORC must regulate the periodic activa-
tion of origins in the S phase (Fig. 42.14). In some
metazoan cells, ORC behavior is more complex—for
example, the largest subunit, Orc1, is degraded during
part of the cell cycle.
ARS elements often contain binding sites for other
sequence-specific DNA binding proteins, including tran-
scription factors. For example, a transcription factor
called ARS-binding factor 1 (ABF-1) binds to the B3
sequence within the ARS1 element (Fig. 42.5). Deletion
of the ABF-1 binding site only slightly reduces the ability
of ARS1 to act as a replication origin in vivo and other
transcription factors can substitute for ABF-1.
In addition to their role in DNA replication, several
ORC subunits also regulate heterochromatin formation
and transcription (see Chapters 8 and 10). This crosstalk
between the machinery used for transcription and DNA
replication may explain why regions of chromosomes
with actively transcribed genes typically replicate early
in the S phase (see the next section below). In some
metazoan cells, the Orc6 subunit also functions during
cytokinesis, apparently via interactions with cytoskel-
etal filament proteins independent of its role at replica-
tion origins.
EdU PCNA (t + 30 min) Merge
5 µm
FIGURE 42.6  SUPERRESOLUTION VIEW OF ACTIVE REPLI-
CONS IN A HELA (HENRIETTA LACKS) CELL. EdU (red) was used
to label sites of active replication for 15 minutes followed by washing
out for 15 minutes. Then antibody recognizing proliferating cell nuclear
antigen (PCNA) was used to stain all active replicons in the cell.
Thousands of replicons can be seen, and it is clear that in the 30
minutes between the labelling of the red and green channels, many
new origins of replication have been activated. Scale bar = 5 µm. OMX
superresolution. (Microscopy by Vadim Chagin and Cristina Cardoso,
Technical University of Darmstadt, Germany.)
Replication Origins in Mammalian Cells
Less is known about the structure and function of mam-
malian origins of DNA replication than about ARS ele-
ments in budding yeast. Attempts to develop a mammalian
equivalent to the yeast ARS assay had few successes.
Over the years approximately 30 metazoan origins of
replication were identified by painstaking methods, but
more recently, high throughput methods, including the
sequencing of short nascent strands of replicating DNA
have identified thousands of mammalian replication
origins. Superresolution microscopy can now resolve the
thousands of replicons active at any one time in a human
cell (Fig. 42.6).
Overall, the chromatin landscape influences mam-
malian origins. Important factors include DNA sequence,
DNA modifications, chromatin structure, and nuclear
organization. Although high throughput sequencing
revealed no sequences as specific as the yeast ARS ele-
ments, many origins are associated with short regions of
G-rich DNA. These G-rich regions can form specialized
structures that have fewer nucleosomes and are there-
fore more accessible to the DNA replication machinery.
Indeed, replication origins tend to be located near gene
promoters, where the density of nucleosomes is low.
Such actively transcribed regions of the genome are
packaged into euchromatin with modified histones and
tend to replicate earlier during S phase than regions of
the genome that are not transcribed.
A typical mammalian replicon encompasses about
130 kb and contains approximately four or five licensed
origins-only, one of which is typically used. The first
replicon to be mapped lies just downstream of the
hamster gene for dihydrofolate reductase, an enzyme
that is essential for biosynthesis of thymidine. Investiga-
tors selected cells with this chromosomal region ampli-
fied as hundreds or even thousands of copies (Fig. 42.7)
and looked for the first regions of the amplified DNA to
 CHAPTER 42  n  S Phase and DNA Replication 731

Mapping a cellular replication origin

Add high CYTOPLASM
concentration Cdt1 Geminin
of methotrexate Cells die
Geminin degraded Cdk phosphorylation
Cdks inactive triggers other CMG
Cultured cells components to assemble
Add gradually Prereplication
Mcm2-7
increasing concentration Cells live in high complex
double
of methotrexate over concentration of hexamer
ORC Cdt1
many generations methotrexate 3’
Cdc6 5’

Cdc45 3’ 5’

GINS
Loop domain containing CMG helicases
Restriction
dihydrofolate reductase gene NUCLEUS point
unwind DNA
Normal Chromosome with G1 S
chromosome amplified domain

Base of FIGURE 42.8  COMPONENTS OF THE PREREPLICATION

Potential OBRs
domain (origins of bidirectional COMPLEX BEFORE AND AFTER THE INITIATION OF DNA
replication) REPLICATION. CMG, Cdc45, Mcm2–7, and GINS (Go-Ichi-Ni-San).
DHFR gene
(For reference, see PDB files 3JA8 [for CMG helicase with Cdk45
bound], 3JC5 [for Mcm2–7], and 5F9R [for ORC].)

Dormant Dormant
origins Active origins
Dormant
Active origins
origin origin
DHFR gene β γ Next gene
–30 –20 –10 0 10 20 30 40 50 60
Number of bases (kb)
Initiation zone
FIGURE 42.7  MAP OF DNA REPLICATION LANDSCAPE NEAR
THE DIHYDROFOLATE REDUCTASE (DHFR) GENE. Normal cells
are killed by exposure to methotrexate, but it is possible to select
resistant cell lines by growing them in progressively increasing concen-
trations of the drug, selecting at each stage for cells that survive. Use
of this procedure on hamster cells resulted in a cell line that contains
approximately 1000 copies of a 230,000-bp chromosomal domain
containing the DHFR gene. This region of DNA is replicated using
origins found within a 55,000-bp region adjacent to the DHFR gene.
Most initiation occurs at two specific origins, called β and γ, but other
dormant origins are scattered throughout the entire 55,000-bp region.
replicate. They found that DNA replication can initiate
with low efficiency at a number of sites distributed
across a broad region of approximately 55,000 bp. Two
of these sites, termed Ori-β and Ori-γ (Fig. 42.7) account
for approximately 20% of all initiation in the region.
The emerging view is that the replication machinery
is highly conserved between budding yeasts and verte-
brates but that the location of replication origins is more
flexible in vertebrates. This might reflect both the differ-
ing chromatin landscapes across metazoan genomes (see
Chapter 8) and the diverse range of cell cycles required
to make a complex metazoan.
Origin Licensing and Assembly of
the Prereplication Complex
To preserve the integrity of the genome, each origin of
replication must “fire” no more than once per cell cycle.
We now have a reasonable understanding of the various
solutions to this problem that have been reached by
yeasts and vertebrates.
Recall that yeast ORC is stably bound to replication
origins throughout the cell cycle. However, ORC is not
70 the trigger for DNA replication. Rather, it acts as a
“landing pad” for assembly of a prereplication complex
of other proteins that initiates DNA replication.
Formation of the prereplication complex “licenses”
each origin for a single initiation event as follows. During
late anaphase or very early G1 phase, several proteins,
including Cdc6 and Cdt1, bind to the ORC complex at
origins of replication (Table 42.1). Before the onset of
the S phase ORC-Cdc6-Cdt1 loads a double hexamer of
Mcm proteins (minichromosome maintenance) on the
origin DNA (Fig. 42.8). Mcm proteins were identified in
a screen for genes of budding yeast that are required for
the stability of small artificial chromosomes. Six of these
Mcm genes encode a structurally related group of pro-
teins, termed Mcm2–7, that is required for DNA replica-
tion. ORC-Cdc6–Cdt1 uses ATP hydrolysis to crack open
two hexameric Mcm2-7 rings so they can wrap around
DNA in a head-to-head orientation.
Replication starts when each Mcm2–7 hexamer asso-
ciates with the GINS (Go-Ichi-Ni-San; 5-1-2-3 in Japanese)
complex and the Cdc45 protein to form the replicative
CMG helicase (Cdc45-Mcm-GINS). This helicase forms
the core of the replisome and uses ATP hydrolysis to
separate DNA strands (Fig. 42.8). As the two CMG heli-
cases move away from each other, the origin is converted
from a licensed to an unlicensed state. Because origin
licensing by Mcm2–7 loading can only occur prior to
entry into S phase, origins only fire once per cell cycle.
In mammals, licensing occurs in the early G1 phase
before passage of the restriction point (see Chapter 41).
At the exit from mitosis, destruction of cyclins and syn-
thesis of inhibitory proteins inactivates Cdks. This creates
a window of time between anaphase and the restriction
732 SECTION X  n  Cell Cycle
TABLE 42.1  Biochemical Activities Required for Replication of DNA in Eukaryotes
Activity
Origin recognition
Prereplication complex
Origin activation
DNA unwinding (helicase)
Stabilization of single-stranded DNA
Replicative polymerases
Processivity factor
PCNA loader
Closing Factors
Removal of RNA primer
Ligation of discontinuous DNA fragments
Releasing superhelical tension
Disentangling daughter strands
ATPase, adenosine triphosphatase; CMG, Cdc45, Mcm2–7, and GINS; GINS, Go-Ichi-Ni-San; PCNA, proliferating cell nuclear antigen; RPA, replication
protein A.
point for licensing replication origins (Fig. 42.9). Mam-
malian cells pass the restriction point when the levels of
Cdk2–cyclin E, and subsequently, Cdk2–cyclin A rise
(see Fig. 40.17). This prevents the relicensing of replica-
tion origins until after the next mitosis. In yeasts, the
single Cdk that is complexed with B-type cyclins inhibits
origin licensing. Experimental inactivation of CdkCdc2
during the G2 phase in the fission yeast Schizosaccharo-
myces pombe demonstrated the importance of Cdk
activation: Cells lacking CdkCdc2 activity loaded Mcm2–7
onto already replicated DNA and then carried out further
rounds of “illegal” DNA replication without division. Cdk
regulation of origin licensing during G1 phase actually
provides one explanation for oncogenic stress (see
Chapter 41), in which inappropriate activation of onco-
genes leads to cell-cycle arrest followed by death or
senescence. Inappropriate oncogene activation can lead
to premature stabilization of cyclins D and cyclin E,
resulting in premature activation of Cdks. This, in turn,
can lead to an insufficient number of replication origins
being licensed, thereby inhibiting the cell’s ability to
deal with replication stress (see later) and its associated
DNA damage.
Name of Protein
ORC (origin recognition complex; five of six subunits are AAA ATPases)
Cdc6 (recruits Mcm2–7)
Cdt1 (recruits Mcm2–7)
Cdc7-Dbf4
Cdk (in human, Cdk2-cyclin A) phosphorylation of Sld2/RecQ4 (yeast/human names)
and Sld3/treslin recruits Dbp11/TopBP1 and initiates CMG assembly to trigger the
actual start of replication
CMG helicase is made up of:
• Mcm2–7 (assemble double hexamer before other CMG components)
• Cdc45 and the GINS complex (4 proteins)
RPA (binds single-stranded DNA)
DNA polymerase α/primase (no editing function) starts synthesis of the leading
strand and each Okazaki fragment
DNA polymerase δ replicates the lagging strand
DNA polymerase ε replicates the leading strand (both δ and ε have 3′–5′ exonuclease
editing capability)
PCNA (ring-shaped clamp that slides along the DNA. Keeps polymerases δ and ε
attached to the template strand so that they make longer chains; coordination of
cell-cycle control and replication; role in repair)
RF-C (Binds primer: template junction. AAA ATPase. Loading factor for PCNA,
important for polymerase switch)
Fen1 5′–3′ exonuclease
Dna2 helicase and endonuclease
RNase H
DNA ligase I
DNA topoisomerase I
DNA topoisomerase II
Different cell types use various combinations of several
different pathways to downregulate the activity of the
licensing system once cells enter S phase. In metazoans,
the most important pathways reduce Cdt1 activity by
degradation in S and G2 phases. After the cell passes
the restriction point, Cdks phosphorylate Cdt1. This
promotes its ubiquitylation by the SCFSkp2 and degrada-
tion. In addition, the key replisome component prolif-
erating cell nuclear antigen (PCNA) recruits another
ubiquitin ligase that causes Cdt1 degradation during S
phase. In some cell types, Cdks phosphorylate Cdc6 and
Orc1, leading to their ubiquitylation and degradation.
Vertebrates use a protein called geminin as an alter-
native regulator of origin licensing by Cdt1 (Fig. 42.8).
Geminin binds to Cdt1 and prevents it from loading Mcm
proteins onto DNA. The anaphase-promoting complex/
cyclosome (APC/C; see Fig. 40.15) triggers degradation
of geminin, keeping its concentration very low from
anaphase through the late G1 phase, allowing prereplica-
tion complexes to assemble. Accumulation of geminin
starting in the S phase inhibits the assembly of new
prereplication complexes until after the next mitosis.
Yeasts control origin licensing without geminin.

Cdk2-cyclin E
Origin licensing Cdk2-cyclin A
Cdk activity
Increased
Cdk4/6-cyclin D
G1 S
Restriction point
APC activity
Increased
Geminin
APC/CCdh1 Emi1
SCFSkp2
SCFβ-TrCP
APC/CCdh1 SCFβ-TrCP SCFSkp2 Other SCF
Geminin Emi1 p27Kip1 Cyclin D
Cdc6 Cdc25A Orc1 DP1
Cyclins Cdt1
FIGURE 42.9  PROTEIN DEGRADATION REGULATES DNA
REPLICATION. Degradation of geminin, Cdc6, Cdc25A, and cyclins
during the G1 phase keeps Cdk activity low and allows prereplication
complex formation. Degradation of p27Kip1 and inactivation of the
APC/CCdh1 by Emi1 allows the activation of Cdks to levels sufficient for
the initiation of the S phase. Once cells enter the S phase, the G1/S
regulatory machinery (cyclins D and E and the E2F cofactor DP1) is
degraded. Degradation of Cdt1 and accumulation of geminin block
reassembly of prereplication complexes. APC, anaphase-promoting
complex; SCF, Skp2 (S-phase kinase-associated protein), cullin, and
F-box proteins.
A third way to regulate origin licensing is to sequester
molecules required to assemble the prereplication
complex in the cytoplasm following the onset of S phase.
This was first suggested by studies of DNA replication
in Xenopus egg extracts (see Fig. 40.8) in which intact
nuclei replicate their DNA only once but re-replicate if
the nuclear membrane is disrupted. In different cell
types, nuclear exclusion of ORC, Cdc6, Cdt1, or Mcm2–7
all contribute to limiting licensing during S and G2 phases.
Components of the prereplication complex are absent
from nondividing differentiated cells. In fact, detection
of these proteins with antibodies in cells from cervical
smears can be used as a sensitive method for the early
detection of cancer cells (Fig. 42.10).
Signals That Start Replication
A classic experiment (Fig. 42.11) demonstrated that (a)
a cytoplasmic inducer triggers the transition into the S
phase and (b) this inducer triggers DNA replication in a
G1 nucleus but not in a G2 nucleus. The inducer is a
combination of protein kinases, including Cdk–cyclin
pairs, as well as a specialized kinase, Cdc7-Dbf4. In
mammals, Cdk2–cyclin E activity peaks at the G1/S transi-
tion (Fig. 42.9) and phosphorylates pRb, thereby opening
the restriction point “gate.” This allows the E2F/DP
dimer to function as a transcription factor and stimulate
CHAPTER 42  n  S Phase and DNA Replication 733
HUMAN CERVICAL EPITHELIUM STAINED WITH ANTI-MCM5
A. Normal B. Low-grade C. High-grade
lesion lesion
FIGURE 42.10  SECTIONS OF HUMAN CERVIX STAINED
WITH ANTIBODIES TO MCM5. A, Normal G0 cells in this stratified
epithelium lack Mcm5 and other replication proteins. B–C, Cancer
cells express Mcm5 at higher levels as they become more malignant.
Bound antibodies were detected with peroxidase coupled to a sec-
ondary antibody (brown reaction product) and lightly counterstained
with hematoxylin (blue). (Modified from Williams GH, Romanowski P,
Morris L, et al. Improved cervical smear assessment using antibodies
against proteins that regulate DNA replication. Proc Natl Acad Sci
U S A. 1998;95:14932–14937.)
the transcription of genes involved in DNA replication
(see Fig. 41.9). In addition to cyclin E itself, E2F drives
expression of cyclin A, Cdc25A, enzymes required for
synthesis of DNA precursors (dihydrofolate reductase,
thymidine kinase, and thymidylate synthase), origin-
binding proteins (Cdc6, Orc1, Cdt1 and its inhibitor
geminin), and two components of the replication
machinery (DNA polymerase α and PCNA; see Fig. 42.9).
In the S phase, the SCFSkp2 ubiquitin ligase complex
targets the Cdk inhibitor p27Kip1 for destruction by pro-
teasomes (see Fig. 40.16). SCF gets its name from three
of its components: Skp2, cullin, and F-box proteins (see
Fig. 40.16). Skp2, which is short for “S-phase kinase-
associated protein,” was identified in a complex with
Cdk2–cyclin A. SCF recognizes and ubiquitylates many of
its substrates only after they are phosphorylated by Cdk2-
cyclin A. Cdk2–cyclin A also targets E2F/DP and cyclin E
for degradation when cells enter the S phase (Fig. 42.9).
For DNA replication to start, the paired strands of the
double helix must be separated so DNA polymerase can
recognize the bases and begin synthesizing the daughter
strand. The multi-subunit CMG DNA helicase uses ATP
hydrolysis to peel apart the paired strands of the DNA
double helix. Prior to S phase each origin of replication
734 SECTION X  n  Cell Cycle
A can initiate synthesis de novo without the need for a
+
Cell in S phase Cell in G1 phase
(cell with two nuclei)
B to which DNA polymerase α adds another 20 to 25
100
% G1 cells that enter S phase
50 Advancement
due to inducer
0
0 4 8 12
Hours after fusion
FIGURE 42.11  CELL FUSION EXPERIMENT REVEALS THE
EXISTENCE OF A POSITIVE INDUCER OF THE S PHASE.
A, Synchronized cells in different stages of the cycle were fused to
yield two nuclei in a single cytoplasm. B, If the fusion involved G1 and
S cells, the G1 nucleus was induced to enter the S phase sooner than
expected. If the fusion involved S and G2 cells, the G2 nucleus failed
to rereplicate its DNA (not shown). (Modified from Rao PN, Johnson
RT: Mammalian cell fusion: studies on the regulation of DNA synthesis
and mitosis. Nature. 1970;225:159–164.)
is licensed by assembly of a double hexamer of Mcm2–7
complexes. Cdc7 kinase with its associated regulatory
subunit Dbf4 phosphorylates the Mcm complex. Next,
Cdks phosphorylate other proteins that recruit GINS
and Cdc45 to the phosphorylated Mcm2–7 hexamer,
thereby producing the CMG helicase (Table 41.1 and
Fig. 42.12A). Subsequent activation of the helicase initi-
ates replication.
Mechanism of DNA Synthesis
Replication initiates when the activated CMG helicase
starts to separate the DNA strands, which move outward
in both directions as the replisome assembles at the
origin of bidirectional replication. The newly unwound
DNA binds the single-strand DNA-binding protein, RPA
(replication protein A), ensuring that the separated
strands do not base-pair with one another again (Fig.
42.12B). Table 42.1 describes several other proteins that
also bind at this time. Box 42.1 provides an introduction
to DNA replication in E. coli.
The separated DNA strands are ready for replication,
but DNA synthesis always involves addition of an incom-
ing nucleoside triphosphate to a free 3′ OH group at the
terminus of a preexisting nascent polynucleotide (Fig.
42.1). In the absence of a nascent DNA chain, how does
DNA polymerase get started? This problem is solved on
the lagging strand by a DNA-dependent RNA polymerase
called a primase, which, like other RNA polymerases,
3′OH group. In eukaryotes, all DNA chains are started by
Heterokaryon a complex of DNA polymerase α and a primase subunit,
collectively known as Pol α/Primase. Primase synthe-
sizes an RNA chain of approximately seven nucleotides
Fusion of nucleotides of so-called initiator DNA (iDNA) (Fig.
S and G1 cells
42.12D–E). These initiating reactions are potentially
G1 cell alone risky, because DNA polymerase α lacks proofreading
ability. To avoid errors created by mismatching of an
incoming base, the RNA primer and most or all the iDNA
laid down by Pol α/Primase are subsequently replaced.
Once Pol α/Primase has done its job, two further
essential factors act to complete replisome assembly. A
pentameric protein complex called replication factor
C (RFC) binds the 3′ end of the iDNA. RFC uses energy
16 from ATP hydrolysis to load the trimeric protein PCNA
onto the DNA (Fig. 42.12E–G). The PCNA trimer is
doughnut-shaped, and when the DNA is inserted into its
central hole, it is topologically locked onto the DNA. RFC
binding and PCNA loading displace Pol α/Primase from
the DNA, and PCNA then recruits DNA polymerases δ
and ε to the DNA. With PCNA acting as a sliding platform,
DNA is reeled through the replisome and polymerase
ε synthesizes DNA continuously on the leading strand
(Fig. 42.12G).
On the lagging strand, polymerase δ synthesizes DNA
in bursts of approximately 250 bp, each initiated by Pol
α/Primase and roughly correlating with the passage of
one nucleosome by the replication fork. The replisome
contains a chromatin remodeling activity that removes
nucleosomes from the DNA ahead of the fork and
replaces them after the fork passes.
Both polymerases δ and ε have associated exonuclease
activities. This enables them to proofread the newly
synthesized DNA and correct most mistakes that they
have made. The combination of selecting the correct
nucleotides and efficient correction of mistakes may
explain the amazing fidelity of DNA replication, with
typically only one error per 109 bp polymerized.
Locally, the final steps of DNA replication are removal
of the RNA primer (and probably iDNA) and ligation of
adjacent stretches of newly synthesized DNA. Removal
of the primer can be accomplished in two ways (Fig.
42.12H). On one hand, an RNA exonuclease called
RNase H can chew in from the 5′ end of the primer.
However, this enzyme cannot remove the last ribonucle-
otide that is joined to iDNA. That requires the nucleases
Fen1 (Flap endonuclease) or Dna2. Fen1 requires a
helicase to peel the RNA (and possibly the iDNA) away
from the template, creating a sort of flap. Fen1 then
cleaves at the junction where the flap is anchored to the
DNA template, removing the oligomer of unwanted
nucleotides in one step. Alternatively, the Dna2 nuclease
can do the whole job itself because it also has helicase
 CHAPTER 42  n  S Phase and DNA Replication 735

A. Activation of origin F. RFC binds iDNA, evicting polymerase α / primase,

loading PCNA
Cdc7
Dbf4
Cyclin A
Cdk2
RFC PCNA
ORC ATP

Mcm2-7
double hexamer
Cdc6 and Cdt1 leave Polymerase α and primase leave

G. PCNA recruits polymerase ε to leading strand

B. Binding of Cdc45, GINS and RPA
Polymerase ε
RPA Cdc45
GINS
5’

3’ Active
CMG helicase

C. Cdc45 recruits polymerase α / primase H. Processive DNA

synthesis starts

RFC PCNA
Nascent
DNA

Primase
Polymerase α

I. Primer and
D. Synthesis of RNA primer i-DNA removal

RNA RNase H
primer

Fen1

J. DNA synthesis completed by polymerase ε

E. Synthesis of i-DNA followed by ligation of strands;
p97/Cdc48 removes
Mcm2-7 and replisome
RNA
primer disassembles
5’ i-DNA p97/Cdc48

3’

FIGURE 42.12  MAIN EVENTS OF DNA REPLICATION ON THE LEADING STRAND. For a more detailed description, including events on
the lagging strand, see the text. (For reference, see PDB files 1A76 [for Fen1], 1SXJ [for RFC/PCNA], 2ZXX [for Cdt1], and 3CF2 [for p97/Cdc48].)

activity. Most of these maturation/processing enzymes
are recruited by interacting with the PCNA trimer. The
individual interactions are transient while PCNA remains
as a stable loading platform.
Following removal of initiator RNA, the Pol δ/PCNA
complex extends the upstream nascent chain until it
runs into the downstream 5′ end created by Fen1. DNA
ligase I then joins the two stretches of DNA together
(Fig. 42.12I).
When the DNA is ligated, the replisome must be disas-
sembled. This is an active process triggered by ubiquity-
lation of the Mcm7 subunit of the CMG helicase. The
AAA-ATPase p97/Cdc48 (which also extracts proteins
from the endoplasmic reticulum in the endoplasmic
reticulum-associated degradation [ERAD] pathway; see
Chapter 23) recognizes this ubiquitin and then actively
separates the Mcm2–7 hexamer from Cdc45 and GINS,
causing the replisome to fall apart.
Higher-Order Organization of DNA
Replication in the Nucleus
The term S phase gives the impression that all DNA
replicates more or less synchronously, but this is far from
true. At any given time during the S phase, only 10% to
15% of the replicons actively synthesize DNA. Some
replicate early, others late. This pattern of replication
is not random; some segments of DNA consistently
736 SECTION X  n  Cell Cycle
BOX 42.1  DNA Replication in Escherichia coli
The DNA replication system of Escherichia coli has been
reconstituted entirely from purified components. Analysis of
this system reveals many similarities with eukaryotic replica-
tion, indicating that this process is highly conserved. E. coli
DNA replication can be subdivided into three phases: initia-
tion, elongation, and termination. Thus far, at least 28
polypeptides are known to be involved.
Initiation
E. coli chromosomal DNA replication initiates within a
245-bp region, termed oriC. This region contains four 9-bp
binding sites for the E. coli initiator protein, DnaA. Nearby
are three repeats of a 13-bp A/T-rich sequence. oriC also
contains specific binding sites for two small histone-like
proteins called HU and IHF. Replication is initiated with the
cooperative binding of 10 to 20 DnaA monomers to their
specific binding sites (Fig. 42.13). To be active, these mono-
mers must each have bound ATP. Binding of DnaA permits
unwinding of the DNA at the 13-bp repeats, in a reaction
A R1 R2 R3 R4 oriC DNA
13-mers DnaA sites
DnaA + ATP
HU or IHF
B parallel with PCNA and RFC in eukaryotes is striking. Activi-
DnaC
+ ATP DnaB•DnaC
DnaB complex
DnaC
C the fork moving at a rate of approximately 1000 bp per
SSB
ATP
ADP
DnaA
D Termination
FIGURE 42.13  FACTORS INVOLVED IN THE INITIATION OF
DNA REPLICATION IN ESCHERICHIA COLI. A, DNA sequences
at OriC. B, Unwinding of the origin. C, Binding of helicase. D, The
template, now ready for binding of DNA polymerase. ADP, adenos-
ine diphosphate; ATP, adenosine triphosphate; SSB, single-stranded
DNA binding protein. (Modified from Baker TA, Wickner SH. Genet-
ics and enzymology of DNA replication in Escherichia coli. Annu Rev
Genet. 1992;26:447–477.)
replicate early in the S phase, whereas others consis-
tently replicate late in the S phase.
Up to 1000 sites of replication, called replication
foci are active at any one time during the S phase in a
mammalian cell nucleus (Figs. 42.6 and 42.14B–C and
E–F). Given that each of these replication foci is active
for only about 30 minutes out of the 8- to 10-hour S
phase, a cell will replicate DNA at approximately 10,000
that requires the histone-like proteins. Next, DnaC binds to
DnaB and escorts it to the unwound DNA. DnaB is the key
helicase that will drive DNA replication by unwinding the
double helix, but it binds DNA poorly on its own in the
absence of its DnaC escort. Once DnaB has docked onto
the DNA, DnaC is released, and the helicase can then start
to unwind the DNA, provided that ATP, SSB, and DNA gyrase
are present. SSB is a single-stranded DNA binding protein
that stabilizes the unwound DNA, and DNA gyrase is a
topoisomerase (see Chapter 8) that removes the twist that
is generated when the two strands of the double helix are
separated.
Elongation
As in eukaryotes, E. coli DNA replication involves a leading
strand, with the daughter DNA synthesized as a single con-
tinuous molecule, as well as a lagging strand, with the DNA
synthesized as discontinuous Okazaki fragments. All daugh-
ter strands are started by an RNA primase that deposits
primers of 11 ± 1 nucleotides. The enzyme that actually
synthesizes the DNA is the polymerase III holoenzyme,
which has at least 10 subunits. This contains polymerase and
proofreading subunits and is held to the DNA by a doughnut-
like “sliding clamp” (β). The β is loaded onto the DNA by a
pentameric complex in a process that requires ATP. The
ties specific for the lagging strand include RNase H, which
removes the RNA primers; DNA polymerase I, which fills in
the gaps left behind by primer removal; and DNA ligase,
which links the Okazaki fragments together. DNA replication
in E. coli is significantly faster than it is in eukaryotes, with
second. This higher speed is presumed to be at least partially
attributable to the absence of nucleosomes on the bacterial
chromosome, but the eukaryotic enzymes may also be
intrinsically slower.
A specialized termination zone is found on the circular E.
coli chromosome opposite oriC. This zone contains binding
sites called ter sites, to which the ter binding protein binds.
This protein appears to block the movement of DNA heli-
cases, such as DnaB, thereby stalling the DNA replication
fork. Following termination of replication, a specialized
topoisomerase, the product of the parC and parE genes,
is required to separate the daughter chromosomes from
one another.
of these foci. There are roughly 60,000 origins in a mam-
malian cell, so each replication focus represents five or
six replication origins that are activated coordinately.
The replication foci correspond to structural domains of
chromosomes that are now called TADs (topologically
associating domains) (see Fig. 8.13). The function of
each TAD, measured by replication timing, chromatin
composition and transcriptional output, can change
 CHAPTER 42  n  S Phase and DNA Replication 737

FIGURE 42.14  VISUALIZATION OF DNA REPLICATION

A. BrdUTP
WITHIN THE NUCLEUS. A, BrdUTP is introduced into DNA in place
O– O– of dTTP to label newly replicated DNA. The incorporated BrdU (bro-
P
O O modeoxyuridine) molecules are detected with fluorescent antibodies.
O– Incorporated B, In a related approach, green-dUTP (deoxyuridine triphosphate) and
P O into cellular DNA
O O Br red-dUTP, when added together, show the many sites of DNA replica-
O– NH by replication
P tion in a cell nucleus. Because both UTP (uridine triphosphate) analogs
O O CH are incorporated simultaneously into the DNA, the sites of replication
2 O N O
appear yellow. C, Green-dUTP is followed by red-dUTP added 3 hours
H H
H H later. The later sites of DNA replication show very little overlap with the
Fed to cells
OH H earlier sites. D, Mitotic chromosome from a cell that was labeled early
in the S phase with IdU (green), and then 4 hours later with CldU (red).
B C D The late-replicating and early replicating regions of the chromosome
are segregated into discrete bands. E, CldU (green) added early in the
S phase and IdU (red) added 4 hours later show relatively little overlap.
F, CldU (green) added early in the S phase and IdU (red) added 6
hours later show no overlap. The large red blocks of labeling seen with
the IdU are characteristic of the pattern of replicating heterochromatin
seen late in the S phase. Bodipy-TR-dUTP, a red fluorescent form of
5 µm 5 µm dUTP; BrdUTP, bromodeoxyuridine triphosphate; CldU, chlorine-
Fluorescein-dUTP Fluorescein-dUTP IdU dUTP; Fluorescein-dUTP, a green fluorescent form of dUTP; IdU,
Bodipy-TR-dUTP Bodipy-TR-dUTP CIdU iodine-dUTP. All are used in place of dTTP (deoxythymidine triphos-
at same time 3 hrs later 4 hrs later phate) in DNA synthesis. (B–C, Courtesy P.R. Cook, University of
Oxford, United Kingdom. From Manders EMM, Kimura H, Cook PR.
Direct imaging of DNA in living cells reveals the dynamics of chromo-
E F
some formation. J Cell Biol. 1999;144:813–821, copyright The
Rockefeller University Press. D, Courtesy A.I. Lamond, University of
Dundee, United Kingdom. From Ferreira J, Paolella G, Ramos C, et al.
Spatial organization of large-scale chromatin domains in the nucleus:
a magnified view of single chromosome territories. J Cell Biol.
1997;139:1597–1610, copyright The Rockefeller University Press.
E–F, From Ma H, Samarabandu J, Devdhar RS, et al. Spatial and
temporal dynamics of DNA replication sites in mammalian cells. J Cell
Biol. 1998;143:1415–1425. Copyright 1998 The Rockefeller University
Press.)

5 µm
CIdU added for 2 min CIdU added for 2 min
IdU added 4 hrs IdU added 6 hrs
later for 5 min later for 5 min
coordinately during differentiation. Thus, TADs with
similar function tend to end up close to each other
forming larger compartments—for example, euchroma-
tin and heterochromatin.
Evidence for the replication of clusters of replicons
within the nucleus was first obtained by fiber autoradi-
ography. Cells were fed radioactive precursors for DNA
synthesis and then examined by electron microscopy.
(For an explanation of this technique, see Fig. 40.3.)
Clusters of replicating DNA regions were observed.
Several methods are available to observe the spatial
distribution of DNA replication during the S phase. One
can employ a pulse of nucleotide base analogs that are
incorporated into DNA and later detected by fluores-
cence microscopy. For example, thymidine analogs such
as BrdU (bromodeoxyuridine triphosphate), IdU (iodo-
deoxyuridine), and CldU (chlorodeoxyuridine) can be
detected in fixed cells by reaction with fluorescent
antibodies (BrdU is called by its correct name BrdUTP
in Fig. 42.14). Alternatively analogs labeled with a
5 µm
fluorescent dye allow the newly replicated DNA to be
observed directly in living cells.
The time at which each replicon fires during the S
phase can be seen clearly by synchronizing cells at the
beginning of the S phase, releasing them from cell-cycle
arrest, and then exposing them to BrdU at various times
thereafter. This experiment reveals very distinctive pat-
terns of DNA synthesis occurring at different times
during the S phase (Fig. 42.14B–C and E–F). Early on,
euchromatin replicates throughout the nucleus. Later,
replicating regions are concentrated around nucleoli
and other areas of constitutive and facultative hetero-
chromatin (see Chapter 8). Toward the end of the
S phase, replication is largely concentrated in blocks
of heterochromatin. These observations show that
DNA replication occurs throughout the nucleus, wher-
ever DNA is located. DNA does not move to a small
number of discrete sites to be replicated (as was once
thought).
The timing of replication of particular replication
origins was studied in detail in budding yeast using a
modification of the BrdU labeling method. First, a
738 SECTION X  n  Cell Cycle

procedure was developed whereby all cells in a popula- density) during the S phase. This experiment demon-
tion entered S phase synchronously. Next, the shift in strated that each ARS element replicates at a character-
the density of the DNA following BrdU incorporation istic time during the S phase. More recently, genome-wide
was used to distinguish DNA that had replicated from maps of replication timing have been constructed by
DNA that had not (Fig. 42.15). Incorporation of BrdU isolating newly replicated DNA at various times during S
into DNA makes the newly synthesized daughter DNA phase and identifying the chromosome regions involved
strand heavier, allowing its separation from the parental by high-throughput DNA sequencing.
DNA by centrifugation on a cesium chloride density The most striking aspect of these patterns of DNA
gradient (see Chapter 6). It was then relatively simple to synthesis is their reproducibility from one cell cycle to
take DNA probes from different regions of the chromo- the next. For example, regions of DNA labeled early in
some and determine when each replicated (changed its the S phase overlap little or not at all with DNA labeled

A 1 2 3 4 5 = Restriction enzyme
* cutting site
Origin

1 2 3 4 5
Initiation
H:L
3 H:H
1 2 4 5
Replication H:L
G force
H:H

3
1 2 4 5 Isolated DNA
digested with
Heavy: Heavy:Heavy + Heavy:Light + Heavy: restriction enzyme
Heavy Heavy:Light Heavy:Heavy Heavy
Heavy:
Light

B C D
100 100 100 Centromeres
replicate early
Amount of DNA

% Replicated
% In H:L peak

a
1,5 3
1,5 b
50
50 50
Telomeres
replicate late
3
2,4
0 0 0
HL HH 0 2 4 6 8 0 14 28 42 56
Top Density Bottom Time of replication (min) Time in S phase (min)

FIGURE 42.15  MEASUREMENT OF THE TIME OF REPLICATION OF PARTICULAR CHROMOSOMAL REGIONS IN SACCHARO-
MYCES CEREVISIAE. A–C, This protocol is based on a classic density shift experiment of Messelson and Stahl that proved that DNA replication
is semiconservative. S. cerevisiae cells are grown for several generations in a medium containing 13C and 15N so that their DNA is fully substituted
with heavy isotopes. At the beginning of the experiment, the cells are synchronized so that they enter the S phase in a single wave. At the same
time, the heavy (H) isotope medium is removed and replaced with “light medium” (L) containing 12C and 14N. At various times after the initiation
of the S phase, aliquots of cells are removed, and the DNA is isolated. The DNA is then cleaved with restriction enzymes so that the chromosomes
are cut into many fragments. DNA from each time point is then subjected to CsCl density gradient centrifugation. When any local region of DNA
is replicated, its density alters from heavy/heavy to heavy/light. After very short incubations with light isotopes, only DNA near the origin of replication
will be heavy/light; all other DNA will be heavy/heavy. These two populations of molecules are separated from one another by density gradient
centrifugation. To examine the timing of replication of a specific gene, a cloned segment of DNA corresponding to the region of interest is used
to probe (by DNA hybridization) the heavy/heavy and heavy/light peaks from each gradient. The time of replication of each locus is the time at
which the restriction fragment being detected by DNA hybridization moves from the heavy/heavy peak to the heavy/light peak. The numbers in
panels B and C refer to the numbered regions of the chromosomes shown in A. D, Data from a replication timing experiment show that in
budding yeast, centromeres replicate early in the S phase and telomeres replicate late. To generate curve a, fractions from a gradient like that
shown in panel B were hybridized to a cloned centromere region. To generate curve b, fractions from the same gradient were hybridized to a
cloned telomere region probe. Note that in mammalian cells, centromeres replicate late and telomeres replicate earlier. (Based on the work of the
laboratory of B.J. Brewer and W.L. Fangman. For reference, see Meselson M, Stahl FW. The replication of DNA in Escherichia coli. Proc Natl
Acad Sci U S A. 1958;44:671–682.)
 CHAPTER 42  n  S Phase and DNA Replication 739

3 hours later (Fig. 42.14C and E). However, DNA labeled
at corresponding points of the S phase in two successive
cell cycles superimposes almost entirely. This strongly
suggests that particular TADs initiate DNA synthesis
during preferred “windows” during S phase.
At least three mechanisms may explain the sequence
of replication patterns for different chromosomal regions:
1. Local chromatin structures, established as a result of
gene expression influence the time of replication,
particularly in metazoans. Thus, transcriptionally
active loci (where transcription factors are already
bound) have a head start over other regions of the
chromosomes, permitting them to initiate DNA repli-
cation first. In general, euchromatin (which has low
DNA methylation and high histone acetylation) repli-
cates first, followed by facultative heterochromatin
(including the inactive X) and, finally, the constitutive
pericentric heterochromatin (which has heavy DNA
methylation and low histone acetylation). This mecha-
nism can explain why a particular locus may replicate
at different times in two cell types. For example, one
origin in mammalian genomes is located just upstream
of the DNA encoding the β-globin gene (encoding a
protein subunit of hemoglobin). This region of more
than 200-kb of DNA replicates early in erythroid cells
that express the β-globin gene, but later in other cells
where the gene is inactive.
2. The higher-order packing of chromatin in the nucleus
may influence when origins replicate. It is likely that
replication origins fire in a sequence that is imposed
on them at the same time that the TAD organization
of interphase chromosomes is reestablished during
mitotic exit and early G1 phase.
3. Limiting amounts of certain essential proteins may
contribute to the sequential replication of different
chromatin domains. These proteins accumulate
on replisomes assembled on early-firing replicons
and only become available to later-firing replicons
following completion of DNA synthesis at the early
replicons and disassembly of those replisomes.
Replication Stress and
the Intra-S Checkpoint
The textbook-smooth DNA double-helix is actually lit-
tered with problems and obstacles, including DNA
damage, ribonucleotides inserted by mistake into the
DNA, and awkward secondary structures caused, for
example, by base-pairing within single DNA strands as
opposed to between strands. In addition, if cell-cycle
regulation is perturbed, cells can simply run out of either
the deoxynucleotide triphosphate (dNTP) precursors
that they need to synthesize DNA or a number of key
proteins that are also present in very low amounts. The
replication fork may stall as result of these problems and
often needs help to complete replication.
An intra-S checkpoint responds to stalled replication
forks, probably as a result of detecting excessive single-
stranded DNA (Fig. 42.16). Replication forks stall if they
encounter a damaged DNA base, bases that the repli-
some cannot “read” or a DNA secondary structure that
it cannot unfold. DNA damage can result from ultraviolet
light, mutagens or chemicals such as aldehydes produced
as a by-product of ethanol metabolism.
Replication blockage leads to a condition known
as replication stress in which single-stranded DNA
accumulates. Replication stress with stalled forks can
also result from inappropriate activation of genes that
promote cell cycle progression (oncogenic stress; see
Chapter 41). Possible reasons include insufficient licens-
ing of origins during the G1 phase, depletion of nucleo-
side triphosphate pools, or insufficient pools of essential
replication factors. Surprisingly, cells make a number
of essential components—including the dNTPs—in
amounts just sufficient for replication, so there is little
margin for error.

dsDNA Unreplicated RPA

breaks DNA DNA damage
*
RPA

ATR actived by Stalled fork Fork approaches

excess ssDNA from activated
(ATM)2 2x ATM stabilizes stalled dormant origin
active replication forks
Chk1/Chk2

Cdc25A
Block initiation of new replication foci
Degraded
Block cell cycle progression

FIGURE 42.16  REPLICATION STRESS AND THE INTRA-S CHECKPOINT. If DNA breaks are detected, the ATM (ataxia-telangiectasia
mutated) kinase activates downstream kinases Chk1 and Chk2, leading to phosphorylation of Cdc25A and its subsequent ubiquitin tagging and
degradation. This blocks the initiation of new replication forks as well as cell-cycle progression more generally. If DNA persists in unreplicated
form, or if replication forks stall, the ATR (ataxia-telangiectasia and Rad3–related) kinase activates a similar downstream response. ATR stabilizes
stalled replication forks to give nearby dormant origins time to fire and replicate the DNA downstream of the block.
740 SECTION X  n  Cell Cycle
Transcription Processing of mRNA
increases increases
Primary Mature mRNA
transcript exported to
cytoplasm
Histone
gene 3' ends of pre-mRNA
remain behind
NUCLEUS
FIGURE 42.17  THREE MECHANISMS THAT ELEVATE HISTONE EXPRESSION DURING THE S PHASE.
In conditions of replication stress, the ATM (ataxia-
telangiectasia mutated) and ATR (ataxia-telangiectasia
and Rad3–related) kinases activate the DNA damage
response (see Box 43.1). The activities of these kinases
and their downstream effectors result in the degradation
of Cdc25A, the phosphatase that triggers entry of the cell
into mitosis. The resulting inactivation of Cdks during
the S phase prevents replication initiation within other
unreplicated domains.
A key aspect of this response unique to S phase is that
ATR-activated by the presence of excessive levels of
single-stranded DNA associated with RPA protects the
stalled forks from disassembly, known as replication
fork collapse. This response is critical, because replica-
tion forks with unwound and nicked DNA molecules can
turn into breaks if the fork disassembles before replica-
tion is complete and all the DNA is ligated. Error-prone
mechanisms tend to repair DNA damage that arises
during replication stress. The resulting mutations are
thought to contribute to oncogenic transformation.
Of course, stalled replication forks must not only be
protected, but they must also somehow be rescued. At
moderate levels of replication stress, ATR activation
blocks the activation of new replication foci, but allows
dormant origins to fire near stalled forks in replication
domains that are already active. This results in the arrival
of a converging fork on the downstream side of the
damaged DNA, leaving only a very small gap that can be
repaired by a specialized DNA polymerase in a process
known as translesion synthesis. Thus, in the real world
where replication forks routinely encounter problems
that cause them to stall, a pool of dormant licensed
replication origins is essential for integrity of the genome.
Synthesis of the Histone Proteins
Chromatin contains approximately equal masses of DNA
and core histones. Human cells require about 62 × 106
copies of each core histone, assuming a genome size of
6.2 × 109 bp and 200 bp per nucleosome. Because
approximately 90% of histone transcription occurs
during the S phase, enormous amounts of these proteins
Stability of the mRNA
(on free ribosomes)
increases
Degrading
enzymes
are made during a relatively brief period. Histone synthe-
sis apparently keeps pace, in part, because there are
approximately 40 sets of histone genes.
Synthesis of histones during the S phase is tightly
coupled to ongoing DNA replication and a controlled
supply of histones is essential for normal replication. If
replication is blocked either by addition of drugs or by
temperature-sensitive mutants, histone synthesis declines
abruptly shortly thereafter, possibly because nucleosome
deposition appears to be linked to lagging strand synthe-
sis. Indeed, the chaperone that assembles histones into
nucleosomes is associated with the replisome. This link
between histone synthesis and DNA replication appears
to involve at least three processes (Fig. 42.17).
First, transcription of the histone genes rises three-
fold to fivefold as cells enter the S phase. Each histone
gene has a cell-cycle-responsive element in its promoter
to which a transcription factor binds specifically during
the S phase.
Second, the processing of histone messenger RNAs
(mRNAs) increases six- to 10-fold as cells enter the S
phase. Histone mRNAs are not polyadenylated, and the
primary transcripts are considerably longer than the
mature forms. Processing of the 3′ end of histone pre-
mRNAs involves the U7 small nuclear ribonucleoprotein
(snRNP) (see Chapter 11), a portion of which recognizes
histone mRNA and base-pairs with it during processing.
Cell-cycle-dependent regulation of processing appears to
involve changes in the accessibility of the necessary
portion of U7 small nuclear RNA (snRNA). This region
is inaccessible in G0 cells but becomes accessible
when cells that reenter the cycle and begin the S phase.
The mechanism for this change in RNA conformation
is not known.
Third, changes in the stability of the mRNA also regu-
late histone synthesis. Normally, the level of histone
mRNA on free polysomes drops rapidly by approximately
35-fold as cells enter the G2 phase. If DNA synthesis is
interrupted during the S phase, a region at the 3′ end of
the mature message targets the mRNA for degradation.
If this region is removed from the 3′ terminus of the
histone mRNA, the normal link between ongoing

replication and mRNA stability is lost. Furthermore, this
sequence, transposed onto the 3′ terminus of a globin
mRNA, renders that mRNA sensitive to degradation if
DNA synthesis is blocked. Degradation of histone mRNA
requires ongoing protein synthesis, and it has been
speculated that histones themselves participate in the
control.
As discussed in Chapter 8, specialized variant forms
of histones are synthesized and inserted into the chro-
matin outside of S phase. These histones are encoded
by mRNAs with introns and normal poly(A) tails and
are therefore not processed by the specific S phase–
associated pathway (see Chapter 11). Their insertion
into chromatin is typically correlated with RNA tran-
scription rather than DNA replication and involves spe-
cific chromatin-remodeling factors.
Other Events of the S Phase
Although the bulk of attention on the S phase focuses
on the duplication of the chromosomes, centrosomes
also duplicate at this time. Duplication of the centro-
somes is essential for stability of the genome, because
they set up the poles of the mitotic spindle that is
responsible for accurate partitioning of the replicated
chromosomes. Interestingly, Orc1 functions indepen-
dent of DNA replication with cyclin A to limit centriole
duplication to once per cell cycle. (See Fig. 34.17 for a
discussion of centrosome duplication.)
With the completion of DNA replication and duplica-
tion of the centrosomes, the cell is ready to divide. As
the levels of Cdk activity rise toward the threshold that
is sufficient to trigger mitotic entry and other factors
necessary for mitosis accumulate, the cell continues to
screen the integrity of the DNA to ensure that the
CHAPTER 42  n  S Phase and DNA Replication 741
genome has been replicated completely and that no
harmful DNA damage is present. These checks, together
with other ongoing preparations for mitosis, are the
principal events of the G2 phase (see Chapter 43).
ACKNOWLEDGMENTS
We thank Hiro Araki, Julian Blow, Cristina Cardoso,
David Gilbert, and Julian Sale for suggestions on revi-
sions to this chapter.
SELECTED READINGS
Boos D, Frigola J, Diffley JF. Activation of the replicative DNA helicase:
breaking up is hard to do. Curr Opin Cell Biol. 2012;24:423-430.
Costa A, Hood IV, Berger JM. Mechanisms for initiating cellular DNA
replication. Annu Rev Biochem. 2013;82:25-54.
Deegan TD, Diffley JF. MCM: one ring to rule them all. Curr Opin
Struct Biol. 2016;37:145-151.
Hills SA, Diffley JFX. DNA replication and oncogene-induced replicative
stress. Curr Biol. 2014;24:R435-R444.
Labib K. How do Cdc7 and cyclin-dependent kinases trigger the initia-
tion of chromosome replication in eukaryotic cells? Genes Dev.
2010;24:1208-1219.
McIntosh D, Blow JJ. Dormant origins, the licensing checkpoint, and
the response to replicative stresses. Cold Spring Harb Perspect Biol.
2012;4:a012955.
Méchali M, Yoshida K, Coulombe P, Pasero P. Genetic and epigenetic
determinants of DNA replication origins, position and activation.
Curr Opin Genet Dev. 2013;23:124-131.
O’Donnell M, Langston L, Stillman B. Principles and concepts of DNA
replication in bacteria, archaea, and eukarya. Cold Spring Harb
Perspect Biol. 2013;5:a010108.
O’Donnell M, Li H. The eukaryotic replisome goes under the micro-
scope. Curr Biol. 2016;26:R247-R256.
Rhind N, Gilbert DM. DNA replication timing. Cold Spring Harb Per-
spect Biol. 2013;5:a010132.
Zeman MK, Cimprich KA. Causes and consequences of replication
stress. Nat Cell Biol. 2014;16:2-9.
This page intentionally left blank
 CHAPTER 43 

G2 Phase, Responses to DNA

Damage, and Control of Entry
Into Mitosis

T he G2 phase is the gap between the completion of
DNA replication and the onset of mitosis. This chapter
begins with the biochemical basis for the G2/mitosis (M)
transition and discusses how the G2/M checkpoint delays
this transition if DNA damage is detected. Finally, the
chapter introduces the major pathways that cells use to
repair damaged DNA.
Enzymology of the G2/Mitosis Transition
The transition from the G2 phase into mitosis is the most
profound morphologic and physiological change that
occurs during the life of a proliferating cell. Entry into
mitosis is controlled by a network of stimulatory and
inhibitory protein kinases and phosphatases, presided
over by Cdk1–cyclin B1. (Chapter 40 introduced the
components involved in the G2/M transition.)
Cdk1, the driving force for entry into mitosis, is
present at a constant level throughout the cell cycle. The
Wee1/Myt1 phosphorylates Cdk1
on T14 & Y15 (inhibitory)
multifaceted regulation of Cdk1 (see Fig. 40.14) includes
binding of cyclin cofactors, inhibition and activation
by phosphorylation, binding of inhibitory molecules,
changes in subcellular localization and changes in the
activities of competing protein phosphatases (Fig. 43.1).
These various factors are finely balanced, until a positive
feedback loop enables the cells to make an abrupt and
decisive entry into mitosis.
Mammals have at least three B-type cyclins: B1, B2,
and B3. Cyclin B1 is essential for triggering the G2/M
transition. Cyclin B1, newly synthesized during the latter
part of the cell cycle, binds Cdk1 and shuttles it in and
out of the nucleus. Importin β carries the Cdk1–cyclin
B1 complex into the nucleus, and then Crm1 rapidly
exports it back to the cytoplasm (see Chapter 9). Cdk1–
cyclin B2 associates with the Golgi apparatus during
interphase and might function in Golgi disassembly
during mitosis (see Fig. 44.4). Cyclin B3 may function
only during meiosis in mammals.

Cyclin B1 Y15 Cyclin B1

Cyclin B1
Wee1 Myt1 T 14
T161 T161
Cdk1 Cdk1 Cdk1 Cdk1 Cdk1
Inactive Inactive Inactive Active Inactive
kinase kinase CAK kinase Cdc25A,B,C kinase kinase
CAK phosphorylates Cdc25 Cyclin B1
Cyclin B1 Cdk1 on T161 (stimulatory) dephosphorylates degraded
T14 and Y15

S G2 M G1

FIGURE 43.1  REGULATION OF CDK1 BY CYCLIN BINDING AND PROTEIN PHOSPHORYLATION FROM THE LATE S PHASE
THROUGH MID-MITOSIS. (For reference, see Protein Data Bank [PDB; www.rcsb.org] file 1X8B and Squire CJ, Dickson JM, Ivanovic I, et al.
Structure of human Wee1A kinase: kinase domain complexed with inhibitor PD0407824. Structure. 2005;13:541–550.)

743
744 SECTION X  n  Cell Cycle

As cells approach the G2/M transition, phosphoryla-
tion simultaneously activates and inhibits Cdk1 bound to
cyclin A or B. Cdk-activating kinase (CAK-actually Cdk7–
cyclin H), located in the nucleus, phosphorylates Cdk1
on T161 (Fig. 43.1). This triggers a refolding of the active
site cleft that allows the enzyme to bind substrates (see
Fig. 40.13). This phosphorylation activates Cdk1-cyclin
kinases in the nucleus (Fig. 43.1).
At the same time, Wee1 and Myt1 kinases phosphory-
late T14 and Y15 to turn the enzyme off. Wee1 in the
nucleus phosphorylates Cdk1 on Y15 adjacent to the
adenosine triphosphate (ATP)-binding site. This stops
the kinase from using ATP to phosphorylate substrates.
While Cdk-cyclin complexes are in the cytoplasm, they
are phosphorylated on T14 and Y15 by Myt1 kinase associ-
ated with the Golgi apparatus and endoplasmic reticu-
lum. The role of Wee1 as a mitotic inhibitor is clearly
demonstrated in Schizosaccharomyces pombe: overex-
pression of Wee1 delays or prevents the entry of cells
into mitosis (Fig. 43.2). Together, Wee1 and Myt1 ensure
that Cdk1 remains inactive as it shuttles into and out of
the nucleus (Fig. 43.3).
This combination of stimulatory and inhibitory phos-
phorylations holds Cdk1–cyclin complexes poised for a
burst of activation.
That burst occurs when one of three Cdc25 protein
phosphatases removes the inhibitory phosphates from
T14 and Y15 (Figs. 43.1 and 43.8). Cdc25s are “dual-
specificity” protein phosphatases (see Fig. 25.5) that
remove phosphates from serine (S), threonine (T), and
tyrosine (Y) residues. Of the three Cdc25 phosphatases,
only Cdc25A is indispensable for life. It functions at both
the G1/S and G2/M transitions, whereas Cdc25B and
Cdc25C have roles only in the G2/M transition.
Because Cdc25 phosphatases trigger mitotic entry,
their regulation is both important and elaborate.
Cdc25A and Cdc25C are held inactive during interphase
by mechanisms involving stimulatory and inhibitory

A. Wee1  B. Wee1+ C. Wee1+ (3x) D. Wee1+ (5x)

FIGURE 43.2  EFFECT OF CHANGING CELLULAR LEVELS OF WEE1 PROTEIN ON CELL-CYCLE PROGRESSION IN FISSION
YEAST. A, Cells that lack functional Wee1 protein enter mitosis too soon in the cell cycle and are smaller than wild-type cells (B). C–D, Cells
that express excess Wee1 protein are too effective at inactivating Cdk1 and are severely delayed in their ability to enter mitosis (hence their larger
size). (From Russell P, Nurse P. Negative regulation of mitosis by Wee1+, a gene encoding a protein kinase homolog. Cell. 1987;49:559–567.)

AT
ADP
14-3-3 Cyclin B1 Myt1
docking (Active) Y15 Cyclin B1 Myt1
site Cyclin B1 docking site (Inactive)
T14 blocked
T161
Cdc25C Cdk1 NES
(Inactive) (Inactive)
CYTOPLASM
NUCLEUS 14-3-3 AT
ADP
14-3-3 Cyclin B1
Y15
Cyclin B1 T161
Wee1 T14 Wee1
14-3-3 (Active) T161 Cdk1 NES
Cdc25C Cdk1 NES (Inactive) (Active)
(Inactive) (Inactive)
Cdc25A Cyclin B1 nuclear
(Inactive/unstable) export signal inactivated

S phase G2 phase Mitosis

FIGURE 43.3  CDK REGULATION IN INTERPHASE AND MITOSIS. Inhibitory phosphorylation and shuttling of components between the
nucleus and cytoplasm regulate Cdk1 activity in interphase and mitosis. Cdc25, which would remove the inhibitory phosphates, is held inactive
until its activation by Polo kinase and Cdk1-cyclin B stimulates mitotic entry (not shown here). ADP, adenosine diphosphate; ATP, adenosine
triphosphate; NES, nuclear export sequence. (Based on an original figure by Helen Piwnica-Worms. For reference, see PDB file 1YWT.)
 CHAPTER 43  n  G2 Phase, Responses to DNA Damage, and Control of Entry Into Mitosis 745
Interphase
Low Cdc25C activity, cytoplasmic
14-3-3 Phosphorylated
S216 binds
14-3-3 protein
Cdc25C
14-3-3
Cdc25A Cyclin B1/Cdk1
docking site
Low Cdc25A protein levels due to proteolysis
Reduced interactions with Cdk1-cyclin B1
due to 14-3-3 binding at the Cdc25
C-terminus
FIGURE 43.4  REGULATION OF CDC25A AND CDC25C ACTIVITY IN INTERPHASE AND MITOSIS. (Based on an original figure by Helen
Piwnica-Worms.)
phosphorylation, binding to a 14-3-3 adapter protein,
alterations in their subcellular localization, and ubiquitin-
mediated proteolysis (Fig. 43.4).
Phosphorylation on a serine residue of Cdc25 creates
a binding site for a 14-3-3 adapter protein that interacts
with the phosphoserine and several flanking amino acids
(see Fig. 25.10). Association with 14-3-3 interferes with
Cdc25A binding to Cdk1–cyclin B1. Phosphorylation at
other sites also targets Cdc25A for ubiquitination by
SCFβTrCP and destruction by proteasomes. Cdc25C has a
nuclear export sequence (see Fig. 9.17), so Cdc25C
bound to 14-3-3 is primarily cytoplasmic (Fig. 43.3).
Following the theme of phosphorylation having both
positive and negative effects, full activation of Cdc25s
requires phosphorylation of other residues in the amino-
terminal region. A protein kinase called Polo (see next
paragraph) initiates this phosphorylation of Cdc25,
followed by more extensive phosphorylation by
Cdk1–cyclin B1, the substrate of Cdc25. This action of
Cdk1–cyclin B1 on Cdc25 creates a powerful positive
feedback amplification loop that provides a burst of Cdk
activity and triggers entry into mitosis (Fig. 43.7).
A molecular trigger is required to start the ampli-
fication cycle in which Cdk1–cyclin B1 and Cdc25
activate each other. Members of the Polo family of
protein kinases are candidates for this role, by activat-
ing Cdc25. These kinases possess amino acid motifs
called “polo boxes” that bind to protein partners after
they have been “primed” by phosphorylation by another
kinase. Interestingly, the most common “priming
kinases” for polo are Cdk–cyclin pairs. This creates
another positive amplification loop that allows for
additional levels of control and rapid activation of Cdk–
cyclin complexes. In addition to activating Cdks by phos-
phorylating Cdc25, polo family kinases are involved in a
variety of mitotic events, including formation of a bipolar
spindle, cytokinesis, and passage through certain cell-
cycle checkpoints.
Mitosis
Enhanced Cdc25C activity, nuclear,
S216 phosphorylation blocked
Y15
Cyclin B1 Cdc25C Cyclin B1
T14
T161 T161
Cdk1 Cdk1
(Inactive) (Active)
Cdc25A
Cdc25A accumulates due to increased stability
Enhanced interaction with Cdk1-cyclin B1
complexes due to loss of 14-3-3 binding
Enhanced activity due to
mitotic-specific phosphorylation
For cells to enter and complete mitosis, protein phos-
phatase 2A (PP2A, a protein serine/threonine phospha-
tase; see Fig. 25.6) must be inactivated. PP2A removes
the phosphates that Cdk1 puts on its target proteins. If
PP2A is not inhibited, cells enter mitosis, but they then
slip back into interphase. PP2A inhibition involves three
steps. Newly active Cdk1 phosphorylates a protein
kinase called Greatwall (a name from Drosophila genet-
ics). Greatwall phosphorylates a small target protein that
then binds to the substrate-binding pocket of PP2A,
acting as a competitive inhibitor. Cdk1 activation at the
onset of anaphase eventually leads to dephosphorylation
of the small protein and activation of PP2A, thereby
allowing cells to exit mitosis.
Changes in Subcellular Localization at
the G2/M Transition
Late in G2, phosphorylation inactivates the nuclear
export signal of cyclin B1 (see Chapter 9). As a result,
Cdk1–cyclin B1 accumulates in the nucleus within 5
minutes (Figs. 43.3 and 43.5). Cdc25C also stops shut-
tling at the G2/M transition, probably as a result of Polo
kinase phosphorylation. The Cdk1–cyclin B that accumu-
lates in the nucleus is thought to be already active, so
the reason for this rapid nuclear localization is not
entirely clear. The concentration of Cdk–cyclin B com-
plexes together with Cdc25A and Cdc25C in the con-
fined volume of the nucleus may contribute to the final
burst of Cdk1–cyclin B1 activation.
Cdk1 Activity and the Initiation
of Prophase
Cdk2–cyclin A plays a critical role during the S phase
(see Chapter 42), but also helps trigger the G2/M transi-
tion. Cdk–cyclin A activity peaks at G2/M, before the
peak of Cdk1–cyclin B1 activity, and inactivation of
746 SECTION X  n  Cell Cycle
cyclin A in Drosophila or mammalian cultured cells
arrests the cell cycle in G2. Furthermore, G2 cells enter
mitosis prematurely if injected with active Cdk2–cyclin
A complexes just completing S phase. Finally, microin-
jection of a selective inhibitor of Cdk–cyclin A causes
prophase cells to return rapidly to interphase; chromo-
somes decondense, rounded prophase cells flatten, and
the interphase microtubule network returns.
Cdk activity regulates several events during the G2-to-
prophase transition. In the cytoplasm, the half-life of
microtubules drops dramatically from approximately 10
minutes to approximately 30 seconds late in G2 (see
Table 44.1). This, coupled with an enhanced ability of
centrosomes to initiate microtubule polymerization,
completely transforms the organization of the microtu-
bule cytoskeleton. Centrosomes take on the appearance
of spindle poles and migrate apart over the surface of
the nucleus. While this is happening, chromatin begins
to condense in the nucleus. A protein complex called
condensin is required for this condensation to begin
during prophase (see Fig. 8.18). Cdks activate condensin
by phosphorylation of two of its subunits in late G2.
G2 phase Prophase Metaphase
FIGURE 43.5  CYCLIN B1 LOCALIZATION DURING MITOSIS.
Cyclin B1 moves rapidly from the cytoplasm into the nucleus at the
onset of prophase and subsequently associates with the spindle
during mitosis. (Courtesy Christina Karlsson and Jonathon Pines,
Wellcome/CRC Institute, Cambridge, UK.)
Cyclin B1 Cyclin B1
Cdk1
CYTOPLASM
Shuttling
NUCLEUS
Cdk1 Cdk1
Cdk1
Cyclin A
Cyclin A
Cdk2 Cdk2
G1 S G2
FIGURE 43.6  CDK1 REGULATION. Locations and patterns of activation of Cdk1 complexed to cyclin A versus cyclin B across the cell cycle.
These events occur while most Cdk1–cyclin B1 is in
the cytoplasm. It appears most likely, therefore, that
Cdk1–cyclin A triggers at least the nuclear events of
prophase (Fig. 43.6). Commitment to mitosis appears
to be irreversible only after Cdk1–cyclin B1 enters
the nucleus.
Recap of the Main Events of
the G2/M Transition
Synthesis of cyclin B1 in the latter portion of the S and
G2 phases leads to assembly of Cdk1–cyclin B heterodi-
mers that shuttle into and out of the nucleus, spending
most of their time in the cytoplasm associated with
microtubules. In late G2 phase, Cdk1–cyclin A activity
initiates mitotic prophase, beginning with changes in
microtubule dynamics and chromosome condensation.
Several events trigger entry into the active phase of
mitosis. Cdc25A accumulates and no longer binds 14-3-3
proteins, allowing it to interact more effectively with
Cdk1–cyclin B. The phosphoserine-binding site for 14-3-3
proteins on Cdc25C is dephosphorylated, allowing
Cdc25C to accumulate in the nucleus. In addition, phos-
phorylation of cyclin B1 blocks its export from the
nucleus and promotes its import, thus causing Cdk1–
cyclin B1 to accumulate rapidly in the nucleus. The
inhibitory kinase Wee1 is also phosphorylated, causing
its activity to drop. Cdc25A and Cdc25C activate Cdk1–
cyclin B1 by removing inhibitory phosphates on T14 and
Y15. This starts in the cytoplasm and then may be stimu-
lated as the proteins concentrate in the nucleus. There,
the action of Cdk1–cyclin B1 on the nuclear lamina trig-
gers nuclear envelope breakdown and drives the cell
into mitosis. Fig. 43.7 highlights the positive feedback
loop between Cdc25 and Cdk1–cyclin B that drives the
cell into mitosis.
G2/M transition events:
Microtubule stability drops
Cyclin B1
Centrosomes migrate apart
Chromosomes condense
Cdk1 Kinetochore assembly starts
Cyclin B1
Cyclin B1 Cyclin B1
degraded
Cdk1
Cyclin A Cyclin A
degraded
Cdk1
Prophase M
 CHAPTER 43  n  G2 Phase, Responses to DNA Damage, and Control of Entry Into Mitosis 747

Cell-cycle arrest in Rad9+ and rad9 cells

Cdc25 phosphatase with DNA damage
dephosphorylation
cdc13tsRad9+ cdc13tsRad9-
Wee1 and Myt1
phosphorylation

T161 Cells on
Inactive Active Cdk1 CAK
kinase protein kinase plates
Myt1,
Activated by Polo kinase Cdc25 Wee1
and amplified by protein protein
positive feedback phosphatase kinases
from Cdk1–cyclin B
Bud

S G2 M
e
DNA otyp
damage pe phen
FIGURE 43.7  SUMMARY OF THE CDK1 FEEDBACK REGULA-
W ild-ty Nucleus
TION MECHANISM AT THE G2/M TRANSITION.
G2 arrest

Why did such an elaborate system evolve to regulate
the G2/M transition? The answer appears to lie in
the exquisite sensitivity provided by the interlocking
network of stimulatory and inhibitory activities. On the
one hand, this network ensures a rapid, almost explo-
sive, final transition into mitosis. On the other, it pro-
vides a number of ways to delay the G2/M transition if
the cell detects damage to chromosomes. Attempting
mitosis with chromosomal damage can lead to cell death
or contribute to cancer.
G2/M Checkpoint
Separation of sister chromatids during mitosis is a poten-
tial danger point for a cell. After DNA is replicated each
chromosome consists of paired sister chromatids held
together by cohesin. Therefore, if the DNA is damaged,
the cell can use information present in the undamaged
chromatid to guide the repair process. However, once
sisters separate, this corrective mechanism can no longer
operate. In addition, if a cell enters mitosis before com-
pleting replication of its chromosomes, attempts to sepa-
rate sister chromatids damage the chromosomes. To
minimize these hazards, a checkpoint operates in the G2
phase to block mitotic entry if DNA is damaged or DNA
replication is incomplete.
Just as DNA damage can arrest the cell cycle in G1
phase, damaged or unreplicated DNA also halts the cell
cycle temporarily in the G2 phase. Interestingly, the G1
checkpoint—which can be activated by a single DNA
break in human cells—is more sensitive than the G2/M
checkpoint, which requires 10 to 20 breaks to block
cell-cycle progression. The G2/M checkpoint may be less
sensitive then the G1 checkpoint, because G2 cells are
already primed to enter mitosis. Consequently, human
cells can enter mitosis with limited amounts of damaged
or unreplicated DNA. These problem regions can be
No delay
Phe
n
Cells otype o
k f
with eep divi rad9 mu
fragm ding tan
ente and d t
d nu i
clei e
FIGURE 43.8  GENETIC IDENTIFICATION OF THE G2/M
CHECKPOINT. Cells defective in the G2/M checkpoint (Rad9 mutants
of budding yeast) cannot delay their entry into mitosis in the presence
of damaged DNA and therefore divide themselves to death. Budding
yeast Rad9 is an ortholog of the ATM (ataxia-telangiectasia mutated)
adapter protein 53BP1 (see text). Confusingly, budding yeast Rad9 is
not related to fission yeast Rad9, which gives the 9-1-1 complex its
name (see text). (Courtesy Ted Weinert, University of Arizona, Tucson.)
detected and repaired in the daughter cells after division
(see later).
Studies of radiation-induced G2 delay in budding
yeast identified a major cell-cycle checkpoint that is sen-
sitive to the status of the cellular DNA. Cells defective in
this checkpoint are more sensitive than wild-type cells
to radiation injury because they continue to divide,
despite the presence of broken or otherwise damaged
chromosomes (Fig. 43.8). The cells die, presumably from
chromosomal defects or loss. In metazoans, the G2/M
checkpoint delays entry into mitosis until the damage is
either fixed, triggers cell suicide by apoptosis, or causes
cells to enter a nonproliferating (senescent) state. The
checkpoint works by modulating the activities of the
components that control the G2/M transition.
DNA Damage Response
Considering that it is the blueprint for life, DNA
is remarkably accident-prone, and organisms have an
elaborate network of mechanisms to repair those acci-
dents. This complex network is known as the DDR
748 SECTION X  n  Cell Cycle

(DNA damage response). This section discusses a few
key components of the DDR, and Box 43.1 (see later)
explains several mechanisms that repair damaged DNA.
The minimal machinery of a DNA damage checkpoint
(see Fig. 40.4) involves SENSORS that detect DNA damage,
TRANSDUCERS (usually protein kinases) that produce a
γH2AX
BRCA1
G2 nucleus
G1 nucleus
FIGURE 43.9  INDUCTION OF DNA DAMAGE FOCI USING A
HIGH-ENERGY LASER. Laser-induced DNA damage results in acti-
vation of the DDR with production of γH2AX (red) at damage sites
followed by binding of other factors, including BRCA1 (green). DNA is
blue. The left cell is not yellow because BRCA1 is not present in G1-
phase cells. Micrograph taken 30 minutes after induction of damage.
(Micrograph courtesy Martin Mistrik and Jiri Bartek.)
biochemical signal as a result of the detected damage,
and EFFECTORS (both protein kinases and transcriptional
activators) that coordinate repair pathways to block cell-
cycle progression and repair the damage.
When DNA is damaged, several proteins concentrate
at the damage site within seconds to minutes, forming a
focus that activates the G2/M checkpoint and repairs the
damage. The use of high-energy lasers to “draw” patterns
of DNA damage onto cell nuclei revolutionized our
understanding of the DDR by allowing a minute-by-
minute mapping of the events during focus formation
and leading to DNA repair (Fig. 43.9).
If a single strand of DNA is broken, the SENSOR enzyme
poly(adenosine diphosphate [ADP]-ribose) polymerase,
binds to the broken end and immediately starts polymer-
izing a chain of ADP-ribose (which it makes from nico-
tine adenine dinucleotide [NAD]). Several proteins bind
to poly(ADP-ribose) chains and recruit the critical TRANS-
DUCER ATM kinase (ataxia-telangiectasia mutated) to the
break together with its partner NBS1, a subunit of the
MRN complex (Fig. 43.10A). ATM is usually present in
the nucleus as an inactive dimer. Interactions with MRN
separate the dimer into ATM monomers that are acti-
vated by MRN docked to the broken end of a DNA
molecule.
The MRN complex is a SENSOR that detects double-
strand DNA breaks. In this case, the NBS1 subunit of
MRN recruits ATM kinase to the break site. MRN is also
a nuclease with a key role in repairing those breaks. Its
job is to chew back (resect) one DNA strand in a 5′ →
3′ direction at the site of a DNA break, leaving a stretch

A B DNA damage

DNA breaks
DNA strand
with damage
removed
ATM
dimer (inactive)
MRN complex RPA
binds to DNA break binds ssDNA

AT
ATM ADP ATRIP
autophosphorylation
ATRIP binds
ATM monomer to RPA
Active ATM binds ATR
(active) MRN complex

ATRIP

γ-H2AX ATR active

ATR
phosphorylated

FIGURE 43.10  MECHANISMS FOR LOCALIZING ATM AND ATR TO SITES OF DNA DAMAGE. ATR, ataxia-telangiectasia and Rad3
related; ssDNA, single-stranded DNA.
 CHAPTER 43  n  G2 Phase, Responses to DNA Damage, and Control of Entry Into Mitosis 749

of single-stranded DNA that becomes coated with RPA, ovarian cancer coexists with breast cancer. BRCA1 is a
the protein that also coats single-stranded DNA at repli- very large, complex protein with numerous roles in DNA
cation forks (Fig. 42.12). RPA then attracts a protein repair that are still being determined.
called ATRIP plus its partner, the second key TRANSDUCER, ATM (Table 43.1) is dispensable for life, though
ATR kinase (ataxia-telangiectasia and Rad3 related). people lacking it have ataxia-telangiectasia—a disorder
ATM and ATR stay bound to the chromatin, which is associated with degeneration of cerebellar neurons, dila-
remodeled by reactions involving protein phosphoryla- tion of blood vessels, a very high predisposition for
tion, acetylation/deacetylation, methylation, and ubiqui- cancer, and a number of other symptoms. The NBN gene
tylation. The response is very complex. For example, is mutated in humans with Nijmegen breakage syn-
several hundred phosphorylation events have been drome, a rare inherited disorder featuring chromosomal
detected during the DDR. These chromatin changes are instability and a predisposition to cancer. ATR is essen-
likely part of a response to suppress transcription so that tial for life, presumably because it has a key role in ensur-
RNA polymerase does not collide with the damage and ing that replication forks are stabilized until all the
cause more problems. The histone modifications associ- single-stranded DNA created during S phase is replicated.
ated with the DDR generally tend to create “open” chro-
matin that is more accessible to the DNA repair ATM/ATR
machinery. Repair foci are often found just adjacent to
heterochromatin, and indeed, DNA repair appears to be
more efficient in euchromatin than heterochromatin. H2AX Chk1 Chk2 MDM2
ATR tends to stay on the single-stranded DNA close activated activated sequestered
to the break, but ATM spreads along the chromatin
γ-H2AX p53 Other
through a cycle of reactions involving its most famous Chromatin effects:
stabilized kinases
substrate, the specialized histone isoform H2AX (Figs. Cohesin binding Cdc25A
Focus formation
43.9 and 43.11). H2AX phosphorylated by ATM is known Recruitment of
inhibited p21 14-3-3σ
repair machinery degraded transcribed transcribed
as γ-H2AX. γ-H2AX forms very rapidly in a focus of modi-
fied chromatin immediately surrounding the damage. Wee1 Cdc25C
This amplifies and spreads the response to damage, activated inhibited
sequestered
because γ-H2AX binds a SENSOR protein, MDC1 that Cdk1–cyclin A
inhibited
recruits more ATM and repair factors. The response Cdk1–cyclin B1
spreads along the chromatin as ATM creates more γ- inhibited
H2AX, so that after a few minutes the γ-H2AX focus S G2 M G1
covers about a megabase of DNA.
MDC1 has two BRCT domains that bind the phos- FIGURE 43.11  THE G2/M CHECKPOINT BLOCKS THE G2/M
phorylated site on γ-H2AX. BRCT domains were discov- TRANSITION FOLLOWING ACTIVATION OF ATM AND/OR ATR
BY DNA DAMAGE. Dotted lines show activities that are switched off
ered in BRCA1, one of the first genes whose mutations by the checkpoint. The dashed line between ATM/ATR and the kinase
were linked to familial breast cancer. BRCA1 is mutated that inhibits Cdc25C indicates that this pathway is not yet known.
in 90% of families where inherited predisposition to (Based on an original figure by Helen Piwnica-Worms.)

TABLE 43.1  Key DNA Repair Gene Defects Associated With Human Disease
Human Disease Pathway Defective Genes*
Ataxia-telangiectasia (AT) Checkpoint ATM
Seckel syndrome Checkpoint ATR
Xeroderma pigmentosum NER XP-A, XP-B, XP-C, XP-D, DDB-2, XP-F, XP-G, POLH
Cockayne syndrome NER XP-B, XP-D, CSA, CSB
Trichothiodystrophy NER XP-D
Hereditary nonpolyposis colon cancer MMR MSH2, PMS2, MLH1
AT-like disorder DSB repair MRE11
Nijmegen breakage syndrome DSB repair NBN
Breast cancer predisposition HR BRCA1, BRCA2
LIG4 syndrome NHEJ LIGIV
Severe combined immune deficiency NHEJ ARTEMIS
ATM, ataxia-telangiectasia mutated; ATR, ataxia-telangiectasia and Rad3 related; DSB, double-strand break; HR, homologous recombination;
MMR, mismatch repair; NER, nucleotide excision repair; NHEJ, nonhomologous end joining.
*This list is an outline. For an updated list of the human DNA repair genes known to date, the reader is referred to http://sciencepark.mdanderson.org/labs/
wood/.
750 SECTION X  n  Cell Cycle

Table 43.1 shows only a few of the diseases associated
with mutations in proteins involved in repairing DNA
breaks. (See Box 43.1 for a discussion of the major DNA
repair pathways.) It is likely that new components of this
pathway remain to be identified.
From the DNA Damage Response to
the G2/M Checkpoint
ATM and ATR at damage foci cooperate with adapters to
phosphorylate and activate the two key EFFECTOR kinases

BOX 43.1  DNA Repair in Vertebrates*

Every human cell experiences approximately 105 DNA
damage events each day. These are mostly repaired accu-
rately, so only about 100 mutations are passed on in each
new human generation. In cancer, the spontaneous muta-
tion frequency can be at least 200-fold higher. Cell division
must not occur with inaccurately replicated or damaged
genomes, as this may cause cell death or heritable mutation.
A number of systems have evolved to repair particular forms
of DNA damage sustained by the cell (Fig. 43.12). These
repair mechanisms act in concert with the apoptotic machin-
ery to ensure that the cell will die if the DNA damage cannot
be repaired (see Chapter 46). DNA damage checkpoints are
a critical component of the cellular response to DNA damage
(see Fig. 40.4), as they impose a delay in the cell cycle during
which cells have a chance to repair their genomes. Promis-
ing new strategies for cancer treatment include use of agents
that actually cause DNA damage, with the goal of over-
whelming the already defective defenses in cancer cells, and
causing them to activate cell death pathways.

Base Excision Repair

Bases in DNA can become oxidized, reduced, alkylated, or
deaminated owing to endogenous activities or environmen-
A. Type of DNA B. Repair
lesion pathway tal stress. Damaged bases are cut away from the DNA sugar–
Base damage: phosphate backbone by a damage-recognizing glycosylase,
U deamination of leaving an abasic site (Fig. 43.13A). Abasic sites, which also
cytosine to uracil
can be generated directly by DNA damage, are then removed
Base by cleavage of the sugar–phosphate backbone mediated
excision by certain glycosylases and endonucleases. The missing
repair
sequence is then reconstructed from its complementary
G Base damage: strand by DNA polymerase β, with DNA ligase III-Xrcc1
methylation of
guanosine completing the repair by sealing the gaps in the backbone.

Nucleotide Excision Repair

Incorporation Mismatch
C of mismatched repair Bulky DNA adducts caused by chemical agents or environ-
A
base mental stress (particularly UV radiation from sunlight) are
excised by a complex, though well-understood, reaction
T Formation of UV Nucleotide (Fig. 43.13B). Defects in nucleotide excision repair genes
photoproduct: excision
6-4 thymidine cause the human genetic disease xeroderma pigmento-
dimer repair
T sum (XP), which is characterized by hypersensitivity to
sunlight and predisposition to skin cancer. Eight proteins
encoded by genes mutated in xeroderma pigmentosum
(Table 43.1) take part in nucleotide excision repair, provid-
3' OH Double-strand Double-strand ing one of the best examples in which human genetics has
break break repair
helped to unravel a complicated biological process. Recog-
3' OH • Homologous
recombinational nition of the DNA lesion involves the heterotrimeric rep-
G C repair lication protein RPA, XPA, and XPC, and the nine-subunit
• Nonhomologous
end joining transcription factor TFIIH, which contains XPB and XPD.
ATP-dependent unwinding of the DNA by XPB and XPD
forms a preincision complex. XPG, which replaces XPC
in the complex, makes an incision six to nine bases 3′ of
the damaged base, and XPF-Ercc1 cuts 20 to 25 bases 5′ of
DNA double the damage site. This releases a short single-stranded DNA
helix expanded fragment containing the damaged DNA. After excision, DNA
polymerases δ or ε fill in the gap by copying the undamaged
strand. Prokaryotes have a similar system of adduct recogni-
FIGURE 43.12  INTRODUCTION TO DNA REPAIR. Examples tion, removal, and repair involving the UvrA, UvrB, and UvrC
of DNA damage and the repair pathways that respond to different proteins; however, the enzymes that are involved are not
types of lesion. conserved between kingdoms.
 CHAPTER 43  n  G2 Phase, Responses to DNA Damage, and Control of Entry Into Mitosis 751

BOX 43.1  DNA Repair in Vertebrates*—cont’d

A. Base excision repair B. Nucleotide excision repair C. Mismatch repair

DNA damage DNA damage Mismatch

RPA, XPA, XPC

Glycosylase Recognition TFIIH (contains XPB Recognition MSH2/MSH6 dimer
removes
damaged base and XPD)
Nick
Abasic site
ATP-dependent
unwinding by RPA, XPA, XPC, TFIIH Sliding clamp
DNA polymerase β XPB, XPD searches for nick Msh2/Msh6 dimer plus
DNA ligase III-Xrcc1 (Okazaki frag.) Mlh1/Pms2 dimer
form sliding clamp
Preincision
complex forms
May travel thousands
of bases
Exonuclease
XPF-Ercc1 nicks XPG nicks 6–9 bp degrades back Exo1
20–25 bp to 5' to 3' to mismatch

DNA polymerase δ, ε
DNA polymerase δ, ε

FIGURE 43.13  PATHWAYS FOR THE REPAIR OF BASE DAMAGE, BULKY ADDUCTS SUCH AS THYMIDINE DIMERS FORMED
BY ULTRAVIOLET LIGHT, OR MISMATCHED BASES. For detailed descriptions, see the text. The inset in panel A shows human
3-methyladenine DNA glycosylase complexed to DNA. This enzyme scans the DNA for bases that are not strongly H-bonded, uses its
“finger” to swing them up into the pocket for scanning, and, if they are damaged, catalyzes excision of that base. (Inset illustration by Graham
Johnson [www.fivth.com] for the Howard Hughes Medical Institute, copyright 2004, all rights reserved. For reference, see PDB file 1BNK
and Lau AY, Scharer OD, Samson L, et al. Crystal structure of a human alkylbase-DNA repair enzyme complexed to DNA: mechanisms for
nucleotide flipping and base excision. Cell. 1998;95:249–258.)

Mismatch Repair Double-Strand Break Repair

Errors in DNA replication missed by the proofreading activity
of the DNA polymerase are recognized by a dimer consisting
of the MSH2 and MSH6 proteins. When a mismatch is
detected, this heterodimer undergoes an ATP-dependent
transition to a sliding clamp and recruits a second heterodi-
mer, consisting of MLH1 and PMS2 (Fig. 43.13C). To distin-
guish between the original (“correct”) sequence and the
newly synthesized DNA strand, this sliding clamp complex
can then translocate along the DNA until a break is reached,
such as that found between Okazaki fragments. The broken
strand is therefore identified as the newly synthesized DNA
strand. The mismatch repair complex then recruits the exo-
nuclease EXO1 and degrades the newly synthesized DNA
strand all the way back to the misincorporated base. The
resultant long, single-stranded region is stabilized by binding
of RPA and eventually filled in by the replicative DNA poly-
merases δ and/or ε. A second type of “mismatch” involves
mistaken incorporation of ribonucleotides into DNA by
either DNA polymerases δ and ε. Specialized ribonuclease H
(RNAse H) enzymes detect and cleave DNA/RNA hybrids and
remove the ribonucleotides.
DNA double-strand breaks can be caused by ionizing radia-
tion or radiomimetic drugs or arise spontaneously after
replication. They are particularly hazardous forms of damage,
as they carry the risk of losing chromosomal material or,
if misrepaired, causing chromosomal translocations. Two
major pathways repair double-strand breaks. Homologous
recombinational repair uses undamaged DNA as a template
for the accurate repair of double-strand breaks, this sequence
usually being derived from the sister chromatid after replica-
tion. Nonhomologous end joining (NHEJ) repairs double-
strand breaks with no requirement for homology. NHEJ is
the predominant activity that repairs double-strand breaks in
the G1 and early S phase, whereas homologous recombina-
tion becomes more important in the late S and G2 phase.
Homologous recombinational repair is normally extremely
accurate, but NHEJ often introduces errors, as it can join
both related and unrelated DNA ends together. Both path-
ways require the activity of the MRN protein complex
(Mre11/RAD50/Nbs1), which localizes to DNA double-strand
breaks and is also found at telomeres. The exonuclease activ-
ity of this complex resects (chews back) broken DNA ends

Continued
752 SECTION X  n  Cell Cycle

BOX 43.1  DNA Repair in Vertebrates*—cont’d

to provide single-stranded DNA substrates for the repair
systems. These mechanisms repair also repair double-strand
breaks during genome editing (see Fig. 6.16).
The key protein required for homologous recombina-
tional repair in cells is Rad51, the eukaryotic homologue
of Escherichia coli RecA (Fig. 43.14A). Rad51 forms an
extended nucleoprotein filament on single-stranded DNA,
replacing RPA, which acts like a placeholder when single-
stranded DNA is produced. Rad51 catalyses the search for
homologous sequences, strand pairing, and strand exchange.
A number of proteins, including BRCA1 and BRCA2, control
the activity of mammalian Rad51 in homologous recombina-
tional repair. Inactivation of BRCA1 and BRCA2 predisposes
to cancer. The helicase Rad54 facilitates strand invasion,
when the single-stranded region forces its way into the com-
plementary DNA duplex on the undamaged sister chromatid.
Following invasion of the recombining DNA strands, poly-
merase activity extends the DNA beyond the site of the
double-strand break, leading to the formation of a Holliday
junction (Fig. 43.14B). Resolution of the Holliday junction

A. Homologous recombinational repair C. Nonhomologous end joining

DNA break

Double-strand break
Binds ends
Recruits DNA- Ku70/
dependent Ku80 ring
protein kinase
5' to 3' resection MRN complex
Rad50/Mre11/Nbs1 MRN complex
5' 3'
Aligns broken ends Other repair factors

3' 5'
5' 3'

3' 5' DNA ligase IV/

Rad51 Ends sealed Xrcc4
Initial strand invasion
New DNA synthesis

B. Holliday junction structure

3' 5'
5'
Second end capture, = Resolvase cutting sites 3'
synthesis, ligation

Holliday
junction

Holliday junction resolution 3'

5' 3' 5'

5' 5' 3'

3' +
5' 3'
Repair complete
3' 5'

FIGURE 43.14  PATHWAYS FOR THE REPAIR OF DNA DOUBLE-STRAND BREAKS. A double-strand break is recognized by the
MRN complex, which recruits ATM (ataxia-telangiectasia mutated). The break is then bound by repair factors involved in either the homolo-
gous recombination or nonhomologous end-joining pathway of DNA repair. A, Homologous recombination pathway of DNA repair. The MRN
complex in conjunction with other nucleases chews back (resects) the DNA at a break, leaving a single-stranded overhang that is stabilized
by RPA (not shown). This recruits ATR (ataxia-telangiectasia and Rad3 related) to the damage site. It is believed that the MRN complex also
plays a role in keeping the broken ends close to one another. Next, RAD51 forms a nucleoprotein filament on the single-stranded DNA,
displacing the RPA. The Rad51 nucleoprotein filament then initiates homology searching and repairs the DNA break by inserting the extended
single-stranded DNA into homologous sequences (usually on the sister chromatid [blue]) and allowing homologous recombination and DNA
repair/resynthesis to occur. Capture of the second single-stranded DNA end allows the formation of a joint molecule with a double Holliday
junction. Resolution of this Holliday junction structure results in accurate, templated repair of the double-strand break. B, A Holliday junction
formed by four complementary oligonucleotides complexed to the enzyme Cre (not shown). The Holliday junction is a dynamic structure
(arrows) that can migrate along the DNA. C, Nonhomologous end-joining pathway of DNA repair. This pathway is initiated by break recogni-
tion by the Ku70/Ku80 heterodimer, which recruits DNA-PK and tethers the broken ends. The breaks are then processed in a reaction
involving the MRN complex and other repair factors. DNA-PK’s precise role is not yet entirely clear. Next, DNA ligase IV/XRCC4 is recruited
to the processed double-strand break, which is ligated back together. (B, For reference, see PDB files 3CRX and 2CRX and Gopaul DN,
Guo F, Van Duyne GD: Structure of the Holliday junction intermediate in Cre-loxP site-specific recombination. EMBO J 1998;17:
4175–4187.)
 CHAPTER 43  n  G2 Phase, Responses to DNA Damage, and Control of Entry Into Mitosis 753
BOX 43.1  DNA Repair in Vertebrates*—cont’d
and filling-in of the repaired DNA sequences results in com-
plete repair of the lesion. One of two known human Holliday
junction resolvases is the Gen1 nuclease, a relative of the
Fen1 flap endonuclease that functions during DNA replica-
tion (see Fig. 42.12). (The other more complex resolvase
is the product of four genes.) Cells must have one or the
other of these enzymes or they die during mitosis because
of an inability to separate DNA strands that have undergone
repair in the previous cell cycle. Note that Fig. 43.14 shows
only a subset of the proteins involved in homologous recom-
binational repair. It is likely that new factors remain to
be identified.
NHEJ is initiated at a DNA double-strand break by binding
of the Ku70 and Ku80 heterodimer as a ring that binds the
*By Ciaran Morrison, National University of Ireland, Galway.
Chk1 and Chk2. Phosphorylation of the ATM adapter,
53BP1 (p53-binding protein 1), recruits Chk2 for activa-
tion by ATM. The trimeric 9-1-1 complex is required
for Chk1 activation by ATR. This complex, which gets
its name from its subunits Rad9, Hus1, and Rad1, resem-
bles proliferating cell nuclear antigen (PCNA), the
doughnut-shaped processivity factor that is indispens-
able during DNA replication (see Fig. 42.12). PCNA is
loaded onto DNA by replication factor C (RFC, a penta-
meric AAA-ATPase) and anchors DNA polymerases and
other factors to DNA. A similar ATPase composed of one
special subunit, Rad17, plus the four small subunits of
RFC loads the 9-1-1 complex onto DNA at or near sites
of damage. Mutants in those four RFC subunits are defec-
tive in G2/M checkpoint control in yeasts, Drosophila
and Caenorhabditis elegans. RPA stimulates loading of
the 9-1-1 complex at damage sites, making it specific for
regions of single-stranded DNA.
Phosphorylation releases Chk1 and Chk2 from chro-
matin, so they can diffuse throughout the nucleus and
cell to implement the DDR. They also trigger the G2/M
checkpoint response by altering the cell cycle machinery
and inducing the transcription of key EFFECTORS. In some
cases their actions trigger cell death by apoptosis.
Activation of Chk1 is important to establish the G2/M
checkpoint response because Chk1 phosphorylates the
Cdc25A and Cdc25C protein phosphatases thereby
blocking cell-cycle progression (Fig. 43.11). Phosphory-
lation produces binding sites for a 14-3-3 protein that
blocks Cdc25A from activating Cdk1–cyclin B. Chk1
phosphorylation also targets Cdc25A for ubiquitin-
mediated proteolysis ensuring that levels of Cdc25A
remain low.
The transcription factor p53 (see Fig. 41.15), another
EFFECTOR of the G2/M checkpoint, is phosphorylated and
activated following DNA damage (Fig. 43.11). Activated
catalytic subunit of the DNA-dependent protein kinase,
stimulating other repair factors and aligning the broken
ends of the DNA (Fig. 43.14C). A complex of the protein
XRCC4 with DNA ligase IV seals the ends of the broken
DNA. NHEJ is also necessary for V(D)J recombination
and therefore for the development of the immune system
(see Fig. 28.10).
Given the importance of accurate transmission of the
genetic material, deficiencies in DNA repair and checkpoint
genes are associated with a number of diseases (Table 43.1).
Note that several DNA repair activities are essential for
life, so their inactivation has not been described in any
human diseases.
p53 stimulates transcription of the Cdk inhibitor p21.
Although p21 is best known for promoting cell-cycle
arrest in G1 cells as part of the G1 DNA damage check-
point, it can also act in G2. Expression of p21 is an effec-
tive way of blocking the initiation of prophase, because
it inhibits Cdk1–cyclin A approximately 100-fold better
than it inhibits Cdk1–cyclin B1.
Active p53 also drives the expression of 14-3-3σ, an
adapter protein that interferes with shuttling of Cdk1–
cyclin B1 between the nucleus and cytoplasm (Fig.
43.11). Binding of 14-3-3σ maintains the Wee1 inhibitory
kinase in a more active state, ensuring that the Cdk1–
cyclin B1 complex remains inactive. Disruption of the
gene for 14-3-3σ is fatal for cells with DNA damage.
Instead of activating their G2/M checkpoint, they enter
an aberrant state with characteristics of both mitosis and
apoptosis, and then die.
Typically, G2/M checkpoint activation has one of three
outcomes. If DNA damage is so extensive that it cannot
be repaired, the cell either enters a non-proliferating state
known as senescence or commits suicide by apoptosis
(see Chapter 46). Less-serious damage can be repaired
by one of the systems described in Box 43.1.
Transition to Mitosis
The complex web of stimulatory and inhibitory activities
in the G2 phase poises Cdk1–cyclin B in a state ready for
the explosive burst of activation that triggers the G2/M
transition. These complex regulatory pathways are the
basis of the G2/M checkpoint control that prevents cells
from segregating their chromosomes if genomic DNA
cannot meet stringent quality control standards. Eventu-
ally, however, if all goes well, Cdk1–cyclin A and Cdk1–
cyclin B1 are activated, and the cell embarks on mitosis,
probably the most dramatic event of its life.
754 SECTION X  n  Cell Cycle
ACKNOWLEDGMENTS
We thank Anton Gartner, Ciaran Morrison, and Ashok
Venkitaraman for their suggestions on revisions to this
chapter.
SELECTED READINGS
Ciccia A, Elledge SJ. The DNA damage response: making it safe to play
with knives. Mol Cell. 2010;40:179-204.
Hartwell LH, Weinert TA. Checkpoints: Controls that ensure the order
of cell cycle events. Science. 1989;246:629-634.
Jackson SP, Bartek J. The DNA-damage response in human biology and
disease. Nature. 2009;461:1071-1078.
Mehta A, Haber JE. Sources of DNA double-strand breaks and models
of recombinational DNA repair. Cold Spring Harb Perspect Biol.
2014;6:a016428.
Melo J, Toczyski D. A unified view of the DNA-damage checkpoint.
Curr Opin Cell Biol. 2002;14:237-245.
Morgan DO. The Cell Cycle: Principles of Control. London: New
Science Press; 2007.
Parrilla-Castellar ER, Arlander SJ, Karnitz L. Dial 9-1-1 for DNA damage:
the Rad9-Hus1-Rad1 (9-1-1) clamp complex. DNA Repair (Amst).
2004;3:1009-1014.
Paull TT. Mechanisms of ATM activation. Annu Rev Biochem. 2015;84:
711-738.
Pearl LH, Schierz AC, Ward SE, et al. Therapeutic opportunities within
the DNA damage response. Nat Rev Cancer. 2015;15:166-180.
Polo SE, Jackson SP. Dynamics of DNA damage response proteins at
DNA breaks: a focus on protein modifications. Genes Dev. 2011;25:
409-433.
Smits VA, Medema RH. Checking out the G(2)/M transition. Biochim
Biophys Acta. 2001;1519:1-12.
Wieser S, Pines J. The biochemistry of mitosis. Cold Spring Harb Per-
spect Biol. 2015;7:a015776.
Wood RD, Mitchell M, Sgouros J, et al. Human DNA repair genes.
Science. 2001;291:1284-1289.
 CHAPTER 44 

Mitosis and Cytokinesis

M itosis is the division of a somatic cell (a vegetative

Prophase Early prometaphase
cell in yeast) into two daughter cells. The daughters are CDK activity high
usually identical copies of the parent cell, but the process
can be asymmetrical. For example, division of some stem
cells gives rise to one stem cell and another daughter cell
that goes on to mature into a differentiated cell. See Box
41.2 for examples.
Traditionally, mitotic events are subdivided into six Late prometaphase Metaphase
phases: prophase, prometaphase, metaphase, ana-
phase, telophase, and cytokinesis (Fig. 44.1). The
dramatic reorganization of both the nucleus and cyto-
plasm during the mitotic phases is brought about by
activation of a number of protein kinases, including
Cdk1–cyclin B–cks (abbreviated here as “Cdk1 kinase”;
Anaphase Telophase
see Chapter 40). After activation by Cdc25 phosphatase,
APC/C activity high
Cdk1 kinase accumulates in the nucleus, where it joins
Cdk1–cyclin A–cks, which was activated somewhat
earlier (see Fig. 43.6). These two Cdk1 kinase complexes
operate as both master controllers and workhorses that
directly phosphorylate many proteins whose functional
and structural status is altered during mitosis. Their
Early cytokinesis Late cytokinesis
progressive inactivation following the correct attach-
ment of the chromosomes to spindle microtubules drives
the orderly exit of cells from mitosis.
Mitosis is an ancient process, and a number of varia-
tions emerged during eukaryotic evolution. Many single-
celled eukaryotes, including yeast and slime molds,
undergo a closed mitosis, in which spindle formation FIGURE 44.1  OVERVIEW OF THE PHASES OF MITOSIS.
and chromosome segregation occur within an intact APC/C, anaphase-promoting complex/cyclosome.
nuclear envelope to which the spindle poles are
anchored. This chapter focuses on open mitosis, as
Prophase
used by most plants and animals, in which the nuclear
envelope disassembles before the chromosomes segre- Prophase, the transition from G2 into mitosis, begins
gate. Fig. 44.2 summarizes some of the important events with the first visible condensation of the chromosomes
during the various mitotic phases. and disassembly of the nucleolus (Fig. 44.3). In the

755
756 SECTION X  n  Cell Cycle

A. Interphase B. Prophase C. Prometaphase D. Metaphase

Nucleus
Centrosome
Chromosomes
NE

Microtubules

Centrosomes separate Begins with nuclear Chromosomes align

Chromosomes condense envelope (NE) break-down on spindle equator
Chromosomes attach
to spindle

E. Anaphase A F. Anaphase B G. Telophase H. Cytokinesis

NE

CS

Midbody
CF CF CS remnant
CS
Pole
Sister chromatids separate Organized central spindle (CS) Cleavage furrow (CF) constricts Chromosomes decondense
and move to poles assembles Nuclear envelope (NE) Interphase microtubule
Poles (arrows) separate reassembles network reforms
Cleavage Furrow (CF)
assembles Daughter cells separate

FIGURE 44.2  KEY EVENTS OF MITOSIS. A–C, Prophase–prometaphase: Cdk1 kinase triggers condensation of replicated sister chromatids,
disassembly of the nuclear envelope and Golgi, and a dramatic reorganization of the cytoskeleton. As the barrier between the chromosomes and
cytoplasm is abolished, microtubules contact the condensed chromosomes and attach at the kinetochores (see Fig. 8.21). Interaction of kineto-
chores with dynamic microtubules culminates with the chromosomes aligned at the midplane of the bipolar spindle. D–F, Metaphase–anaphase:
Once all chromosomes achieve a bipolar attachment to the spindle, an inhibitory signal is silenced, leading to activation of a proteolytic network
that destroys proteins responsible for holding sister chromatids together and also inactivates Cdk1 by destroying its cyclin B cofactor (see Fig.
40.15). Sister chromatids separate and move toward opposite spindle poles, which themselves move apart. G–H, Telophase–cytokinesis: Targeting
of nuclear envelope components back to the surface of the chromatids leads to the reformation of two daughter nuclei. In most cells, the two
daughter nuclei and the surrounding cytoplasm are partitioned by cytokinesis. (Micrographs courtesy William C. Earnshaw.)

cytoplasm, the interphase network of long microtubules
centered on a single centrosome (see Fig. 34.18) is
converted into two radial arrays of short microtubules
called asters. Most types of intermediate filaments disas-
semble, the Golgi apparatus and endoplasmic reticulum
fragment, and both endocytosis and exocytosis are
curtailed.
Nuclear Changes in Prophase
Chromosome condensation, the landmark event at the
onset of prophase, often begins in isolated patches of
chromatin at the nuclear periphery. Later, chromosome
condense into two threads termed sister chromatids
that are closely paired along their entire lengths. Although
chromosome condensation was first observed more than
a century ago, the biochemical mechanism remains a
mystery. Protein kinases trigger mitotic chromosome
condensation and onset of condensation is correlated
with phosphorylation of histones H1 by Cdk1 kinase and
H3 by Aurora-B protein kinase. However, chromosomes
still condense when both of these phosphorylation
events are blocked. It is possible that a combination of
histone modifications promotes mitotic chromatin con-
densation (see Fig. 8.3).
Two pentameric protein complexes, condensin I
and condensin II are major regulators of mitotic chromo-
some architecture. These complexes share the SMC2 and
SMC4 (structural maintenance of chromosomes) ABC
adenosine triphosphatases (ATPases), but have two dif-
ferent sets of three auxiliary proteins (see Fig. 8.18).
 CHAPTER 44  n  Mitosis and Cytokinesis 757

A. Prophase Chromosome B
condensation
Phosphorylated begins
condensin enters nucleus
Histone H3 Duplicated
phosphorylation centrioles begin
begins to separate
Cell surface Microtubule half-
markers life decreases
internalized and asters form
Intracellular membrane DNA
Cell begins to Microtubules
networks remodeled round up Centrosomes

FIGURE 44.3  INTRODUCTION TO PROPHASE. A, Summary of the major events of prophase. B, Distribution of DNA (blue), microtubules
(red), and γ-tubulin (centrosomes [green]) in a prophase human cell. (B, Images were recorded by Dr. Melpomeni Platani on the University of
Dundee’s School of Life Sciences Imaging Facility OMX 3DSIM Microscope and stored and processed in OMERO.)

Condensins are required to disassemble the topologically

associating domains (TAD) of interphase chromatin (see
Fig. 8.13) and promote the formation of the linear array
of loops that characterizes mitotic chromosomes (see
Fig. 8.14). The decisive experiment was to target SMC2
for rapid degradation, with the result that no well-
organized mitotic chromosomes formed. The chromatin
compacted but was less organized. Slower depletion of
condensins by RNA interference (RNAi) or conditional
gene disruption gave less-dramatic results.
Condensin complexes can encircle DNA and also
promote its supercoiling in vitro, but how these activi-
ties help them to orchestrate the changes in chromatin
architecture is not known. Condensin II is nuclear during
interphase, and has an important role in prophase chro-
mosome formation following its activation by phos-
phorylation. Condensin I acts both in prophase and
prometaphase in further compacting the chromosomes.
DNA topoisomerase II and other scaffold proteins also
function during mitotic chromosome formation, but
condensin appears to play the major role.
Cytoplasmic Changes in Prophase
Most of the cytoskeleton reorganizes during prophase.
The microtubule array changes from an extensive
network permeating the cytoplasm into two dense,
radial arrays of short, dynamic microtubules around the
duplicated centrosomes (see Fig. 34.17). Each of these
asters eventually becomes one pole of the mitotic
spindle. During prophase, the two asters usually migrate
apart across the surface of the nuclear envelope, signal-
ing the start of spindle assembly (Fig. 44.3).
Mitotic microtubules behave like interphase microtu-
bules in many ways (see Chapter 34). They are mostly
nucleated at their minus ends, they grow by addition of
tubulin subunits at their plus ends, and they undergo
random catastrophes during which they rapidly shorten.
To a large extent, the prophase changes in microtubule
organization can be explained by two simple biochemi-
cal changes: (a) increased microtubule-nucleating activ-
ity of centrosomes and (b) altered dynamic instability
TABLE 44.1  Comparison of Microtubule Dynamics
in Interphase and Mitotic Newt Lung Cells
Parameter Interphase Mitosis
Elongation rate 7 µm/min 14 µm/min
Elongation time before 71 s 60 s
catastrophe
Shortening rate 17 µm/min 17 µm/min
Probability of rescue 0.046/s 0
from catastrophe*
Length 100 µm 14 µm
*Most cellular microtubules grow constantly by addition of subunits to
their free ends but they occasionally stop growing and begin shrinking
rapidly (a “catastrophe”). Unless shrinking is reversed (a “rescue”), the
microtubule completely disappears.
Data from Gliksman NR, Skibbens RV, Salmon ED: How the transition
frequencies of microtubule dynamic instability regulate microtubule
dynamics in interphase and mitosis. Mol Biol Cell. 1993;4:1035–1050.
properties of the microtubules (Table 44.1; also see
Chapter 34). Interphase microtubules have a high prob-
ability of recovering from catastrophes, so they grow
quite long. Mitotic microtubules grow more rapidly
but exist only transiently, because rescues are rare fol-
lowing a catastrophe. Thus, they usually shorten all
the way back to the centrosome, with little chance of
rescue. These differences in dynamic instability can be
reproduced in vitro in mitotic and interphase cellular
extracts. They appear to arise, at least in part, from
counterbalancing interactions between microtubule-
associated proteins that promote microtubule stability,
and kinesin-13 (see Fig. 36.13), which promotes micro-
tubule disassembly.
Other cytoskeletal elements that disassemble during
prophase include many, but not all, classes of interme-
diate filaments (including the nuclear lamins) (see
Fig. 35.1A) and specialized actin filament structures,
such as stress fibers. However, the junctional complexes
between adjoining cells are maintained in epithelial
cells. As a result of the cytoskeletal reorganization, most
cells round up during prophase. This is particularly
evident for animal cells that are cultured on a flat
758 SECTION X  n  Cell Cycle

substrate, but cells in tissues also change their shape
dramatically during mitosis.
RNA transcription of the chromosomes stops during
mitosis except for highly specialized transcription at
centromeres. Phosphorylation of components of the
transcriptional machinery by Cdk1 kinase appears to be
responsible for this shutoff. Cdk1 kinase phosphoryla-
tion of ribosomal elongation factor 2a (EF2a) also stops
most (but not all) ongoing protein synthesis and assem-
Interphase Mitosis
Golgi Ministacks
Golgi stacking GM130
proteins
cyclin B–p9
p115
bly of new ribosomes. Phosphorylation of several nucleo-
lar proteins leads to disassembly of the nucleolus.
The Golgi apparatus and endoplasmic reticulum frag-
ment and disperse during prophase (Fig. 44.4). Several
kinases, including Cdk1 drives Golgi apparatus disas-
sembly, the first step being fragmentation into smaller
ministacks following phosphorylation of Golgi stacking
proteins and tethers. Later steps are still being investi-
gated. Many lines of evidence argue that Cdk1 phos-
phorylation of key components prevents the fusion of
transport vesicles back into Golgi stacks (see Chapter
21), the net result being that the Golgi buds away into
small vesicles that disperse throughout the mitotic cell
cytoplasm. Other evidence suggests that an imbalance of
vesicle flow between the Golgi and the endoplasmic
reticulum results in the Golgi being absorbed into the
endoplasmic reticulum during mitosis. Whatever the
Cdk1–
mechanism of its disassembly, Golgi reassembly begins
again during late anaphase/early telophase, following
inactivation of Cdk1 kinase.

FIGURE 44.4  GOLGI APPARATUS DYNAMICS IN INTER-
PHASE AND MITOSIS. Disassembly in mitosis is driven by phos-
phorylation of components blocking fusion of Golgi membranes.
Prometaphase
In cells that undergo an open mitosis, prometaphase
begins abruptly with disassembly of the nuclear envelope
(Fig. 44.5). Microtubules growing outward from the
spindle poles penetrate holes in the nuclear envelope,
make contact with the chromosomes, and attach to them

A. Early prometaphase C. Late prometaphase E. Kinetochore attachments

to the spindle
2 4
1 Tension

3 5
(+) ends

1. Nuclear envelope disassembles 4. Chromosome slides rapidly poleward Amphitelic (correct)

2. Microtubules grow and shrink in aster
3. Kinetochore captures microtubule
along microtubule
5. Microtubule from opposite pole is
captured by sister kinetochore
6. Chomosome attached to both poles
congresses to middle of spindle

B D
Syntelic (errors)

DNA
Microtubules
Centrosomes Merotelic (errors)

FIGURE 44.5  INTRODUCTION TO PROMETAPHASE. A, Summary of the key events of early prometaphase. B, Distribution of DNA (blue),
microtubules (red), and γ-tubulin (centrosomes [green]) in early prometaphase human cells. C, Summary of the key events of late prometaphase.
D, Distribution of DNA, actin, microtubules, and centrosomes in late prometaphase PtK1 cells. E, Terms used to describe the orientation of
kinetochore attachments to the mitotic spindle. (B and D, Images were recorded by Dr. Melpomeni Platani on the University of Dundee’s School
of Life Sciences Imaging Facility OMX 3DSIM Microscope and stored and processed in OMERO.)
 CHAPTER 44  n  Mitosis and Cytokinesis 759

at specialized structures called kinetochores (see Fig.
8.19). Interactions of the two opposing kinetochores of
paired sister chromatids with microtubules from oppo-
site poles of the spindle ultimately result in alignment of
the chromosomes in a group midway between the poles.
An important cell-cycle checkpoint (see Chapter 40)
known as the spindle assembly checkpoint (SAC)
delays the onset of chromosome segregation until all
kinetochores are attached to microtubules.
Nuclear Envelope Disassembly in Prometaphase
Nuclear envelope disassembly involves the removal of
two membrane bilayers coupled with disassembly of the
nuclear pores and the fibrous nuclear lamina mesh-
work that underlies the inner bilayer (Fig. 44.6). Phos-
phorylation causes the nucleoporin Nup98 to dissociate
from nuclear pores. This removes the permeability
barrier between nucleus and cytoplasm. Phosphoryla-
tion of other proteins causes the pore to disassemble to
soluble subcomplexes. Phosphorylation of the nuclear
lamins at two sites flanking the coiled-coil causes the
lamina network to disassemble into subunits. Interaction
between microtubules and dynein associated with the
nuclear envelope can rip holes in the envelope, although
this is not required for nuclear envelope disassembly.
Nuclear envelope membranes are dispersed in the
cytoplasm from prometaphase until telophase (Fig.
44.6), but the mechanism is not settled. Some experi-
ments suggest that the nuclear membranes break up into
small vesicles that disperse in the cytoplasm. Other
experiments suggest that the nuclear envelope is
absorbed into the endoplasmic reticulum, which remains
as an extensive tubular (or flattened cisternal network—
another source of discussion) throughout mitosis.
Further experiments are required to answer this ques-
tion, and both mechanisms could contribute. Lamin B
remains associated with the dispersed nuclear envelope,
whereas lamins A and C and many proteins of the nuclear
pore complexes disperse as soluble subunits.
During prophase, kinetochores transform from non-
descript balls of condensed chromatin into structures on
the surface of the chromosomes. By early prometaphase,
the characteristic trilaminar disk structure (see Fig. 8.19)
can be seen. Each sister chromatid has a kinetochore.
Sister kinetochores are located on opposite faces of
the mitotic chromosome.
Organization of the Mitotic Spindle
The mature metaphase spindle is a bilaterally sym-
metrical structure with centrally located chromosomes
flanked by arrays of microtubules radiating from the
poles (Fig. 44.7).
Three predominant classes of microtubules are
present in the metaphase spindle (Fig. 44.12). Kineto-
chore microtubules have their plus ends embedded in
the kinetochore and their minus ends at or near the
spindle pole. They characteristically form bundles, called
kinetochore fibers, which contain anywhere from 1
microtubule in the budding yeast to more than 200
microtubules in some higher plants. Each human kineto-
chore binds approximately 20 microtubules. Up to
approximately 80% of the approximately 2200 spindle
microtubules in humans may be present in kinetochore
fibers, but not all microtubules in those fibers stretch all

A. Interphase B. Mitosis C D. Lamins A and C E. Lamin B

Nuclear Endoplasmic
membrane: reticulum
Outer
Inner
HO PO4– O PO4– O
Lamina
Interphase O O
disassembly C C
OCH3 O–
Nuclear
lamina Cdk1– G2 / M G2 / M
cyclin B–p9
Condensing
chromosomes
PO4 PO4
O O
Eggs? Somatic C C
cells? Earliest OCH3 O–
prometaphase OH O PO4– OH O PO4–

Lamin B

+ or

Chromosomes Vesicles derived Lamin B

from the nuclear dispersed through the Soluble Subunits on
Metaphase Lamina Lamina
envelope endoplasmic reticulum subunits membrane vesicles

FIGURE 44.6  DISASSEMBLY OF THE NUCLEAR ENVELOPE DURING MITOSIS. A–B, Two contrasting models to explain the fate of the
nuclear envelope during the transition from interphase to mitosis in a higher eukaryote. C, Micrographs showing solubilization of lamin A fused
to green fluorescent protein (GFP) (green) during mitosis. DNA is blue. Scale bar is 10 µm. D–E, Reversible disassembly of lamins A, C, and B
is driven by posttranslational modifications of the lamin polypeptides. (C, Courtesy William C. Earnshaw.)
760 SECTION X  n  Cell Cycle

A. Metaphase–forces balanced, spindle length stable B. Anaphase–forces elongating the spindle dominate

Cytoplasmic dynein moves

toward minus (–) end
Microtubule disassembly at kinetochores
Microtubule (+)
plus flux moves sister chromatids toward poles
(+) (+)
(+) (+)
(+) Flux
(+) (+)

(+)
Ordered central
(+) spindle assembles
(–) (–) (–) (+) (–) (+)
(–) (–) (–) (–)
(–) (–)
(–) (–) (–) (–) (–)
(+) Kinesin-13
(+)
(+) (–)
(+) (–)
(+)

(–) (+)
(–) (+) NuMA
Bipolar plus-end–directed kinesin-5 dominates
Inward force from minus-end–directed kinesin-14 and microtubule to elongate spindle, pushing poles apart
disassembly at poles by kinesin-13 is balanced by outward force
from kinesin-5 plus dynein stretching poles apart
Microtubule assembly at kinetochores and disassembly at poles causes tubulin subunit flux along kinetochore microtubules

FIGURE 44.7  ROLE OF MOTOR PROTEINS IN SPINDLE DYNAMICS. Mitotic spindle structure depends on microtubule assembly/
disassembly plus balanced forces that slide microtubules relative to one another and to pull the poles together or push them apart. A, In
metaphase, the structure is at steady state. Forces that tend to elongate the spindle, including cytoplasmic dynein (which moves toward
microtubule minus ends, pulling the poles out toward the cell cortex) and bipolar kinesin-5 (which moves toward microtubule plus ends, pushing
the poles apart), are counterbalanced by cohesion between sister chromatids and kinesin-14, which moves toward microtubule minus ends (and
pulls the poles together) and microtubule disassembly at the spindle poles. Dynein and its associated protein, NuMA (nuclear mitotic apparatus),
also help to organize a focused spindle pole. B, In anaphase, sister chromatids separate, the balance of kinesin activity shifts, microtubule disas-
sembly at the poles declines, and the spindle undergoes a dramatic elongation. During anaphase, bipolar kinesin-5, chromokinesin KIF4A, and
protein regulated in cytokinesis 1 (PRC1) also have important roles in organizing the central spindle, which is essential for subsequent assembly
and function of the cleavage furrow.

the way from the kinetochores to the spindle poles.
Interpolar microtubules are distributed throughout
the body of the spindle and do not attach to kineto-
chores. Many interpolar microtubules penetrate between
and through the chromosomes and extend for some
distance beyond them. Thus, the central spindle contains
a large number of interdigitated antiparallel microtu-
bules. Tracking these spindle microtubules by electron
microscopy revealed a tendency for the interdigitated
microtubules of opposite polarity to pack next to one
another. During late anaphase, these antiparallel micro-
tubules bundle to form a structure called the central
spindle that plays important roles in signaling during
cytokinesis. Astral microtubules project out from the
poles and have a role in orienting and positioning the
spindle in the cell through interactions with the cell
cortex in somatic cells. All microtubules within each
aster have the same polarity, with their minus ends
proximal to the pole. Each unit of a spindle pole, with
its associated kinetochore and interpolar and astral
microtubules, is referred to as a half-spindle.
Spindle structure is largely determined by a combina-
tion of microtubule dynamics plus the action of at least
seven different types of kinesins and cytoplasmic dynein
(see Chapter 36). These motors often work in opposition
to one another. Furthermore, forces exerted by motors
can influence the dynamic assembly/disassembly of
microtubules. As a result, the highly dynamic spindle
changes shape as a delicate balance of forces shifts
between the various motors. For example, inactivating
one or more kinesins with drugs or switching a
temperature-sensitive mutant to the nonpermissive
temperature can cause the spindle to collapse rapidly
on itself.
Spindle Assembly
In metazoans, spindle assembly starts in prophase with
the separation of the asters. In most cells, each aster is
organized around a centrosome, consisting of a centriole
pair and associated pericentriolar material. γ-Tubulin
ring complexes in the pericentriolar material efficiently
nucleate microtubules (see Fig. 34.16), so each centro-
some acts as a microtubule organizing center
(MTOC). By the end of prophase, the spindle consists of
two asters linked by a few interpolar microtubules.
Cytoplasmic dynein at the cell cortex exerts an outward
force separating the centrosomes, whereas kinesin-14
motors (which move toward microtubule minus ends)
on the interpolar microtubules exert a counterbalancing
force holding the asters together.
This balance of forces changes when the nuclear
envelope breaks down. Bipolar kinesin-5 motors phos-
phorylated by Cdk1 kinase concentrate in the central
spindle, where they crosslink adjacent antiparallel

interpolar microtubules. Kinesin-5 moves toward the
plus ends of microtubules, so when attached to two
adjacent antiparallel microtubules, its action will cause
them to slide and push the spindle poles apart (Fig.
44.7). However, the two half-spindles do not separate
because they are physically linked via the chromo-
somes, with sister kinetochores attached to opposite
spindle poles.
Also at this time, the asters mature into focused
spindle poles. The pericentriolar material efficiently
nucleates the assembly of new microtubules with their
minus ends at the pole. Microtubule assembly at two
other sites also contributes to spindle morphology. At
kinetochores, a complex derived from interphase nuclear
pores recruits γ-tubulin. This locally nucleates microtu-
bules that grow by inserting subunits at the kinetochores,
pushing the minus ends with γ-tubulin outwards toward
the spindle poles. These preformed kinetochore fibers
are then incorporated into the spindle. In the central
spindle, microtubules are nucleated on the walls of other
microtubules by γ-tubulin recruited by the multi-subunit
augmin complex. The action of augmin creates branched
microtubules throughout the central spindle, contribut-
ing to an even fir tree-like distribution of microtubules.
The daughter microtubules may be released from their
nucleation site, free at both ends, and move toward the
spindle poles as part of the flux of tubulin from the
center of the spindle toward the centrosomes.
The microtubule array focuses at the poles partly
because centrosomes tether microtubules, and partly
due to the concerted action of various motors and micro-
tubule crosslinking proteins such as dynein and nuclear
mitotic apparatus (NuMA) protein. NuMA is released
from the nucleus when the nuclear envelope breaks
down, and it accumulates near the poles at the minus
ends of microtubules.
Large cells that lack centrosomes, such as eggs, form
spindles by an alternative pathway that also functions in
the background in cells with centrosomes (Fig. 44.8).
This mechanism hijacks the nuclear trafficking system to
Unstable microtubule
g radient
TP Microtubule
G
stabilized by
n-
Ra
Ran-GTP
gradient
Stabilized Motors sort
microtubules microtubules,
accumulate on which bind to
chromosomes kinetochores
FIGURE 44.8  ASSEMBLY OF A BIPOLAR SPINDLE IN THE ABSENCE OF CENTROSOMES. A gradient of Ran-guanosine triphosphate
(GTP) stabilizes microtubules around chromosomes, which contain high concentrations of bound Ran GEF RCC1. This releases spindle assembly
factors from importin α and β. Microtubules that accumulate around the chromosomes are sorted, organized, and focused to make poles by
motors and (−) end-binding proteins such as NuMA. These spindle poles lack prominent astral microtubules.
CHAPTER 44  n  Mitosis and Cytokinesis 761
enable chromosomes to control the activity of key
spindle assembly factors. The nuclear import receptors
importin α and β bind these factors, as though they were
going to transport them into the nucleus. This blocks
their spindle assembly activity. During mitosis, chromo-
somes bind high levels of RCC1, the guanine exchange
factor for Ran (Ran-GEF in Fig. 9.18). This RCC1 creates
a gradient of the active guanosine triphosphatase
(GTPase) Ran-GTP (guanosine triphosphate) around the
chromosomes. During interphase, Ran-GTP is confined
to the nucleus, where it releases importins from their
cargo. In mitosis the chromosome-associated cytoplas-
mic Ran-GTP gradient locally liberates the spindle assem-
bly factors including motor proteins and NuMA from
sequestration by importins. They then stabilize nearby
microtubules and organize them into a bipolar spindle.
If centrosomes are removed or destroyed experimen-
tally in cells about to enter mitosis, somatic cells can also
use motor proteins to organize microtubules into bipolar
spindles that lack asters but are otherwise remarkably
normal. Most treated cells manage to complete mitosis
successfully but normal mammalian cells then either
arrest in the next cell cycle prior to replicating their DNA
or commit suicide. Both outcomes depend on the pres-
ence of the important tumor suppressor protein p53 (see
Fig. 43.11). Thus, centrosomes are not required to form
spindles, but they contribute to cell-cycle progression in
many cells. This dependence on centrosomes is not
universal; Drosophila, for example, can live without
centrosomes.
Chromosome Attachment to the Spindle
Dynamic microtubules of prometaphase asters scan the
cytoplasm effectively “searching” for binding sites that
will capture and stabilize their distal plus ends. Captured
microtubules are approximately fivefold less likely to
depolymerize catastrophically than free microtubules.
When catastrophes do occur, the microtubules depoly-
merize back to the pole, recycling tubulin subunits for
incorporation into other, growing microtubules.
Other motors and
(–) end-binding proteins
organize spindle poles
(note absence of asters)
762 SECTION X  n  Cell Cycle

Breakdown of the nuclear envelope makes the con-
densed chromosomes accessible to the microtubules.
Chance encounters allow kinetochores to capture
microtubule plus ends. Capture probably involves the
nine-component KMN network, which includes the rod-
shaped Ndc80 complex (see Fig. 8.21) that binds along
the sides of microtubules near their plus ends. Another
member of the complex, the scaffolding protein Knl1
(its name in vertebrates—the “K” of KMN; see later),
anchors Ncd80 in the kinetochore.
Historically, it was thought that forces generated by
bipolar attachment of the kinetochores of sister chro-
matids center chromosomes midway between the two
spindle poles. This hypothesis was based on the observa-
tion that when a kinetochore first attaches to a microtu-
bule, the chromosome moves along the side of that
microtubule toward the spindle pole (Fig. 44.9). Subse-
quent capture of a microtubule emanating from the
opposite spindle pole by the sister kinetochore would
provide a counterforce pulling the chromosome in the
opposite direction. Chromokinesin family motor proteins
distributed along the chromosome arms were also
thought to contribute to the gradual movement of the
chromosome toward the middle of the spindle. These
movements are accompanied by coordinated shrinkage
of the microtubules at the leading kinetochore and
growth of microtubules at the trailing kinetochore.
More recent studies revealed that chromosomes
attached to only one spindle pole can move away from
that pole if the unattached kinetochore associates with
the kinetochore fiber of a chromosome already aligned
at the spindle equator. In this case, the kinetochore of
the mono-oriented chromosome glides toward the
equator, where it is more likely to capture microtubules
emanating from the opposite pole. This motion of one
chromosome along the kinetochore fiber of another
chromosome requires the kinesin-7 motor centromere
protein E (CENP-E) (see Fig. 36.13) associated with the
kinetochore of the moving chromosome. Recognition of
a tubulin posttranslational modification leads CENP-E to
move the chromosome toward the spindle equator,
rather than out into the aster.
The attachment of microtubules to kinetochores can
be reconstituted in vitro from mixtures of chromosomes,
isolated centrosomes, and tubulin subunits. The plus
ends of microtubules grow out from centrosomes and

A E
Spindle
pole
Spindle pole
4 Microtubule
Kinetochore
Distance traveled toward

3
spindle pole (µm)

B 1

–1 Start of
movement
–2
0 20 40 60 80
Time (seconds)

C D
Kinetochore Microtubule
10 µm

FIGURE 44.9  INITIAL CHROMOSOMAL MOVEMENTS DURING PROMETAPHASE. A–B, Capture of a microtubule by the kinetochore
results first in movement along the side of the microtubule toward the pole from which that microtubule originated. These images come from a
study in which living cells, observed by differential interference microscopy, were subjected to rapid chemical fixation just after a chromosome
had attached to the spindle (arrow). C, Attachment of the chromosome to the spindle was confirmed by indirect immunofluorescence staining
for tubulin and thin-section electron microscopy (D). E, The graph shows the movements of the chromosome before and after attachment. (From
Rieder CL, Alexander SP, Rupp G. Kinetochores are transported poleward along a single astral microtubule during chromosome attachment to
the spindle in newt lung cells. J Cell Biol. 1990;110:81–95, copyright The Rockefeller University Press.)
 CHAPTER 44  n  Mitosis and Cytokinesis 763

attach to the chromosomes. Surprisingly, chromosome-
bound microtubules can either lengthen or shorten at
the attached end without detaching from the chromo-
some. Similar experiments with kinetochores isolated
from budding yeast cells showed that kinetochores can
remain attached to a shortening microtubule plus end
even against an applied force of 9 pN (piconewtons).
Physiological levels of tension actually stabilize the
attachments of kinetochores to microtubules in vitro, as
in vivo. This tethering of kinetochores to disassembling
microtubules is essential for chromosome movements
during mitosis.
Correcting Errors in Chromosome Attachment to
the Spindle
The goal of mitosis is to partition the replicated chromo-
somes accurately between two daughter cells. Therefore,
all chromosomes must attach correctly to both spindle
poles (known as amphitelic attachment; Fig. 44.5)
before being segregated. Three other sorts of attachment
are seen: (a) chromosomes with one kinetochore lacking
attached microtubules (known as monotelic attach-
ment; this is a normal intermediate), (b) chromosomes
with both sister kinetochores attached to the same
spindle pole (known as syntelic attachment), and (c)
chromosomes with a single kinetochore attached simul-
taneously to both spindle poles (known as merotelic
attachment). Correcting monotelic and syntelic errors
takes time, and the SAC (see “Finding Time to Fix
Chromosome Attachment Errors: The Spindle Assembly
Checkpoint” below) delays mitotic progression to allow
the correction process to occur.
When syntelic attachments occur, one or both kineto-
chores must detach for the chromosome to achieve a
bipolar orientation. Chromosome attachment to oppo-
site spindle poles is more stable than attachment to a
single pole, because the tension generated by bipolar
attachment (where forces pull a chromosome simultane-
ously toward opposite spindle poles) preferentially sta-
bilizes microtubule connections to both kinetochores.
Merotelic attachments are more dangerous, as the kineto-
chore is under tension and the attachments are therefore
stable. Merotelic attachments are the most common
cause of chromosome segregation errors in cultured
mammalian cells.
Syntelic and merotelic chromosome attachments are
corrected through the action of Aurora B protein kinase,
which forms the chromosomal passenger complex
(CPC), along with inner centromere protein (INCENP),
survivin, and borealin (Fig. 44.10). The other subunits
target Aurora B to its various sites of action during mitosis
and regulate the kinase activity. The complex concen-
trates at inner centromeres (the heterochromatin beneath
and between the two sister kinetochores) during pro-
metaphase and metaphase. At anaphase onset, the CPC
moves to the overlapping interpolar microtubules of the
central spindle and to the cell cortex, where the cleav-
age furrow will form, ultimately winding up in the

A Borealin
B C

DNA
Survivin
Microtubules

D E. CPC Aurora-B F
INCENP

Borealin

Survivin

Histone H3 phosphorylation
Kinetochore targets
Central spindle targets
Cleavage furrow targets

FIGURE 44.10  CHROMOSOMAL PASSENGER COMPLEX (CPC) REGULATES MITOTIC EVENTS. The CPC (green) is present at
centromeres in prometaphase and metaphase (B), but transfers to the spindle midzone at anaphase (C) and midbody at anaphase (D).
E, Diagram of Aurora B protein kinase complexed with INCENP (inner centromere protein), survivin, and borealin with some key targets of the
CPC. F, If CPC function is inhibited (in this case by RNA interference [RNAi] depletion of borealin), chromosome attachment errors are common
and many chromosomes fail to segregate properly in anaphase. Distribution of DNA (blue), microtubules (red), and survivin–green fluorescent
protein (GFP) (green) in human mitotic cells. Inset in A, Distribution of kinetochores (red), and borealin (green) in a prometaphase cell.
(A–D, Micrographs by Sally Wheatley and William C. Earnshaw. F and Inset in A, Micrographs by Ana Carvalho, Reto Gassmann, and William
C. Earnshaw. Insets in A and F, From Gassmann R, Carvalho A, Henzing AJ, et al. Borealin: a novel chromosomal passenger required for stability
of the bipolar mitotic spindle. J Cell Biol. 2004;166:179–191, copyright The Rockefeller University Press. A–C, From Wheatley SP, McNeish IA.
Survivin: a protein with dual roles in mitosis and apoptosis. Int Rev Cytol. 2005;247:35–88.)
764 SECTION X  n  Cell Cycle

intercellular bridge during cytokinesis. The CPC regu-
lates mitotic events from prophase through cytokinesis.
Along the way it contributes to the correction of
chromosome attachment errors and to the operation of
the checkpoint that delays the cell cycle in response to
those errors.
Aurora B corrects chromosome attachment errors by
phosphorylating Ndc80 in the microtubule-binding KMN
complex (see Fig. 8.21). Aurora B phosphorylation
strongly inhibits Ndc80 binding to microtubules, causing
the kinetochore to release attached microtubules. When
a chromosome is correctly attached to both spindle
poles, tension stretches the kinetochore away from the
CPC buried in the chromatin beneath. This can stabilize
chromosome–microtubule interactions by preventing
the kinase from phosphorylating Ndc80.
Finding Time to Fix Chromosome Attachment Errors:
The Spindle Assembly Checkpoint
Segregation of replicated chromosomes into daughter
cells is extremely accurate. For example, budding yeasts
lose a chromosome only once in 100,000 cell divisions.
The frequency of chromosome loss may be 20-fold to
400-fold higher for human cells grown in culture. To
achieve even this level of accuracy, most cells delay
entry into anaphase until all chromosomes have achieved
amphitelic attachment to the spindle. This delay is
caused by the spindle assembly checkpoint (SAC), which
senses the completion of microtubule binding to kineto-
chores at metaphase (Fig. 44.11). The spindle checkpoint
differs from DNA damage checkpoints in that its default
setting is “on” as cells enter mitosis. It is silenced only
when every chromosome is properly attached to the
spindle.
The SAC involves the products of the mitotic arrest–
defective (MAD) genes, the budding-uninhibited-by-
benzimidazole (BUB) genes, the monopolar spindle
(Mps1) kinase and the CPC. The MAD and BUB genes
were identified in yeast genetic screens for cells that
continued to divide (and die) when the spindle was
disassembled by drugs. These genes are conserved
from yeast to humans. SAC proteins accumulate at

A D

C
23 minutes
Time

B
Seat
belt
N

Time Mad1 • Mad2

Destroy free kinetochore 23 minutes
with blast of laser light
APC/C
substrate

C
APC/C APC/C
it

Go
Wa

MCC Cdc20APC/C
Kinetochore•
Mad1 • Mad2 Mad2
APC/C
Microtubule Cdc20APC/C

Checkpoint proteins
not on kinetochore BubR1

Cdc20MCC U Ub Ub
Go! Wait! Ub b

Mad2 MCC
Proteins not to scale relative to kinetochore Substrate

FIGURE 44.11  SPINDLE CHECKPOINT. Signaling by unattached kinetochores stops the cell from entering anaphase until all chromosomes
have made a proper bipolar spindle attachment. A, As long as there is a chromosome that is not properly attached to the spindle (beige cells),
the cell does not enter anaphase. The cell enters anaphase approximately 20 minutes after chromosome attachment is complete (green cells).
B, In a cell with a persistently maloriented chromosome, anaphase entry is delayed (beige cells). If the unattached kinetochore is destroyed with
a high-powered laser, the cell enters anaphase about 20 minutes later. This proves that the unattached kinetochore sends an inhibitory signal.
C, Overview of the spindle assembly checkpoint (see text for details). D, Structure of the Mad1/Mad2 complex.

kinetochores early in mitosis, when the checkpoint is
“on” (ie, during prophase or prometaphase), and are
gradually displaced as microtubules bind and the kineto-
chores come under tension.
The target of the SAC is the APC/CCdc20, the anaphase-
promoting complex/cyclosome (APC/C) ubiquitin-
protein ligase (an E3 enzyme; see Figs. 23.3 and 40.16)
with its substrate recognition factor Cdc20 bound, APC/
CCdc20 ubiquitylates target proteins to mark them for
destruction by proteasomes. Key APC/CCdc20 substrates
are proteins that must be degraded for the cell to move
from metaphase to anaphase, including cyclin B and
securin, an inhibitor of the enzyme that triggers separa-
tion of sister chromatids at anaphase (Fig. 44.16).
During mitosis, Cdk1 activates the APC/C by phos-
phorylating an auto-inhibitory loop, allowing Cdc20 to
bind. The SAC is an additional regulatory circuit that
inactivates APC/CCdc20 until all kinetochores attach to
spindle microtubules. Kinetochores without microtubules
attract proteins that assemble the mitotic checkpoint
complex (MCC), the inhibitor that inactivates APC/CCdc20.
Checkpoint activation starts when Aurora B in the
CPC activates Mps1 kinase, allowing it to phosphorylate
Knl1 in the kinetochore at several sites. Mps1 phos-
phorylation of Knl1 creates a binding site that results in
Mad1 recruitment to the kinetochore. Mad1 then recruits
Mad2 to form a stable complex (Fig. 44.11). A loop on
Mad2 wraps around Mad1 like a safety belt making
the complex particularly stable. This form of Mad 2 is
known as “closed” Mad2. Mad1/Mad2 can transiently
bind soluble Mad2 molecules (known as “open” Mad2),
load them onto Cdc20 in the closed safety belt conforma-
tion (this loading probably occurs at kinetochores), then
release them to form the soluble MCC of Mad2/Cdc20/
BubR1/Bub3. The MCC associates with the APC/CCdc20,
interfering with binding of cyclin B and other key sub-
strates. As each chromosome becomes attached to both
poles of the spindle it stops producing MCC. When the
last chromosome has achieved a proper attachment, the
last source of MCC is extinguished, and entry into ana-
phase can proceed.
A. Metaphase
Kinetochore
microtubules
Astral
microtubules
Interpolar
microtubules
Cyclin B and
securin degraded Chromosomes oscillate
FIGURE 44.12  INTRODUCTION TO METAPHASE. A, Summary of the major events of metaphase. B, Distribution of DNA (blue), microtu-
bules (red), and gamma tubulin (centrosomes [green]) in a metaphase human cell. (B, Images were recorded by Dr. Melpomeni Platani on the
University of Dundee’s School of Life Sciences Imaging Facility OMX 3DSIM Microscope and stored and processed in OMERO.)
CHAPTER 44  n  Mitosis and Cytokinesis 765
Silencing of the checkpoint involves several pathways.
Protein phosphatase 2A (PP2A) and protein phosphatase
1 (PP1) are recruited to the kinetochore in feedback
loops involving the CPC and other checkpoint compo-
nents. They dephosphorylate Knl1 so that it releases
Mad1. When microtubules bind, cytoplasmic dynein
motors actively strip checkpoint components from the
kinetochore, dragging them away toward the centro-
somes. In yeast, access of Mps1 to its target sites on
Knl1 is physically blocked when microtubules bind.
Exactly how this interaction is regulated in metazoans is
still actively studied. In addition, the SAC appears to
crosstalk with the DNA damage response (see Chapter
43), since DNA damage response components activate
SAC components and vice versa. However, the network
of interactions is very complex and details are still being
worked out.
Experimental inactivation of the spindle checkpoint
causes a catastrophic, premature entry into anaphase,
regardless of the status of chromosome alignment. This
leads to an unequal distribution of sister chromatids and
genetic imbalance between daughter cells known as
aneuploidy. Yeasts can live without the checkpoint
genes, but their loss is lethal for mice, which die early
during embryogenesis. Mice heterozygous for various
checkpoint components show increased aneuploidy.
Humans with mutations in BubR1 have mosaic variegated
aneuploidy syndrome (extra copies or loss of various
chromosomes in a variety of tissues), which is associated
with microcephaly (decreased brain size) and an
increased cancer risk.
Metaphase
When all the chromosomes have attained amphitelic
orientations and moved to positions roughly midway
between the two spindle poles, the cell is said to be in
metaphase (Fig. 44.12). The compact grouping of chro-
mosomes at the middle of the spindle is referred to as
the metaphase plate. In many cells, even though chro-
mosomes remain, on average, balanced at the middle of
B
DNA
Microtubules
Centrosomes
766 SECTION X  n  Cell Cycle

A B C D

Spindle pole
E F G

P AP

AP P

2 µm
2 min
Spindle pole

FIGURE 44.13  KINETOCHORE OSCILLATIONS BETWEEN P (POLEWARD) AND AP (AWAY FROM THE POLE) MOVEMENT DURING
LATE PROMETAPHASE AND ANAPHASE IN PTK1 (RAT KANGAROO) CELLS. A–D, Images showing the movements of several pairs of sister
kinetochores, labeled with green fluorescent protein (GFP)-Cdc20 (green), combined with phase-contrast images of the cell (red). E and G, Higher-
magnification views of sister kinetochores (marked with dashed lines) in prometaphase and anaphase, respectively. F, Kymograph (collage of images
of a vertical strip showing the same two kinetochores at various time points during the movie) showing the movements of these two kinetochores.
Movements toward (P) and away from (AP) spindle poles are indicated. P movement involves microtubule shrinkage at the leading kinetochore and
microtubule growth at the trailing kinetochore (which is undergoing AP movement away from its associated kinetochore). Spindle poles are near
the top and bottom of panels E to G. (Micrographs courtesy E.D. Salmon, University of North Carolina, Chapel Hill.)

the spindle, they jostle one another and undergo numer-

ous small excursions toward one pole or the other
throughout metaphase (Fig. 44.13).
Metaphase can also be defined biochemically as the
time of destruction of cyclin B and securin (Fig. 44.16),
because this begins as soon as the last chromosome
achieves amphitelic orientation. Degradation of cyclin A
begins earlier, at the entry into prometaphase, and is
largely complete before metaphase. Loss of securin initi-
ates a process leading to the separation of sister chroma-
tids and the onset of anaphase.
Microtubule Flux Within the Metaphase Spindle
Although the average length of the kinetochore micro-
tubules is roughly constant during metaphase, the
microtubules change continuously in three ways. First,
there is constant net addition of new tubulin subunits
(approximately 10 subunits per second) to the plus end
of the microtubules, where they are attached to the
kinetochore. Second, a comparable number of tubulin
subunits is continuously lost from the minus end of
the kinetochore tubules at the spindle poles. There­
fore, tubulin subunits slowly migrate through kineto-
chore microtubules from the kinetochore to the pole
(Fig. 44.14). This subunit flux or treadmilling in kineto-
chore microtubules is caused by microtubule depolymer-
ization at the poles driven by kinesin-13 family members.
In addition, tubulin moves toward the poles as by micro-
tubules are transported towards the pole (many nucle-
ated by the augmin complex) within the kinetochore
fiber. All microtubules attached to each kinetochore
A B C
0 sec
Fluorescent tubulin
10 sec speckles move
toward poles
20 sec
P P
P P P P
30 sec
FIGURE 44.14  MICROTUBULE FLUX IN METAPHASE. A, Cells
entering mitosis were injected with tubulin subunits modified chemi-
cally by attachment of a caged fluorescent dye. This dye becomes
fluorescent after being irradiated with UV light. When cells entered
metaphase, the spindle was illuminated with a narrow stripe of UV
light, activating a narrow band of fluorescent tubulin subunits. With
time, these subunits approach the spindle poles (P). Because the
length of kinetochore microtubules is constant during this time, the
labeled tubulin molecules must migrate along the microtubules toward
the pole (arrows). This can occur if new subunits are added to the
microtubule at the kinetochore and old subunits are removed at the
pole. B, Microtubule flux at metaphase in a Drosophila embryo visual-
ized by fluorescence speckle microscopy. Embryos were injected with
very low levels of fluorescent tubulin, which appears as speckles dis-
tributed along the microtubules. If a very sensitive camera is used,
these speckles can be seen to move toward the poles, reflecting the
flux in the underlying microtubules. Scale bar is 5 µm. C, Movement
of labeled tubulin speckles toward the spindle poles. (A, Courtesy
Arshad Desai and the MBL Cell Division Group, Marine Biology Labo-
ratory, Woods Hole, MA; from Mitchison TJ, Salmon ED. Mitosis: a
history of division. Nat Cell Biol. 2001;3:E17–E21. B, Courtesy Paul
Maddox and Arshad Desai, University of California, San Diego.)
 CHAPTER 44  n  Mitosis and Cytokinesis 767

change coordinately in length during chromosomal
oscillations.
Anaphase
The separation of sister chromatids at anaphase is one of
the most dramatic events of the entire cell cycle (Fig.
44.15). Sister chromatids move to opposite spindle poles
(anaphase A), and the poles move apart (anaphase B).
Anaphase is also the time when the mitotic spindle
activates the cell cortex in preparation for cytokinesis.
Two forms of the APC/C (see Fig. 40.15) trigger the
transition from metaphase to anaphase by degrading key
proteins. APC/CCdc20 targets cyclin B for degradation,
causing Cdk activity to fall (see Fig. 40.17). This decline
in Cdk activity allows for activation of APC/CCdh1, because
Cdh1 phosphorylated by Cdk1 kinase cannot bind to the
APC/C. APC/CCdh1 targets polypeptides whose destruc-
tion by the proteasome is required for the cell to exit
from mitosis and return to interphase. APC/CCdh1 remains
active during G1 phase, where it is essential for the
licensing of DNA replication origins (see Fig. 42.28).
Biochemical Mechanism of
Sister Chromatid Separation
Separation of sister chromatids is regulated by the chro-
mosomes themselves, not by the mitotic spindle. Under
certain circumstances, sister chromatids can separate in
the absence of microtubules, ruling out a requirement
for forces from the spindle in the process.
Three factors regulate sister chromatid separation: a
protein complex known as cohesin, a protease known
as separase, and an inhibitor of separase known
as securin (Fig. 44.16). This system is conserved from
yeast to human. Chapter 8 discusses the functions of
cohesin in interphase.
Cohesin is a complex of four proteins that resembles
the condensin complex (see Fig. 8.18). Like condensin,
cohesin has two large subunits from the SMC ATPase
family. These proteins, SMC1 and SMC3, are complexed
with proteins called Scc1 (which has other names
omitted here for simplicity) and Scc3. Additional proteins
are required to stabilize the loading of this complex onto
DNA. Cells with mutations in cohesin components sepa-
rate sister chromatids prematurely in mitosis, resulting
in chaotic chromosome missegregation. This system is
very ancient, as bacteria depend on an SMC-related
protein for orderly chromosome segregation.
A variety of evidence suggests that cohesin forms a
ring with a diameter of 35 nm, large enough to encircle
two sister chromatids like a lasso. In yeast, the complex
functions only if it binds chromosomes during DNA
replication. Cohesin accumulates at preferred sites on
the chromosomes, often near centromeres in budding
yeast or in regions of heterochromatin in fission yeast.
In vertebrates, most cohesin dissociates from the chro-
mosome arms by late metaphase, owing to the action of
the protein kinases Plk1 and Aurora B. Importantly, a
critical fraction remains associated with heterochromatin
flanking centromeres where it is protected from cleav-
age by shugoshin until the onset of anaphase (see follow-
ing paragraphs and Chapter 45).
Sequential cleavage of two key proteins triggers sister
chromatid separation at anaphase. This proteolysis makes
anaphase onset an irreversible transition. The first target,
securin, inhibits the separase protease. After the last
chromosome forms an amphitelic attachment to the
spindle, the spindle checkpoint is silenced. This allows
APC/CCdc20 to tag securin with ubiquitin, leading to its
destruction by proteasomes throughout metaphase. When
securin levels fall below a critical threshold, separase is
unleashed to cleave the Scc1 subunit of cohesin. Cleavage
of Scc1 breaks the cohesin ring, allowing the sister
chromatids to separate triggering the onset of anaphase
(Fig. 44.16B).
Efficient Scc1 cleavage requires that the protein be
phosphorylated near its cleavage site. This allows a
mode of regulation where shugoshin (Japanese for
“guardian spirit”) recruits PP2A to centromeres. PP2A
keeps Scc1 dephosphorylated. This inhibits its cleavage
and protects cohesin until shugoshin is released follow-
ing amphitelic attachment of the chromosome. This

A. Anaphase B
Sister chromatids separate
Cohesin Anaphase A: Chromatids approach
degrades poles (APC/CCdc20 active)
Interdigitated interpolar
microtubules bundled by PRC1
and stem body material to
form central spindle
DNA
Anaphase B: Spindle poles Microtubules
migrate apart (APC/C Cdh1active) Centrosomes

FIGURE 44.15  INTRODUCTION TO ANAPHASE. A, Summary of the major events of anaphase. B, Distribution of DNA (blue), microtubules
(red), and gamma tubulin (centrosomes [green]) in a mid-anaphase human cell. APC/C, anaphase-promoting complex/cyclosome; PRC1, protein
regulated in cytokinesis 1. (B, Images were recorded by Dr. Melpomeni Platani on the University of Dundee’s School of Life Sciences Imaging
Facility OMX 3DSIM Microscope and stored and processed in OMERO.)
768 SECTION X  n  Cell Cycle

Hinge
A. S phase

Replication
fork
Cohesin
complex

Smc3

Smc1
Scc1

SA1

B C D E
Separase
Chromatin
loops Securin
APC/C
Ubiquitin Sister
Cohesin chromatids
Proteasome separate
Sister
chromatids

Active
Mitotic separase
chromosome cleaves Scc1
Metaphase/
anaphase
S phase Early mitosis transition Anaphase

FIGURE 44.16  REGULATION OF SISTER CHROMATID PAIRING BY THE COHESIN COMPLEX. A–B, The cohesin complex forms a
35-nm diameter ring that links sister chromatids during DNA replication. At anaphase onset, degradation of its securin inhibitor liberates active
separase enzyme. C–E, Separase then cleaves cohesin subunit Scc1, and the two sister chromatids are freed to separate from one another and
move toward opposite spindle poles.

mechanism is absolutely essential during meiosis, as
without it, it would not be able to segregate homologous
chromosomes from each other (see Fig. 45.12).
Securin can act as an oncogene in cultured cells
and is overexpressed in some human pituitary tumors.
Overexpression of securin may dis­rupt the timing of
chromosome segregation, leading to chromosome loss
and, ultimately, contributing to cancer progression.
Mitotic Spindle Dynamics and Chromosome
Movement During Anaphase
Anaphase is dominated by the orderly movement of
sister chromatids to opposite spindle poles brought
about by the combined action of motor proteins and
changes in microtubule length. There are two compo-
nents to anaphase chromosome movements (Fig. 44.15).
Anaphase A, the movement of the sister chromatids to
the spindle poles, requires a shortening of the kineto-
chore fibers. During anaphase B, the spindle elongates,
pushing the spindle poles apart. The poles separate
partially because of interactions between the antiparallel
interpolar microtubules of the central spindle and par-
tially because of intrinsic motility of the asters. Most cells
use both components of anaphase, but one component
may predominate in relation to the other.
Microtubule disassembly on its own can move chro-
mosomes (see Fig. 37.8). Energy for this movement
comes from hydrolysis of GTP bound to assembled
tubulin, which is stored in the conformation of the
lattice of tubulin subunits. Microtubule protofilaments
are straight when growing, but after GTP hydrolysis
protofilaments are curved, so they peel back from the
ends of shrinking microtubules (see Fig. 34.6). Several
kinesin “motors” influence the dynamic instability of the
spindle microtubules. Members of the kinesin-13 class,
which encircle microtubules near kinetochores and at
spindle poles, use adenosine triphosphate (ATP) hydro-
lysis to remove tubulin dimers and promote microtubule
disassembly rather than movement.
Kinetochores are remarkable in their ability to hold
onto disassembling microtubules. In straight (growing)
microtubules, the Ndc80 complex is mostly responsible
for microtubule binding. It binds to the interface between
α and β tubulin subunits. This interface bends in curved
(shrinking) microtubules, so Ndc80 cannot bind. This
could allow it to redistribute onto straight sections of
the lattice and thereby move away from the curved
protofilaments at the disassembling end. In metazoans
the Ska complex in the outer kinetochore binds α and β
tubulin subunits away from the interface, so it can bind

to curved (disassembling) protofilaments. At yeast kineto-
chores the Dam1 ring (green in Fig. 8.21) couples the
kinetochore to disassembling microtubules.
Anaphase A chromosome movement involves a com-
bination of microtubule shortening and translocation of
the microtubule lattice that result from flux of tubulin
subunits (Fig. 44.14). The contributions of the two
mechanisms vary among different cell types. When living
vertebrate cells are injected with fluorescently labeled
tubulin subunits, the spindle becomes fluorescent (Fig.
44.17). If a laser is used to bleach a narrow zone in the
fluorescent tubulin across the spindle between the
chromosomes and the pole early in anaphase, the chro-
mosomes approach the bleached zone much faster than
the bleached zone approaches the spindle pole. This
shows that the chromosomes “eat” their way along the
A of the poles involves interaction of the astral microtu-
Photobleached
zone
Time
Microtubule
disassembly
B 42 70 212 422
5 µm
C 40 92 210 436
FIGURE 44.17  CHROMOSOMES MOVE ON SHRINKING
MICROTUBULES DURING ANAPHASE. A, Mitotic cells were
injected with a fluorescently labeled tubulin that was incorporated into
the spindle. Just after anaphase onset, a laser was used to photo-
bleach a stripe (white) across the spindle near the upper pole. The live
cell was monitored over time by fluorescence (B) and phase-contrast
(C) microscopy. In this mammalian cell, the chromosomes approach
the bleached stripe much faster than the stripe approaches the spindle
pole. In other organisms with higher rates of microtubule flux in their
spindles, the bleached zone would also move appreciably toward the
pole. The numbers are time in seconds. (B–C, From Gorbsky GJ,
Sammak PJ, Borisy GG. Microtubule dynamics and chromosome
motion visualized in living anaphase cells. J Cell Biol. 1988;106:1185–
1192, copyright The Rockefeller University Press.)
CHAPTER 44  n  Mitosis and Cytokinesis 769
kinetochore microtubules toward the pole. In these
cells, subunit flux accounts for only 20% to 30% of
chromosome movement during anaphase A, and this flux
is dispensable for chromosome movement. In Drosophila
embryos, in which subunit flux accounts for approxi-
mately 90% of anaphase A chromosome movement, the
chromosomes catch up with a marked region of the
kinetochore fiber slowly, if at all.
Anaphase B appears to be triggered at least in part by
the inactivation of the minus-end–directed kinesin-14
motors, so that all the net motor force favors spindle
elongation. Four factors contribute to overall lengthen-
ing of the spindle: release of sister chromatid cohesion,
sliding apart of the interdigitated half-spindles, microtu-
bule growth, and intrinsic motility of the poles them-
selves (Fig. 44.7). During the latter stages of anaphase B,
the spindle poles, with their attached kinetochore micro-
tubules, appear to move away from the interpolar
microtubules as the spindle lengthens. This movement
bules with cytoplasmic dynein molecules anchored at
the cell cortex.
Anaphase B spindle elongation is accompanied by
reorganization of the interpolar microtubules into a
highly organized central spindle between the separat-
ing chromatids (Fig. 44.15). Within the central spindle,
an amorphous dense material called stem body matrix
stabilizes bundles of antiparallel microtubules and holds
together the two interdigitated half-spindles. Proteins
concentrated in the central spindle help regulate cytoki-
nesis. One key factor, PRC1 (protein regulated in cytoki-
nesis 1), is inactive when phosphorylated by Cdk kinase
and functions only during anaphase when Cdk activity
declines and phosphatases remove the phosphate groups
placed on target proteins by Cdks and other mitotic
kinases. PRC1 directs the binding of several kinesins to
the central spindle. The kinesin KIF4A targets Aurora B
kinase to a particular domain of the central spindle,
where phosphorylation of key substrates then regulates
spindle elongation and cytokinesis.
How can protein kinases such as Aurora B continue
to function during anaphase while protein phosphatases
are removing phosphate groups placed there by Cdks
and, indeed, Aurora B during early mitosis? One answer
is that the phosphatase activity is highly localized, con-
trolled by specific targeting subunits. Cdk phosphoryla-
tion can inhibit targeting subunits such as the exotically
named Repo-Man (recruits PP1 onto mitotic chromatin
at anaphase) from binding protein phosphatase 1 or
localizing to targets, such as chromatin in early mitosis.
When Cdk activity drops, Repo-Man (and other similar
targeting subunits) is dephosphorylated, and now targets
PP1 to chromatin, where it removes phosphates placed
there by Aurora B in the CPC. As long as phosphatases
are not specifically targeted to the cleavage furrow,
Aurora B can continue to control events there during
770 SECTION X  n  Cell Cycle

mitotic exit by phosphorylating key target proteins
required for cytokinesis.
Telophase
During telophase, the nuclear envelope reforms on the
surface of the separated sister chromatids, which typi-
cally cluster in a dense mass near the spindle poles (Fig.
44.18). Some further anaphase B movement may still
occur, but the most dramatic change in cellular structure
at this time is the constriction of the cleavage furrow and
subsequent cytokinesis.
Reassembly of the Nuclear Envelope
Nuclear envelope reassembly begins during anaphase
and is completed during telophase (Fig. 44.19). As in
spindle assembly, Ran-GTP promotes early steps of
nuclear envelope assembly at the surface of the chromo-
somes by releasing key components sequestered by
importin β. These include several nuclear pore com­
ponents, and one of the earliest events in nuclear enve-
lope reassembly involves binding of the nuclear pore
scaffold protein ELYS to chromatin. ELYS can recognize
DNA regions rich in A : T base pairs, so it is likely to
bind directly to the DNA. ELYS then recruits other
components of the nuclear pore scaffold and nuclear
pore trans-membrane proteins. The pore subsequently
matures as various peripheral components and elements
of the permeability barrier are added.
The mechanism of nuclear membrane reassembly is
debated. In cells where nuclear membranes fragments
into vesicles during mitosis, a Ran-GTP–dependent
pathway directs at least two discrete populations of
vesicles to chromatin where they fuse to reform the
nuclear envelope. In cells where the nuclear membrane
is absorbed into the endoplasmic reticulum during
mitosis, reassembly involves lateral movements of mem-
brane components within the membrane network and
their stabilization at preferred binding sites at the periph-
ery of the chromosomes.
Lamin subunits disassembled in prophase are recycled
to reassemble at the end of mitosis. Lamina reassembly
is triggered by removal of mitosis-specific phosphate
groups and methyl-esterification of several COOH side

A. Telophase B
Cleavage plane
specified
Nuclear envelope
reassembles around Organized
chromosomes central spindle
assembles

Poles DNA
continue Microtubules
to separate Centrosomes

FIGURE 44.18  INTRODUCTION TO TELOPHASE. A, Summary of the major events of telophase. B, Distribution of DNA (blue), microtubules
(red), and γ-tubulin (centrosomes [green]) in a telophase human cell. (B, Images were recorded by Dr. Melpomeni Platani on the University of
Dundee’s School of Life Sciences Imaging Facility OMX 3DSIM Microscope and stored and processed in OMERO.)

A B C

0 min 8–10 min 333 nm ≥ 25 min

FIGURE 44.19  SCANNING ELECTRON MICROSCOPY OF THE STAGES OF ASSEMBLY OF MEMBRANE VESICLES ON THE
SURFACE OF CHROMOSOMES IN A XENOPUS EGG CYTOSOLIC EXTRACT. A cell lysate containing membrane vesicles was added to
isolated chromatin from Xenopus sperm, fixed, and then imaged by scanning electron microscopy. Each panel shows the time of incubation prior
to fixation. (Micrographs courtesy of K.L. Wilson, Johns Hopkins Medical School, Baltimore, MD. A and C, From Wiese C, Goldberg MW, Allen
TD, et al. Nuclear envelope assembly in Xenopus extracts visualized by scanning EM reveals a transport-dependent “envelope smoothing” event.
J Cell Sci. 1997;110:1489–1502.)
 CHAPTER 44  n  Mitosis and Cytokinesis 771

chains on lamin B (Fig. 44.6). Together with ELYS, B-type

lamins are among the earliest components of the nuclear
envelope to target to the surface of the chromosomes
during mid-anaphase. Either at this time or shortly there-
after, other proteins associated with the inner nuclear
membrane, including BAF, LAP2, and lamin B receptor
(see Fig. 9.10), join the forming envelope. Later during
telophase when nuclear import is reestablished, lamin
A enters the reforming nucleus and slowly assembles
into the peripheral lamina over several hours in the
G1 phase. If lamin transport through nuclear pores is
prevented, chromosomes remain highly condensed fol-
lowing cytokinesis, and the cells fail to reenter the next
S phase.
Cytokinesis
Cytokinesis divides a mitotic cell into two daughter cells
(Fig. 44.20). Cytokinesis depends on signals to specify
the cleavage plane (Fig. 44.21), assembly and constric-
tion of the contractile apparatus, specific alterations of
the cell membrane, and the final separation (abscission)
of the two daughter cells.
In animals, protozoa, and most fungi, a contractile
ring of actin filaments and myosin-II guides the separa-
tion of daughter cells at the end of mitosis (Fig. 44.2).
Myosin-II pulls on the ring of actin filaments, applying
tension to the plasma membrane, much like contraction
of smooth muscle (see Figs. 39.23 and 39.24). Because

A. Early cytokinesis B. Late cytokinesis C

Chromatin decondenses
New membrane Actomycin
inserted Nuclear substructures
contractile reform
Actomycin ring forms
Interphase microtubule
Midbody array reassembles
begins
to form Midbody

ESCRT III action leads to

separation (abscission) of the two cells

FIGURE 44.20  INTRODUCTION TO CYTOKINESIS. A–B, Summary of the major events of cytokinesis. C, Distribution of DNA (blue),
microtubules (red), and γ-tubulin (centrosomes [green]) in a human cell undergoing cytokinesis. ESCRT, endosomal sorting complexes required
for transport. (C, Images were recorded by Dr. Melpomeni Platani on the University of Dundee’s School of Life Sciences Imaging Facility OMX
3DSIM Microscope and stored and processed in OMERO.)

A. Evidence that the cleavage furrow is positioned B. An organized central spindle is required for
midway between asters in eggs cleavage furrow formation and/or function
TOP VIEW
Glass rod pushed
down into egg

90°

Sand dollar egg Microtubules

Chromosomes

Ectopic furrow Actin

ring

Profilin
Metaphase 1 Cytokinesis 1 Metaphase 2 Cytokinesis 2 Wild type mutant

FIGURE 44.21  IN EGGS, A CLEAVAGE FURROW FORMS MIDWAY BETWEEN SPINDLE ASTERS. IN ANIMAL CELLS, THE CENTRAL
SPINDLE IS IMPORTANT. A, A classic experiment in which a sand-dollar egg is caused to adopt a toroid shape. At cytokinesis 2, the egg
cleaves into four cells, and a furrow forms between the back sides of the two spindles. (For a description of this and other classic experiments
in cytokinesis, see the book by Rappaport in the “Selected Readings” list.) B, Left, A wild-type Drosophila spermatocyte undergoing cytokinesis,
with the contractile ring stained in yellow. Right, In a profilin mutant, no central spindle forms, and the cell fails to form a contractile ring.
(Micrographs courtesy Professor Maurizio Gatti, University of Rome, Italy. B, From Giansanti MG, Bonaccorsi S, Williams B, et al. Cooperative
interactions between the central spindle and the contractile ring during Drosophila cytokinesis. Genes Dev. 1998;12:396–410.)
772 SECTION X  n  Cell Cycle
the contractile ring is confined to a narrow band of
cortex around the equator, it forms a cleavage furrow,
constrict­ing the plasma membrane locally like a purse
string (Fig. 44.20). Signals from the mitotic spindle and
cell cycle machinery control the position of this ring (ie,
the relative sizes of the two daughter cells) and the
timing of its constriction.
Protozoa, animals, fungi, and plants use an evolution-
arily conserved set of components to implement differ-
ent strategies to separate daughter cells. For example,
both fission yeast and metazoan cells use signals from
polo kinase and a Rho-GTPase to direct the assembly of
a contractile ring of actin, myosin-II, and other conserved
components, even though the yeast has a closed mitosis
and the metazoans have an open mitosis. In animal
cells, contractile ring constriction provides the force that
remodels the cortex to generate the two daughter cells.
In contrast, in yeasts, which have a cell wall, contractile
ring constriction is thought to guide the orderly centrip-
etal growth of the cell wall septum, which contributes
force to overcome turgor pressure and invaginate the
plasma membrane. Plants lack myosin-II, so they divide
by targeted fusion of membrane vesicles to build a new
cell wall rather than constricting a cleavage furrow (Box
44.1 and Fig. 44.26). These differences reflect the fact
that widely divergent eukaryotes use variations of similar
themes for cytokinesis. Cytokinesis in prokaryotes is
genuinely different, since completely different proteins
are involved (Box 44.2 and Fig. 44.27).
Although cytokinesis has been studied for more
than 100 years, it has posed a number of challenges due
to its complexity at the molecular level. For example
genetic analysis of fission yeast revealed more than
150 genes that contribute to cytokinesis. RNAi-based
protein knockdown and molecular replacement analy­
sis indicates that similar proteins participate in cytoki-
nesis of Caenorhabditis elegans, Drosophila, and
vertebrate tissue culture cells. Cytokinesis research typi-
cally employs living cells, although progress is being
made toward reconstituting some aspects of the process
in cell-free systems.
Signals Regulating the Position of
the Cleavage Furrow
Elegant experimental data from classic studies on fertil-
ized echinoderm eggs suggest that a cleavage stimulus,
emitted by the mitotic spindle, specifies the position of
the cleavage furrow midway between the poles and
perpendicular to the long axis of the spindle, thereby
ensuring that the cleavage process separates the daugh-
ter nuclei (Fig. 44.21). In fertilized eggs, the poles, with
their large astral arrays of microtubules (see Fig. 6.4B),
were regarded as the source of the cleavage stimulus, as
furrows can be induced to form midway between two
poles, even when no chromosomes are present. In addi-
tion, a signal emitted by the bundled microtubules of the
central spindle appeared to modulate the behavior of the
furrow signaled by the poles. We now know that the
central spindle does emit a positive signal directing a
cleavage furrow to form above it, while the poles con-
tribute by focusing that furrow at a point on the cortex
midway between them.
The molecular nature of the cleavage stimulus is now
beginning to be understood in animals. The following is
a simplified scenario:
1. During anaphase, overlapping microtubules between
the separating chromatids establish an ordered array
known as the central spindle. A key protein compo-
nent of this array is a protein heterodimer known as
centralspindlin. Centralspindlin is normally seques-
tered in the cytoplasm, but phosphorylation by the
CPC enables it to target to the central spindle. Dro-
sophila mutants that fail to form a central spindle
cannot initiate cytokinesis (Fig. 44.21B). In contrast,
C. elegans embryos that lack a central spindle can
initiate but not complete the process.
2. One of the components of centralspindlin recruits a
GEF (guanine exchange factor; see Figs. 4.6 and 4.7)
for the small GTPase RhoA. This Rho-GEF, Ect2, also
has a motif for targeting to the inside surface of the
equatorial plasma membrane.
3. Membrane associated Ect2 locally activates RhoA,
which then stimulates localized actin filament assem-
bly and activation of myosin-II to begin assembly of
the contractile ring.
Signals from the poles of the mitotic spindle contribute,
particularly in large invertebrate embryos, by confining
the zone of active RhoA to a narrow equatorial band
between the separating sister chromatids.
Assembly and Regulation of the Contractile Ring
Exposure of the cell cortex to the cleavage stimulus
culminates in the assembly of a contractile ring consist-
ing of a very thin (0.1 to 0.2 µm) array of actin filaments
attached to the plasma membrane at many sites around
the equator (Fig. 44.22). Polymerization of the actin fila-
ments depends on formins (see Fig. 33.14). Small, bipolar
filaments of myosin-II are interdigitated with actin fila-
ments. The plasma membrane adjacent to this actin-
myosin ring undergoes alterations in its lipid composition
that may help recruit proteins important for the function
of the contractile ring.
Membrane furrowing requires actin and the motor
activity of myosin-II (see Fig. 36.7). In animals, the small
GTPase RhoA regulates actin polymerization by formins
as well as constriction of the ring. Many other proteins
are required for cytokinesis to go to completion. In their
absence, furrowing begins, but the cleavage furrows
ultimately regress, producing binucleated cells. These
supporting proteins include anillin, actin filament cross-
linking proteins, the CPC (Fig. 44.10) and the central-
spindlin complex, among many others. Anillin helps
 CHAPTER 44  n  Mitosis and Cytokinesis 773

A. Early anaphase B. Late anaphase C

INCENP
Myosin II

Actin pointed ends

INCENP
Myosin II

Actin barbed ends

E. Myosin confocal

Central optical section

H. Contractile mechanism I. Equatorial section

G Actin
Myosin II

J. Grazing saggital section

FIGURE 44.22  ORGANIZATION OF THE CONTRACTILE RING. A, Organization of actin at the cell cortex prior to cytokinesis. B, Distribution
of actin and myosin at the start of ring contraction. C, INCENP (inner centromere protein) (red) concentrates at the site where the cleavage furrow
will form just before myosin (green). D, INCENP and myosin concentrate in the contractile ring during contraction. E, Confocal micrograph shows
the distribution of myosin in an optical cross-section contracting contractile ring. F–G, Dividing invertebrate egg with DNA (blue) and actin (red)
in the contractile ring. H, Organization of actin and myosin filaments during cytokinesis. I–J, Electron micrographs showing actin filaments in
the contractile ring. Note the thick filaments that are thought to be myosin-II filaments (red arrowheads) and the thinner actin filaments (yellow
arrows). (C–D, Courtesy William C. Earnshaw. E, I, and J, Courtesy P. Maupin, Johns Hopkins Medical School, Baltimore, MD. F–G, Courtesy
Professor Issei Mabuchi, University of Tokyo, Japan. For reference, see Maupin P, Pollard TD. Arrangement of actin filaments and myosin-like fila-
ments in the contractile ring and actin-like filaments in the mitotic spindle of dividing HeLa cells. J Ultrastruct Res. 1986;94:92–103; Maupin P,
Phillips CL, Adelstein RS, et al: Differential localization of myosin-II isozymes in human cultured cells and blood cells. J Cell Sci. 1994;107:3077–3090;
and Eckley DM, Ainsztein AM, MacKay AM, et al. Chromosomal proteins and cytokinesis. J Cell Biol. 1997;136:1169–1183.)

keep active myosin-II focused into an organized contrac-
tile ring throughout cytokinesis.
The CPC and the centralspindlin complex are both
required for animal cells to assemble the central spindle.
Consequently, if either of the two complexes is elimi-
nated in C. elegans, a contractile ring fails to form. Thus,
they appear to contribute to the cleavage stimulus, and
indeed, both require microtubules to localize to the site
of cleavage furrow formation as originally shown for the
cleavage stimulus. In addition, the CPC regulates the
timely completion of cytokinesis by blocking premature
activation of the ESCRT (endosomal sorting complexes
required for transport) complex, which has a key role in
the final separation of daughter cells (see later).
In fission yeast, with closed mitosis, the nucleus
determines the position of cleavage. Fission yeast assem­
ble a contractile ring along a well-defined pathway by
recruiting proteins from cytoplasmic pools (Fig. 44.23).
During interphase, assemblies of proteins called nodes
form on the inside of the plasma membrane around the
middle of cell. Prior to mitosis, an anillin-like protein
leaves the nucleus and joins these nodes. During pro-
phase, myosin-II, a formin and other contractile ring
proteins join the nodes. When the formin polymerizes
774 SECTION X  n  Cell Cycle

Interphase
-60 Mid1p (anillin-like actin patches
protein) exits nucleus
Anillin-like
Cell wall

Formin Myosin-II
-10 Nodes containing
anillin, myosin-II and formin Profilin
assemble around equator

0 SPBs separate Nodes condense into

a contractile ring of
+5 Anaphase A actin filaments and myosin-II
Time (min)

+10 Anaphase B
elongates mitotic Contractile ring
spindle matures by addition of
actin binding proteins

+30 End Anaphase B

Signal from cell cycle via SIN

pathway triggers constriction of
+40 Constriction begins
contractile ring and deposition of
cell wall material to form a septum

Plasma membrane fusion

completes cytokinesis
+70 Constriction ends

FIGURE 44.23  CYTOKINESIS IN FISSION YEAST SCHIZOSACCHAROMYCES POMBE. During interphase, microtubules (red) position
the nucleus in the middle of the cell. Actin filaments concentrate in small patches (yellow) in the cortex at the two growing ends of the cell (see
Fig. 33.1). The mitotic spindle is inside the nucleus, as the nuclear membrane does not break down during mitosis. As the cell enters mitosis,
an anillin-like protein moves from the nucleus to the equatorial cortex, where it sets up nodes of proteins, including myosin-II and a formin. The
formin grows actin filaments (yellow), and myosin-II pulls the nodes together into a continuous contractile ring. At the end of anaphase a signaling
system consisting of a GTPase and three protein kinases (the septation initiation network [SIN]) triggers constriction of the contractile ring and
associated synthesis of new cell wall to form a septum. The septum is a three-layered structure, with the primary septum flanked by two secondary
septae. Digestion of the primary septum separates the daughter cells. (For reference, see Wu J-Q, Kuhn JR, Kovar DR, et al. Spatial and temporal
pathway for assembly and constriction of the contractile ring in fission yeast cytokinesis. Dev Cell. 2004;5:723–734.)

actin filaments, myosin-II pulls the nodes together into a

ring around the equator of the cell (Fig. 44.23).
Contractile ring assembly in animal cells shares many
properties with fission yeast, but is less completely
understood. The decline in the activity of cell cycle
kinases at the onset of anaphase is part of the trigger,
since they inhibit centralspindlin components through
metaphase. INCENP and anillin move from the inter-
phase nucleus to the cortex around the cell equator in
early anaphase (Fig. 44.22). Formins and profilin polym-
erize some new actin filaments, but preexisting actin fila-
ments are recruited into the contractile ring from
adjacent areas of the cortex. Myosin-II is dispersed
throughout the cytoplasm until anaphase, when it con-
centrates in the cortex, especially around the equator
where the furrow forms. The myosin-II is derived from
various interphase structures including stress fibers (see
Fig. 33.1) that break down during prophase.
Constriction of the Cleavage Furrow
Contractile rings of echinoderm eggs produce enough
force to invaginate the plasma membrane and form the
cleavage furrow, although many details are still being
studied. Constriction of the ring probably involves a
sliding filament mechanism similar to muscle (see Figs.
39.9 and 39.23), but little is known about how the
contractile ring is attached to the plasma membrane.
During the early stages of furrowing, contractile rings
maintain a constant volume, but then disassemble as
they constrict further.
The role of myosin-II as the motor for cytokinesis was
established by microinjection of inhibitory antibodies
into echinoderm embryos and confirmed by genetic
inactivation in the slime mold Dictyostelium. Slime mold
amoebas lacking the myosin-II heavy chain round up
during mitosis and complete nuclear division but cannot
 CHAPTER 44  n  Mitosis and Cytokinesis 775

form a normal cleavage furrow. Mutant cells accumulate

A
many nuclei, because the mitotic cycle continues. Mutant
cells can divide on a substratum using pseudopods to
pull themselves apart into smaller cells.
Constriction of the contractile ring is regulated so that
it does not begin until after the onset of anaphase B,
when sister chromatids are well separated. In fission Midbody ring
yeast, a signaling pathway called the septation initiation B. Abscission (centralspindlin, anillin, cep55)
ESCRT I and ESCRT II
network (SIN) initiates constriction. Much less is known ESCRT III
Midbody
in other cells. Vps4
Microtubules

Abscission
As the contractile ring pulls the cell membrane inward, ESCRT I

the single cell that entered mitosis is gradually trans-

formed into two daughters joined by a thin intercellular
bridge (Fig. 44.20). This process requires a significant
net increase in the surface area of the cell. New plasma
FIGURE 44.24  ABSCISSION IN ANIMAL CELLS. Proteins
membrane is inserted adjacent to the leading edge of associated with overlapping bundles of microtubules form the midbody.
the furrow. The source of the new membrane appears The midbody ring links the midbody to the membrane, and nucleates
to be recycling endosomes (see Chapter 22), so addition the formation of ESCRT (endosomal sorting complexes required for
of membrane to the cleavage furrow is a specialized form transport) III filaments that constrict the membrane to divide the
daughter cells.
of exocytosis. Fusion of vesicles providing the new
membrane depends on specific syntaxins, t-SNAREs
(soluble N-ethylmaleimide-sensitive factor attachment
protein receptors) (see Fig. 21.15) that promote vesicle
fusion along the secretory pathway.
The plasma membrane in the cleavage furrow has a
discrete composition. In budding yeast, this compart-
ment is delineated by rings made from polymers of
septins, a family of GTP-binding proteins recruited by
anillin. Septins are essential for cytokinesis in Saccharo-
myces cerevisiae but not fission yeast.
In most animal cells, constriction of the cleavage
furrow ultimately reduces the cytoplasm to a thin inter-
cellular bridge between the two daughter cells. The
FIGURE 44.25  INCOMPLETE CYTOKINESIS IN A DRO-
intercellular bridge contains a highly ordered, antiparal-
SOPHILA EGG CHAMBER LEAVES CELLS JOINED BY RING
lel array of microtubules derived from the spindle with CANALS. Colocalization of actin (red) and the ring canal protein
a dense knob, the midbody, at its center (Fig. 44.20). HtsRC (green) in the ring canals makes them appear yellow. In the
Isolated midbodies contain more than 160 proteins, with Drosophila egg chamber, ring canals connect nurse cells to each other
approximately one-third involved in various aspects of and to the oocyte. Late in oocyte development, nurse cell contraction
forces their cytoplasmic contents through the ring canals and into the
membrane trafficking.
oocyte. This helps the oocyte gain the stockpile of components that
The midbody is encircled by a dense ring of proteins is needed for early development of the fly embryo. (Courtesy Andrew
that includes centralspindlin, anillin, and a centrosomal Hudson and Lynn Cooley, Yale University, New Haven, CT.)
protein known as Cep55. Interactions between anillin
and membrane-associated septin filaments tether the
membrane to the ring. Cep55 recruits the ESCRT (endo- Ultimately, disassembly of the ESCRT filaments leads to
somal sorting complexes required for transport) III separation of the two daughter cells—abscission.
complex, proteins with important roles in vesicle In some tissues, intercellular bridges remain open as
budding events such as formation of multivesicular ring canals. After several rounds of nuclear division
bodies (see Chapters 22 and 23). In the intercellular with incomplete cytokinesis, the network of cells main-
bridge, ESCRT III forms a helical filament that spirals tains cytoplasmic continuity as each former contractile
around the inner surface of the membrane, becoming ring matures into a larger ring canal. During Drosophila
more and more constricted as it grows away from the oogenesis, four rounds of nuclear division with persis-
midbody (Fig. 44.24). ESCRT III also recruits factors that tent ring canals creates 15 nurse cells, all in continuity
disassemble the bundled microtubules in the intercellu- with the oocytes (Fig. 44.25). The cytoplasmic continu-
lar bridge, allowing the membrane to constrict further. ity through ring canals allows nurse cells to transfer
776 SECTION X  n  Cell Cycle
BOX 44.1  Cytokinesis in Plants
Chromosome segregation is similar in plants and animals, but
cytokinesis is very different because plants lack myosin-II
and do not form a conventional contractile ring (Fig. 44.26).
Myosin-II appeared during evolution in the common ances-
tor of amoebas, fungi and animals, after branching from
plants (see Fig. 2.4B). Plants also lack dynein, so microtubule
dynamics in mitosis are regulated by some of the more than
20 different plus-end– and minus-end–directed kinesins that
are expressed in mitotic cells. In a further difference from
animals, plants also lack centrosomes, and during interphase,
microtubules radiate out from the surface of the cell nucleus
in all directions. In mitosis, the spindle does not focus to
sharp poles at metaphase; instead, it assumes a barrel shape
with broad, flat poles. Early in mitosis, a band of microtu-
bules and actin filaments forms around the equator of the
cell adjacent to the nucleus. This so-called preprophase band
disassembles as cells enter prometaphase. Because the entire
cell cortex is covered by a meshwork of actin filaments,
disassembly of the preprophase band actually leaves an actin-
poor zone in a ring where cytokinesis will ultimately occur.
This is called the cortical division site, and it is marked by
the tethering of specific kinesin motors. In late anaphase,
two nonoverlapping, antiparallel arrays of microtubules
form over the central spindle. This structure, the phragmo-
plast, gradually expands laterally until it makes a mirror-
symmetric double disk of short microtubules oriented
parallel to the spindle axis with their plus ends abutting the
plane of cell cleavage. Golgi vesicles, containing cell wall
Chromosomes Cytokinesis in higher plants Early phragmoplast
Cortical
actin
Preprophase Cortical
band actin-
(microtubules) depleted
zone
Prophase Metaphase
FIGURE 44.26  CYTOKINESIS IN HIGHER PLANTS. See the text for details.
their cytoplasm into the developing egg, thus greatly
increasing its stockpile of proteins and messenger RNAs
available for use in early development. In mammals,
incomplete cytokinesis in the testis results in ring canals
connecting several hundred developing sperm cells.
Exit From Mitosis
To exit from mitosis, cells must inactivate the Cdk1
kinase. This reverses the biochemical and structural
changes that are characteristic of mitosis and prepares
the cell for proliferation in the next cell cycle.
materials (see Fig. 32.13), move along phragmoplast micro-
tubules to the equator, where they fuse due to the action of
cytokinesis-specific soluble N-ethylmaleimide-sensitive factor
(NSF) attachment protein receptor (SNARE) proteins (see
Fig. 21.15), forming a membrane network that becomes the
new plasma membrane and laying down the material that
will become the new cell wall. Dynamin-related proteins also
participate in shaping the newly forming plasma membrane.
Thus, the membrane fusion machinery used for cytokinesis
by eukaryotes likely came from the last eukaryotic common
ancestor. Actin filaments polymerized by formins and
myosin-VIII help position the phragmoplast in the cell. As
the zone of newly deposited membrane expands radially, the
ring of microtubules surrounding it similarly expands. Even-
tually, the new membrane reaches the lateral cell periphery,
and fusion with the plasma membrane separates the two
daughter cells. The cortical division site, not the spindle,
determines the site of cleavage. This was shown by centri-
fuging mitotic cells to displace the spindle from the central
location where it initially formed. Late in mitosis, the phrag-
moplast formed at the midzone of the displaced spindle, but
this phragmoplast then migrated to the plane of the prepro-
phase band, where cytokinesis occurred. Since plant cells
have cell walls and do not move, the orientation of cleavage
planes critically determines the morphology of the organism.
The hormone auxin can influence cleavage, giving rise to
asymmetric division of daughter cells, but the underlying
mechanism is not yet known.
Late phragmoplast
Golgi
vesicles
Early cytokinesis Late cytokinesis Daughter cells
Yeast cells use a signaling pathway to terminate
mitosis, promote contraction of the contractile ring, and
initiate septation. These pathways, called the mitotic exit
network (MEN) in budding yeast and the SIN in fission
yeast, involve a small GTPase and protein kinases. Cdk
kinase activity suppresses the pathway until anaphase,
when Cdk activity drops sharply. The MEN GTPase is
associated with one spindle pole body (the yeast version
of the centrosome), while its key regulator, a GTP
exchange factor, is located in the bud. Elongation of the
mitotic spindle during anaphase B moves the GTPase
into the bud, where it is activated.

BOX 44.2  Cytokinesis in Bacteria
The strategy for cytokinesis in bacteria is similar to that in
animal cells (Fig. 44.27), but the molecules are completely
different. Cleavage of most bacterial cells depends on
a ring of the FtsZ protein (filamentous temperature-sensitive;
mutants in fts genes cannot divide and make long filaments
on cells). This is called the Z ring. FtsZ is the prokaryotic
homolog of eukaryotic tubulins, but it assembles into fila-
ments rather than tubules. As for tubulins (see Fig. 34.4),
FtsZ polymerization requires bound GTP and hydrolysis of
this GTP destabilizes the polymers. Although purified FtsZ
forms rings that use energy from GTP hydrolysis to deform
lipid vesicles, the main function of the Z ring seems to be to
coordinate the assembly of a complex of proteins (divisome)
including an actin homolog FtsA and number of transmem-
brane proteins. The transmembrane proteins synthesize cell
wall materials to form the cleavage furrow.
The Z ring is positioned at the cell equator of Escherichia
coli by the action of three gene products: MinC, MinD, and
MinE (minicell mutants divide at inappropriate locations and
give birth to tiny cells). MinD is an enzyme that recruits MinC
Min C/D Min E Cytokinesis in E. coli
inhibitor Nucleoid
2 minutes
FIGURE 44.27  CYTOKINESIS IN THE BACTERIUM ESCHERICHIA COLI. See the text for details.
The MEN kinases downstream of the GTPase activate
the phosphatase Cdc14p by releasing it from sequestra-
tion in the nucleolus. Cdc14p inhibits Cdk kinase activity
in two ways: (a) it inhibits the degradation of a Cdk
inhibitor protein, and (b) it dephosphorylates Cdh1,
which binds the APC/C and triggers the degradation of
B-type cyclins and other proteins. Cdc14p also triggers
other events during anaphase, including the transfer of
chromosomal passenger proteins to the central spindle.
In metazoans mitotic exit is triggered by the inactiva-
tion of Cdk1 and other mitotic kinases. This transition
is irreversible, in part because cyclins and Aurora
(and other kinases) are degraded. PP2A and its inhibitory
kinase Greatwall (see Chapter 40) replace Cdc14
in mitotic regulation in metazoans. Greatwall activity
requires Cdks, so when Cdk activity declines, PP2A
is released from inhibition. When directed to targets
CHAPTER 44  n  Mitosis and Cytokinesis 777
to the cell cortex, where it inhibits Z-ring formation. MinE
is an antagonist of MinC/MinD action. This system works in
a truly remarkable way. MinE forms a ring at the cell equator
that migrates along the inner surface of the cell membrane
until it reaches the end of the cell, at which point it disas-
sembles. The ring then reforms in the center of the cell and
sweeps toward the other end of the cell. As it moves, MinE
inactivates the MinC/MinD inhibitory complex on the cell
cortex. The inhibitory complex rapidly reestablishes itself on
the cell cortex behind the moving MinE ring. It takes
approximately 2 minutes for each sweep of the MinE ring
along half of the cell, and this cycle is repeated continuously
until the FtsZ ring assembles at the cell center. Bacillus
subtilis uses an alternative mechanism to position the Z ring
for cytokinesis.
Chloroplasts use a homolog of FtsZ for their division, and
FtsZ has been detected in mitochondria of certain primitive
eukaryotes. Mitochondria of higher eukaryotes appear to use
another GTPase, dynamin, to coordinate their fission (see
“Biogenesis of Mitochondria” in Chapter 19).
FtsZ ring
Zone of minimal
Min C/D
throughout cells by their specificity, determining sub-
units PP2A and PP1 remove many of the phosphates
placed on target proteins by the mitotic kinases. Targets
include chromatin, where phosphorylation during
mitosis had displaced factors involved in both gene
activation and repression. Removal of those phosphates
allows the interphase regulation of gene expression to
resume. Dephosphorylation of other targets allows
intermediate filaments to reform, nuclear envelope reas-
sembly plus the resumption of RNA transcription, protein
translation, and membrane trafficking.
ACKNOWLEDGMENTS
We thank David Burgess, Iain Cheeseman, Per Paolo
D’Avino, Arshad Desai, Tatsuo Fukagawa, Gary Gorbsky,
Karen Oegema, Jonathon Pines, and Graham Warren for
778 SECTION X  n  Cell Cycle
their suggestions on revisions to this chapter. We thank
the Dundee Imaging Facility for access to the OMX and
help with microscopy.
SELECTED READINGS
Carmena M, Wheelock M, Funabiki H, et al. The chromosomal pas-
senger complex (CPC): from easy rider to the godfather of mitosis.
Nat Rev Mol Cell Biol. 2012;13:789-803.
Collas P, Courvalin J-C. Sorting nuclear membrane proteins at mitosis.
Trends Cell Biol. 2000;10:5-8.
Glotzer M. Cytokinesis in metazoa and fungi. Cold Spring Harb Per-
spect Biol. 2016;(in press).
Green RA, Paluch E, Oegema K. Cytokinesis in animal cells. Annu Rev
Cell Dev Biol. 2012;28:29-58.
Haeusser DP, Margolin W. Splitsville: structural and functional insights
into the dynamic bacterial Z ring. Nat Rev Microbiol. 2016;14:
305-319.
Jürgens G. Plant cytokinesis: Fission by fusion. Trends Cell Biol. 2005;
15:277-283.
McIntosh JR. Mitosis. Cold Spring Harb Perspect Biol. 2016;(in press).
Müller S, Jürgens G. Plant cytokinesis—no ring, no constriction but
centrifugal construction of the partitioning membrane. Semin Cell
Dev Biol. 2016;53:10-18.
Nasmyth K, Haering CH. Cohesin: its roles and mechanisms. Annu Rev
Genet. 2009;43:525-558.
Qian J, Winkler C, Bollen M. 4D-networking by mitotic phosphatases.
Curr Opin Cell Biol. 2013;25:697-703.
Rappaport R. Cytokinesis in Animal Cells: Developmental and Cell
Biology Series. Cambridge, England: Cambridge University Press; 1996.
Sánchez-Huertas C, Lüders J. The augmin connection in the geometry
of microtubule networks. Curr Biol. 2015;25:R294-R299.
Sharp DJ, Rogers GC, Scholey JM. Microtubule motors in mitosis.
Nature. 2000;407:41-47.
Stukenberg PT, Burke DJ. Connecting the microtubule attachment
status of each kinetochore to cell cycle arrest through the spindle
assembly checkpoint. Chromosoma. 2015;124:463-480.

Meiosis
M eiosis (from the Greek, meaning “reduction”) is a
specialized program of two coupled cell divisions used
by eukaryotes to maintain the proper chromosome
number for the species during sexual reproduction. It
also generates novel combinations of genes. Meiosis is
an ancient process that occurs in virtually all eukaryotes,
including the animal, fungal, and plant kingdoms, and is
thought to have been present in the last eukaryotic
common ancestor.
Sexually reproducing organisms are typically diploid,
with pairs of homologous chromosomes, the two
highly similar but nonidentical copies of each chromo-
some, one inherited from each parent. The number of
chromosomes is halved during meiosis to form haploid
gametes carrying just one set of chromosomes. The
subsequent fusion of male and female gametes restores
the diploid chromosome number. This pairing and sub-
sequent separation of homologous chromosomes is
made possible by genetic recombination, which occurs
during the lengthy and complex prophase of the first
meiotic division.
Each human somatic cell has 23 pairs of chromosomes
(46 in all). Females have 23 homologous pairs, while
males have 22 “autosomal” pairs and two different sex
chromosomes that share a region of homology known
as the pseudoautosomal region. One of each pair is
contributed by each parent in the egg and sperm, respec-
tively. The number of chromosome pairs, 23, is known
as the haploid chromosome number. In animals, the
only haploid cells are gametes (sperm and eggs). At fer-
tilization, haploid gametes fuse to form a zygote, restor-
ing the diploid chromosome number of 46. In plants,
the haploid phase is represented by gametophytes,
which produce ovules and pollen. In most fungi, such
as yeasts, haploid and diploid forms are alternate phases
of the life cycle, and both can propagate by mitosis.
Meiosis changes the genetic makeup of offspring
relative to parents in two ways: the first round of
meiotic segregation produces novel combinations of
CHAPTER 45 
chromosomes from the two parents in each gamete,
and recombination between parental chromosomes
produces novel chromosomes. It works like this.
During meiosis, one round of DNA replication and two
rounds of chromosome segregation reduce the number
of chromosomes from 2n to 1n. Each haploid gamete
is endowed with a random set of the homologous
chromosomes from the two parents. Prior to meiosis
I the chromosomes duplicate (just like mitosis) (Fig.
45.1A), but during the first round of segregation (Fig.
45.1C) the duplicated chromatids remain paired and the
homologous chromosomes separate randomly between
the two daughter cells. Thus each daughter cells ends up
with just one of each pair of homologous chromosomes.
This differs from mitosis where the duplicated chroma-
tids separate, so both daughter cells get the full set of
homologous chromosomes from both parents. During
meiosis II the duplicated chromatids separate and are
partitioned equally between the two daughter cells.
Equally important, homologous chromosomes exchange
DNA sequences during meiotic prophase I, generating
novel chromosomes.
The unique segregational events of meiosis usually
occur in the first division, termed meiosis I (Figs. 45.1
and 45.2). Because it culminates in daughter cells carry-
ing just one set of chromosomes instead of two, meiosis
I is also known as the reductional division. The second
division, meiosis II, is similar in most respects to mitosis:
sister chromatids segregate, and the number of chromo-
somes remains the same (Box 45.1; see also Chapter 44).
Meiosis II is called the equational division.
Meiosis: An Essential Process for
Sexual Reproduction
Sexual reproduction is an important survival strategy
that offers organisms an accelerated mechanism for alter-
ing the genetic makeup of offspring. Without meiosis,
there would be no sex, because fusion of diploid gametes
779
780 SECTION X  n  Cell Cycle

Preparation for meiosis (This step duplicates each chromatid)

A a Centromere Fused sister

centromeres
B b

Premeiotic Meiotic
Two pairs of S phase prophase
homologous
chromosomes

Meiotic prophase (Recombination drives pairing of homologous chromosomes and produces novel chromosomes)
Recombination Chiasmata
nodules
A a A
b a
Meiosis I
B
B b

Leptotene stage Bouquet Zygotene stage Pachytene stage Diplotene stage Diakinesis stage

Meiosis I (Homologous chromosomes randomly separate from one another producing haploid progeny)

Chiasmata A
A b A b
b B
a B B Meiosis I a Meiosis II
a
Interphase
(no S phase)
Metaphase I Anaphase I

Meiosis II (Sister chromatids separate producing one or more gametes)

b B
a

Prophase II Metaphase II Anaphase II Haploid gametes

FIGURE 45.1  OVERVIEW OF THE PHASES OF MEIOSIS. Shown are important structures and the outcome of each stage for a homologous
pair of metacentric chromosomes (A, a) and a homologous pair of telocentric chromosomes (B, b).

would double the number of chromosomes in the
progeny at every generation.
Meiosis I produces random combinations of homolo-
gous maternal and paternal chromosomes. For each
pair of homologs, orientation on the spindle is random
during meiosis I (ie, each homolog has two equivalent
options for the direction to migrate). Thus, for humans
(with 23 pairs of homologous chromosomes), each
gamete has one of 223 (more than 8 million) possible
complements of maternal and paternal chromosomes.
This process does not create new versions of genes,
but it guarantees the offspring will have novel combi-
nations of subtly different (due to polymorphisms)
chromosomes.
Meiosis I also produces novel versions of genes and
chromosomes by recombinational exchange of DNA
segments between homologs. This occurs because to
segregate from one another, each pair of homologous
chromosomes must first find each other. They do this by
undergoing reciprocal recombination (crossover) events
that then hold them together until anaphase of meiosis
I. Chromosomes and the genes they carry vary hugely
between individuals. In humans an average genome
varies from the “reference genome” (see Chapter 7) at
4 to 5 × 106 sites. These include not only polymorphisms
(differences of single base pairs), but also thousands
of longer insertions, deletions, and rearrangements.
Recombination events that result in a crossover and
exchange chromosomal segments produce new chromo-
somes that are a patchwork of segments from the
maternal and paternal homologs. The combined effects
of recombination and random assortment of homologs
in meiosis I yields a vast number of genetically different
gametes. This genetic diversity increases the ability of
eukaryotic populations to adapt to changing environ-
mental conditions.

A. Metaphase I Spindle
Paired sister kinetochores
pole
being pulled toward poles
X
Chiasma Chiasma
Spindle
pole
B. Early Spindle
anaphase I pole
Spindle
pole
FIGURE 45.2  FIRST MEIOTIC DIVISION STAGES FROM THE GRASSHOPPER PYRGOMORPHA CONICA (2N IN MALES = 18
AUTOSOMES + 1 X CHROMOSOME). A, Metaphase I. B, Early anaphase I. C, Late anaphase I. D, Telophase I spermatocytes stained with
lactopropionic orcein. All chromosomes are telocentric (see Fig. 7.2). Seven bivalents shown in the metaphase I spermatocyte have a single
chiasma, whereas the two bivalents at the far right and far left have two chiasmata. The sex chromosome (X) remains unpaired and moves to a
single spindle pole. (Courtesy José A. Suja and Julio S. Rufas, Universidad Autónoma de Madrid, Spain.)
The Language of Meiosis
Meiosis has a language of its own, characterized by a
number of unusual terms, and is easiest to understand
by focusing on the essential biological processes that are
involved. This reduces the process to only three essential
key terms: pairing, homologous recombination, and
segregation. This chapter discusses each step in detail,
so they are defined only briefly here.
Pairing is a two-step alignment of homologous chro-
mosomes with one another in the nucleus. In alignment,
corresponding DNA sequences on the homologous
chromosome find each other among the billions of base
pairs of DNA in the nucleus. In many organisms, early
events of recombination drive the homologous pairing
process. In the second stage, synapsis, the paired
homologous chromosomes become intimately aligned
along their entire lengths with one another separated
by approximately 100 nm. A specialized scaffolding
structure called the synaptonemal complex mediates
this process.
Homologous recombination results in physical
exchange of DNA between homologous chromosomes
(a crossover event) and is a key determinant of chromo-
some behavior during meiotic prophase. Recombination
drives the pairing process in many organisms and can
occur without synapsis under certain circumstances.
Crossover recombination sites are detected by microscopy
CHAPTER 45  n  Meiosis 781
Spindle
C. Late Spindle pole
anaphase I pole
D. Telophase I
X
Paired sister kinetochores
moving toward poles
Spindle
pole Spindle
pole
as chromatin structures called chiasmata (singular:
chiasma, from the Greek, meaning “X-shaped cross”).
Segregation of homologous chromosomes in meiosis
I differs from the segregation of sister chromatids during
mitosis (Box 45.1), because the paternal and maternal
homologous chromosomes segregate randomly to the
two daughter cells. When homologs orient at the meta-
phase plate of the meiosis I spindle, centromeres belong-
ing to the two sister chromatids are fused to form a single
kinetochore that binds microtubules. Cohesion between
chromosome arms distal to chiasmata (ie on the other
side from the centromere; Figs. 45.2 and 45.10) keeps
homologous chromosomes paired with one another
until anaphase of meiosis I, counteracting the bipolar
pulling force of the spindle on the homologs (Fig. 45.2).
At anaphase I the distal cohesion is released from chro-
mosomes allowing the chiasmata to separate, and the
two sister chromatids (at least one of which has under-
gone a crossover exchange) move as a single unit toward
the same spindle pole while the sister chromatids from
other parent move to the other daughter cell. As a result,
the two daughter cells produced in meiosis I have a
haploid number of chromosomes derived randomly from
the two parents, each with two sister chromatids. Each
of the four daughter cells produced in meiosis II has one
sister chromatid for each homologous chromosome (ie,
half the number of chromatids as there are chromosomes
in somatic cells).
782 SECTION X  n  Cell Cycle
BOX 45.1  Important Differences Between Meiosis
and Mitosis
Meiosis involves two cell divisions. The two meiotic
divisions are preceded by a round of DNA replication.
There is no DNA replication between meiosis I and
meiosis II.
The products of meiosis are haploid. The products
of mitosis are diploid.
The products of meiosis are genetically different.
After recombination and random assortment of homologs
in meiosis I, the sister chromatids that segregate in meiosis
II are different from each other. In normal mitosis, sister
chromatids are identical.
Prophase is longer in meiosis I. Proper orientation
and segregation of homologous chromosomes is achieved
thanks to the pairing, synapsis (synaptonemal complex
formation), and recombination that occur in a lengthened
prophase during meiosis I. In humans, prophase in mitosis
takes an hour, whereas meiotic prophase lasts many days
in males and many years in females.
Recombination is increased in meiosis. The
recombination rate is 100- to 1000-fold higher in prophase
I of meiosis than in mitosis. The process has two main
consequences: the formation of chiasmata and the intro-
duction of genetic variation. Chiasmata are structures that
physically link the homologous chromosomes after cross-
over and play an essential role in meiotic chromosome
segregation.
Kinetochore behavior differs in meiosis. During
meiosis I, kinetochores of sister chromatids attach to
spindle microtubules emanating from the same pole.
Homologous kinetochore pairs connect to opposite poles.
In mitosis and meiosis II, sister kinetochores attach to
spindle microtubules coming from opposite poles.
Chromatid cohesion differs in meiosis. Sister
chromatid cohesion is essential for orientation of bivalents
(paired homologous chromosomes) on the metaphase
I spindle. During anaphase of meiosis I, cohesion is
destroyed between sister chromatid arms, and chiasmata
are released to allow segregation of homologs. Cohesion
at sister centromeres persists until the onset of anaphase
II, when it is lost to permit segregation of sisters. In pro-
metaphase of meiosis II, sister chromatids are joined only
by the centromeres, whereas at the beginning of mitotic
prometaphase, sisters are joined all along the arms.
Recombination
Although meiotic recombination is similar to the process
of homologous recombinational repair of double-strand
DNA breaks in somatic cells (review Box 43.1 and Fig.
43.14 as a prelude to studying meiotic recombination),
the two processes differ in two respects. First, meiotic
cells use a specialized enzyme called Spo11 to create
double-strand DNA breaks on purpose. Second, somatic
cells with replicated chromosomes usually repair DNA
breaks using the corresponding DNA sequence on a
sister chromatid as a template. Meiotic cells usually use
a homologous chromosome. The mechanism for this
difference in selectivity is not yet fully understood.
Spo11, together with essential accessory proteins,
generates programmed double-strand DNA breaks early
during meiotic prophase (Fig. 45.3). Similar to type II
DNA topoisomerases (see Fig. 8.16), Spo11 cleaves both
DNA strands in a reaction that produces a covalent
linkage between a tyrosine of the enzyme and the cleaved
phosphodiester backbone. However, Spo11 does not
reseal the breaks; instead it remains attached to one
strand of DNA at the broken end. In mice, Spo11 creates
about 10-fold more DNA breaks than ultimately recom-
bine to produce reciprocal DNA exchanges between
homologous chromosomes or crossovers. Repair of
Spo11-mediated DNA double-strand breaks can also
result in noncrossover events known as gene conver-
sions (Box 45.2 and Fig. 45.3I–J).
DNA double-strand breaks generated by Spo11 are
required for the initial lengthwise alignment of homolo-
gous chromosomes in many organisms, including mice,
plants, and yeast. In mice lacking Spo11, recombination
is not initiated, and synapsis, if it occurs at all, is aberrant,
often involving nonhomologous chromosomes (Fig.
45.4). Gametes in these mutant mice die by apoptosis
early in meiotic prophase. Spo11-induced double-strand
breaks are not required for synapsis of homologous
chromosomes in the nematode Caenorhabditis elegans
and the fruit fly Drosophila melanogaster. How these
organisms pair their homologs without recombination is
still mysterious.
Once the DNA double-strand breaks are produced,
the MRN (Mre11/Rad50/Nbs1) endonuclease nicks the
single-stranded DNA, releasing Spo11. The DNA ends
that lost Spo11 then undergo further processing, as Exo1
exonuclease, chews back the 5′ strands of the double
helix (a process called resection) leaving single-stranded
DNA tails with 3′ termini (Fig. 45.3C; see also Fig. 43.14).
The MRN and Exo1 nucleases also function in somatic
DNA repair.
Next, the Rad51 and Dmc1 proteins drive a search of
the 3′ single-stranded DNA tails for complementary DNA
sequences of the other chromosomes. Rad51 and Dmc1
are related to the Escherichia coli RecA protein used for
homologous recombination in bacteria. Rad51 and Dmc1
coassemble along 3′ single-stranded DNA tails and use
adenosine triphosphate (ATP) hydrolysis to catalyze a
strand exchange reaction with an intact homologous
DNA duplex. The Rad51-and-Dmc1-decorated nucleo-
protein filament disrupts the targeted homologous
double helix, displacing one of the two DNA strands.
This allows formation of new Watson-Crick base pairs
between the invading 3′ single-stranded DNA and the
complementary strand of the target DNA. Following
strand invasion and exchange, new DNA synthesis
 5' 3'
A Cohesin
3' 5'
5' 3'
3' 5' Paired homologous
5' 3' chromosomes
3' 5'
5' 3'
3' 5'

Double-strand break Spo11 cuts DNA, remaining

covalently attached to one strand
B

Red enzymes are

meiosis specific Spo11 released by MRN complex (Rad50/
5' to 3' resection
Exo1 resects one DNA strand Mre11/Nbs1)
Green enzymes also
function in somatic C
repair

Initial strand invasion, Rad51 + Dmc1

DNA synthesis
D

Second end capture,

synthesis, ligation Strand displacement
E H

Formation of double
Holliday junction Strand annealing

F I

Holliday
junction

Holliday junction
= Resolvase cutting sites Synthesis, ligation
resolution

G J

+ +

Crossover Noncrossover

FIGURE 45.3  EVENTS OF RECOMBINATION. Recombination occurs between homologous chromosomes rather than between sister
chromatids. A, Paired homologous chromosomes. Sister chromatids are held tightly together by cohesin, shown here schematically as hoops.
B, Spo11 makes a double-strand break, remaining attached to the DNA. C, Removal of Spo11 and resection of the break. D, First strand invasion.
At this point, the pathway splits in two, one outcome leading to a crossover and the other to a noncrossover. Crossover pathway: E, The
second resected strand establishes base-pairing interactions with the displaced DNA strand of its homologous partner. New DNA synthesis fills
the gaps. F, The resulting molecule contains a double Holliday junction in which the DNA is fully base-paired (see Fig. 43.14B). If the resolvase
(nuclease) cuts the double Holliday junction asymmetrically as shown (ie, one vertical and one horizontal cut), the result is a crossover (G). If the
cuts are symmetrical, a noncrossover molecule is produced. Noncrossover pathway: H, In most cases, the invading DNA, strand is ejected
prior to stabilization and formation of a double Holliday junction. I, DNA gap-filling and ligation yield a noncrossover chromosome (J).
784 SECTION X  n  Cell Cycle
BOX 45.2  Brief Overview of Genetic Terminology
A comprehensive introduction to the field of genetics is
beyond the scope of this text. However, here are a number
of terms used by geneticists that will assist in the understand-
ing of the discussion of genetic recombination and its role
in meiosis (also see Box 6.1).
The genotype of an organism is the combination of
genes present on the chromosomes of that organism. The
phenotype is the physical manifestation of the action of
these gene products (ie, the appearance and macromolecular
composition of the organism). In discussing recombination,
scientists typically refer to the presence or absence of spe-
cific genetic markers. Each genetic marker is a particular
DNA sequence in or around a gene that can be monitored
by examining the phenotypes of the cells that carry it.
A genetic marker might be the presence of a functional
gene, a mutation with altered activity, or simply a polymor-
phism of DNA sequence that has no known functional
consequence.
A haploid organism has one copy of each chromosome.
A diploid organism has two homologous copies of each
chromosome. A diploid organism that is homozygous for a
particular genetic marker has the same sequence of that
particular region of the DNA on both the maternal and
paternal homologous chromosomes. A heterozygous organ-
ism has different forms of the genetic marker on the two
homologous chromosomes. Although the physical events of
genetic recombination occur in both homozygotes and het-
erozygotes, they are most readily detected in the latter.
Two genetic markers located on different chromosomes
will separate from one another in the anaphase of meiosis I
A B
FIGURE 45.4  PAIRING OF HOMOLOGOUS CHROMOSOMES
IS SEVERELY DISRUPTED IN THE SPO11 MUTANT. Pachytene
chromosomes from wild-type mice (A) and mice in which the Spo11
gene has been disrupted (B). SYC3 (axial elements) and centromeres
are red. SYCP1 (in transverse filaments, which are seen only when
synapsis has occurred) is green. (From Baudat F, Manova K, Yuen JP,
et al. Chromosome synapsis defects and sexually dimorphic meiotic
progression in mice lacking Spo11. Mol Cell. 2000;6:989–998.)
50% of the time as a result of the random distribution of
chromosomes to the two spindle poles. If they are on the
same chromosome, they will be linked to one another unless
the chromosome undergoes a genetic recombination event
between them. The greater the separation of two markers
along one chromosome, the more likely it is for such an
intervening recombination event to occur.
Two types of recombination events occur during meiosis
(Fig. 45.3). The first of these—noncrossover events (fre-
quently referred to as gene conversion)—may involve the
loss of one or more genetic markers. Noncrossover events
are the most common outcome of the programmed double-
strand DNA breaks that occur during leptotene. They are
thought to involve the invasion of a double helix by a region
of single-stranded DNA with complementary sequence but
then ejection of this sequence before assembly of a Holliday
junction and completion of recombination.
The second type of recombination event—crossing
over—involves the physical breakage and reunion of DNA
strands on two different chromosomes, typically producing
a balanced exchange of DNA sequences. This is what most
people think of as recombination. In recombination by cross-
ing over, the makeup of genetic markers remains constant;
it is the linkage between different markers that changes.
The normal separation of chromosomes or chromatids is
referred to as disjunction (disjoining). Mistakes in this sepa-
ration are referred to as nondisjunction. Nondisjunction in
meiosis I and II results in the production of gametes with
either too many or too few chromosomes, a condition
known as aneuploidy.
restores sequences that may have been lost or damaged
at the position of the original DNA double-strand break.
Mutants lacking Dmc1 are defective in homologous
chromosome pairing and interhomolog recombination.
As a result, Dmc1 is thought to facilitate the search for
homologous chromosomes as a DNA repair template,
rather than sister chromatids as in somatic DNA repair.
Rad51p and Dmc1p are found in structures called
early recombination nodules that are distributed
along the chromosome axes early in meiosis (Fig. 45.9).
Dmc1 functions only in meiosis, but Rad51 has other
essential functions.
It is now believed that noncrossover events arise
primarily from recombination intermediates that involve
a relatively transient single strand invasion of the
homologous chromosome followed by restorative DNA
synthesis and disassembly of the joint molecule interme-
diate. Crossovers, on the other hand, are thought to arise
predominantly through a pathway that involves stable
branched intermediates known as Holliday junctions
(Fig. 45.3F–G; see also Fig. 43.14B), which are then
cleaved by resolvases such as Gem1 to form (predomi-
nantly) crossover products.
 CHAPTER 45  n  Meiosis 785

Most sexually reproducing organisms depend on Tracking the Homologous Chromosomes

recombination during meiosis to produce haploid
gametes, but fruit flies and yeast have other systems for
segregating homologs in meiosis I. These mechanisms,
collectively known as achiasmate segregation, allow
the segregation of chromosomes that have not under-
gone crossover recombination. One model for the achi-
asmate segregation in flies proposes that nonrecombined
chromosomes remain paired at the end of meiotic pro-
phase owing to stickiness of heterochromatin and, as a
result, segregate properly during anaphase I of meiosis.
In a rare but notable example, the spermatocytes of D.
melanogaster males do not recombine at all, yet still
segregate their chromosomes happily during meiosis I.
This might be regarded as a cruel joke of evolution by
those students who find all the Greek terms of meiotic
nomenclature to be daunting. However, meiosis without
recombination is clearly the exception, and in most
species meiosis depends on recombination in both males
and females.
Through the Stages of Meiotic Prophase I
Pairing and recombination of homologous chromosomes
take place during prophase of meiosis I. Five stages of
meiotic prophase are used to describe the process: lep-
totene, zygotene, pachytene, diplotene, and diakinesis
(Fig. 45.1B).
Leptotene (from the Greek, meaning “thin ribbon”)
starts with the first visible condensation of the chromo-
somes. Paired sister chromatids become visible as linear
arrays of loops flanking a single dense protein-containing
axis (Fig. 45.5A–B). This axis consists of proteins that
play a role in mitotic chromosome structure as well as
proteins specialized for meiotic chromosomes. For
example, the cohesin complex with several specialized
meiosis-specific subunits is a prominent component of
this axial structure (see Fig. 8.18). According to recent
models, recombination begins during leptotene with the
formation of DNA double-strand breaks. By the end of

A. Early leptotene C. Early zygotene E. Pachytene

Sex
chromosomes

B. Late leptotene D. Late zygotene F. Diplotene

Chiasma

FIGURE 45.5  IMMUNOFLUORESCENCE IMAGES OF PROPHASE I SUBSTAGES IN MOUSE SPERMATOCYTES. These images
demonstrate the pairing and synapsis of homologous chromosomes revealed by visualizing the synaptonemal complex proteins SYCP3 (a
component of the axial elements [red]) and SYCP1 (a component of the transverse filaments that is present only when homologs are synapsed
[green]). Centromeres are blue. (Courtesy Paula Cohen, Cornell University, Ithaca, NY.)
786 SECTION X  n  Cell Cycle

A B C D E F G

0 hr 2 hr 8 hr

I Telomere

Nuclear
envelope

FIGURE 45.6  CHROMOSOMAL MOVEMENTS DURING EARLY MEIOTIC PROPHASE. A–G, Pairing of homologous chromosomes during
leptotene in the ascomycete Sordaria. Scale bar is 1 µm in A–F and 5 µm in G. A–B, In early leptotene, homologous chromosomes (visualized
in panels A–F by electron microscope reconstructions of serial-sectioned nuclei) are not yet aligned with one another. C–E, In mid-leptotene,
regions of some homologs begin to align. (In panel D, only the telomeres have aligned. In panel E, the pair of homologs is fully aligned.) F, The
alignment of homologs is complete by late leptotene. G, The alignment of homologs also can be seen by light microscopy using Spo76-GFP, a
component of the chromosome axes. H–I, Stages of formation of the bouquet arrangement in rye. H, Telomeres (green) were detected in nuclei
by in situ hybridization (see Fig. 8.10) after 0, 2, and 8 hours in culture. Chromatin is red. I, Three-dimensional models of the nuclei (nuclear
periphery [red dots], telomere position [green stars]). (A–G, Modified from Tesse S, Storlazzi A, Kleckner N, et al. Localization and roles of Ski8p
protein in Sordaria meiosis and delineation of three mechanistically distinct steps of meiotic homolog juxtaposition. Proc Natl Acad Sci U S A.
2003;100:12865–12870. Copyright 2003 National Academy of Sciences. H–I, Modified from Carlton PM, Cowan CR, Cande WZ. Directed motion
of telomeres in the formation of the meiotic bouquet revealed by time course and simulation analysis. Mol Biol Cell. 2003;14:2832–2843.)

leptotene, homologous chromosomes are aligned loosely
about 400 nm apart (Fig. 45.6D–G).
During leptotene, one or both telomeres attach to the
inner surface of the nuclear envelope and move actively
around the nuclear surface until they coalesce near the
centrosome (spindle pole body in yeasts [Fig. 45.6]).
These movements and clustering of telomeres depend
on cytoplasmic microtubules. Telomeres are linked to
microtubules through a pair of nuclear envelope proteins
known as the LINC (linker of nucleoskeleton and cyto-
skeleton) complex (see Fig. 9.8). Telomere clustering
peaks at the leptotene–zygotene transition with the
chromosomes radiating into the nuclear interior like a
bouquet of flowers, hence the name “bouquet stage.”
Bouquet formation is a nearly universal feature of this
phase of meiosis and the movements of tethered telo-
meres help homologs find each other through physical
alignment. Thus, telomere clustering per se may not be
the goal of this movement. The details vary among dif-
ferent organisms. In fission yeast dynein motors and
microtubule dynamics in the cytoplasm move the telo-
mere cluster from one end of the cell to the other every
10 minutes or so. These “horsetail movements” stretch
the chromosomes parallel to each other. In C. elegans
special chromosome regions known as “pairing centers”
mediate chromosome movement instead of telomeres;
in budding yeast, the telomeres are linked to actin instead
of microtubules, and D. melanogaster may have lost
such a mechanism altogether.
During the transition from leptotene to the zygotene
(Greek, “yoke ribbon”) stage of prophase, clustering
of chromosome ends at the nuclear envelope reaches
its peak, with the “bouquet” arrangement of chromo-
somes. During this stage homologous chromosomes
begin to achieve their maximal alignment as well,
through the initiation of synapsis (Fig. 45.5C–D). Syn-
apsis involves the assembly of the axial element. This
protein scaffold forms part of the synaptonemal
complex when pairing is complete.
In pachytene (from the Greek, meaning “thick
ribbon”), synapsis is complete, with the homologous
chromosome axes joined together along their lengths
by synaptonemal complexes (Fig. 45.5E). During pachy-
tene, crossover-designated recombination intermediates
mature into Holliday junction-containing structures
within the context of the full-length synaptonemal
 CHAPTER 45  n  Meiosis 787

complex. The final resolution of these recombination The earliest pairing events in meiosis involve a ten-
intermediates into crossovers occurs close to the time of dency of homologous chromosome territories to move
synaptonemal complex disassembly, dispersal of the together in the nucleus even before leptotene chromo-
bouquet of chromosomes and exit from pachytene. The some condensation. The mechanism is unknown. As
crossovers then mature into structures called chiasmata double-strand breaks created by Spo11 initiate the
that link homologous chromosomes through meiosis I recombination pathway during leptotene, the condensing
metaphase. homologous chromosomes align with one another at a
Early in diplotene (from the Greek, meaning “double distance of about 400 nm (Figs. 45.6 and 45.7). Genetic
ribbon”), the synaptonemal complex disassembles, analysis in budding yeast revealed that mutants defective
telomeres detach from the nuclear membrane, and chro- in the earliest stages of recombination are also defective
mosomes begin to condense in preparation for division in homolog pairing.
(Fig. 45.5F). The duplicated sister chromatids remain
closely associated, and chiasmata hold the homologous Replicating DNA
Interphase Sister Sister
chromosomes together, although their axes tend to drift chromatid 3 chromatid 4
Sister
apart in the absence of synaptonemal complex. This chromatid 1 Sister
chromatid 2 Cohesin
part of meiotic prophase may last for days or years,
depending on the sex and organism (up to 45 years or
more in female humans).
Oocytes (immature eggs) actively transcribe their Sister chromatids
linked by cohesin
chromosomes during diplotene, as they store up materials in premeiotic
for use during the first few divisions of embryonic devel- S phase
opment. Transcription can be so active that DNA loops
are massively coated with nascent RNA transcripts whose Leptotene
associated proteins are visible by light microscopy in Initiation of
recombination
oocytes of most animals (except mammals). Chromo- Pairing of Chromatid
somes at this stage are known as lampbrush chromo- homologous axis
somes (see Fig. 8.12). chromosomes
Diakinesis (from the Greek, meaning “across move- Zygotene
ment”) is the prometaphase of meiosis I. Following Assembling
central element of Synapsis
nuclear envelope breakdown, homologous chromo- synaptonemal complex
somes shorten and condense. At metaphase I, the biva-
lents (pairs of homologous chromosomes) are aligned Axial
Pachytene (lateral)
at a metaphase plate (Figs. 45.1, 45.2, and 45.12). The elements
two homologs (each a pair of tightly linked sister chro- Transverse
matids) are attached to opposite poles of the meiotic filaments
spindle, which applies force, attempting to pull them Central
apart. Cohesion of the arms distal to chiasmata resists element
these pulling forces. The homologs separate and move
to opposite spindle poles during anaphase I when the
cohesion along the chromosome arms is released. The Diplotene followed
sister chromatids move together to one pole, because by diakinesis
Chromatin
they remain linked by cohesion at their centromeres, Disassembling loops
synaptonemal
where the cohesion complex is protected by a shugoshin complex
protein (see later).
After telophase I, cells enter a brief interkinesis
Chiasma
during which there is no DNA replication. The second
Time

meiotic division is mechanistically similar to mitosis

except that the number of chromosomes is reduced by
half. Additionally, in the eggs of most female vertebrates,
meiosis is arrested at metaphase II until fertilization.

Pairing and Synapsis in More Detail

Pairing describes the side-by-side alignment of homolo-
gous chromosomes at a distance. Homologs are paired FIGURE 45.7  CHROMOSOMAL PAIRING IN MEIOTIC PRO-
in nonmeiotic (somatic) cells in a few organisms, such PHASE. Structural organization of homologous chromosomes and
as the fruit fly D. melanogaster, but not in vertebrates. synaptonemal complex during various stages of meiotic prophase.
788 SECTION X  n  Cell Cycle

The process of homolog alignment almost certainly
involves the invasion of neighboring DNA duplexes by
single-stranded DNA complexed with Rad51 and Dmc1.
Thus, recombination has important roles both in the
exchange of genetic material and in the mechanics of
chromosome behavior during meiotic prophase. Recom-
bination is probably not the only factor driving homolog
pairing, however. Pairing is reduced but not absent in
yeast meiotic cells lacking both Rad51 and Dmc1, and
homologous chromosomes still pair in some systems that
lack recombination (eg, certain D. melanogaster recom-
bination mutants), synaptonemal complex formation
(asynaptic mutants in yeast), or both (eg, normal D.
melanogaster males).
Homolog pairing initiated during leptotene be­
comes much more intimate during synapsis as the
chromosomes are linked by transverse fibers to form
the synaptonemal complex. This structure looks
roughly like railroad tracks linked by transverse bands
(Figs. 45.7 and 45.8). Each of the two outer rails, 90
to 100 nm apart, is the axis of a pair sister chromatids.
They are traditionally termed lateral elements, but
for the sake of simplicity, we refer to them as axial
elements. Thin transverse filaments oriented perpen-
dicular to the axial elements appear to connect homolog
axes to each other and to the central element (the
“third rail”). Synaptonemal complex formation begins
during zygotene at a limited number of sites along the
paired homologous chromosomes where recombination
events will mature into crossovers. By pachytene, a
continuous synaptonemal complex assembles along the
full length of the aligned homologous chromosomes
(Fig. 45.5C–E).
It was once thought that the synaptonemal complex
aligns homologous chromosomes in preparation for
recombination, but it is now clear that homolog pairing
and (in many organisms) the initiation of recombination
precedes synapsis. Thus, synapsis is a downstream
consequence of early steps in recombination in some
well-studied organisms including yeast and mammals.
However, under certain artificial circumstances, even
nonhomologous chromosomes can undergo synapsis.
Another longstanding model proposed that the synapto-
nemal complex promotes the resolution of crossover-
designated recombination intermediates. However, analysis
of budding yeast mutants missing certain synaptonemal
complex proteins indicates that the structure per se is
dispensable for the formation of crossovers and that the
resulting chiasmata can hold homologous chromosomes
paired until anaphase of meiosis I.
What then is the function of the synaptonemal
complex? One possibility is that it may have a key role
in crossover interference (see later), which ensures that
crossovers are distributed broadly across the genome.
Another interesting possibility is that the synap-
tonemal complex communicates information about
meiotic chromosomes (such as homolog alignment and
the formation of crossover-designated recombination

A B C

CE

LE
CE

FIGURE 45.8  ELECTRON MICROGRAPHS OF THE SYNAPTONEMAL COMPLEX. A, Low-magnification view of maize synaptonemal
complexes stained with silver. The lateral (LE) and central elements (CE) are clearly seen. B, A negatively stained cricket synaptonemal complex
following treatment with deoxyribonuclease (DNase). The central element (CE) and transverse filaments (arrow) are visible. C, A whole mount of
a silk moth zygotene chromosome. Cells in meiotic prophase were swollen and then lysed under gentle conditions with detergent. The chromo-
somes were then centrifuged onto thin carbon films so that they could be examined by electron microscopy. The axial elements are easily seen
on this chromosome. Chromatin loops radiate outward from both the unpaired axial elements and the paired lateral elements (where synapsis
has occurred). (A, Modified from Gillies CB. Electron microscopy of spread maize pachytene synaptonemal complexes. Chromosoma.
1981;83:575–591. B, Modified from Solari AJ, Moses MJ. The structure of the central region in the synaptonemal complexes of hamster and
cricket spermatocytes. J Cell Biol. 1973;56:145–152, copyright the Rockefeller University Press. C, From Rattner JB, Goldsmith M, Hamkalo BA.
Chromatin organization during meiotic prophase of Bombyx mori. Chromosoma. 1980;79:215–224.)
 CHAPTER 45  n  Meiosis 789

intermediates) to the cell-cycle pathways that control Several protein components of the axial elements
progression through the substages of meiosis. (sister chromatid axes) have also been identified. One of
these, SYCP3, interacts with both the cohesin complex
(see Fig. 8.18) and Rad51p and Dmc1p. In SYCP3 knock-
Synaptonemal Complex Components out mice, the axial elements are much less prominent, and
Both genetic and biochemical approaches have identi- the axis of the condensed chromosome is about twofold
fied components of the synaptonemal complex. The longer. Other proteins of the synaptonemal complex,
budding yeast protein Zip1 (mammalian SYCP1) com- including SYCP1, do not assemble properly, and as a result
prises the transverse filaments oriented perpendicular to chromosomes in male germ cells lack chiasmata, are
chromosome axes in mature synaptonemal complex, unpaired, and cells die in pachytene/diplotene. Humans
between the axial elements (Fig. 45.9). Mammalian with mutations in genes for cohesin subunits lack chias-
SYCP1 and Zip1 both consist of an extensive coiled-coil mata, fail to complete meiosis, and are infertile.
flanked by two globular domains but lack amino acid
sequence similarity. Altering the length of the Zip1
coiled-coil changes the spacing between axial elements
Chiasmata
in the synaptonemal complex. Chiasmata are specialized chromatin structures that link
homologous chromosomes together until anaphase I
(Figs. 45.1 and 45.10). They form at sites where pro-
Leptotene SYCP3 + grammed DNA breaks generated by Spo11 undergo the
Paired sister SYCP2,
chromatids Cohesin full recombination pathway to generate crossovers.
Axial element It is not known how crossover events, which repre-
(chromosome axis)
sent exchanges of DNA sequence information, are turned
Chromatin loops into chiasmata. The ultrastructure of chiasmata remains
Time

Rad51, Dmc1 a mystery, but presumably each chiasma consists of two

Early recombination
nodule (may be site of
unperturbed sister chromatid arms intertwined with two
noncrossover events) recombinant arms in which the DNA molecules and their
SYCP1
associated protein structures have been spliced. This
Zygotene (yeast Zip1) DNA complex is held in place on the chromosome by
Synaptonemal complex
Zip2/Zip4, Zip3
(yeast synapsis
cohesion of the distal sister chromatid arms between the
assembles from initiation complex) chiasma and the telomeres. Chiasmata too close to telo-
site of crossover (late Mlh1/Mlh3,
recombination nodule)
Msh4/Msh5 meres can be unstable, presumably because the short
SYCE2, length of sister chromatid arms between them and the
TEX12
telomeres is insufficient for stable cohesion. This can
FIGURE 45.9  PROTEINS OF THE SYNAPTONEMAL COMPLEX.
lead to failure of chromosome segregation in meiosis.
Homologous chromosomes and synaptonemal complex showing the A single chiasma can link homologous chromosomes
locations of some protein constituents. together during meiosis I. Humans have 39 such arms

A B C Microtubules
Fused sister
kinetochores
Paired sister
chromatids
(maternal
homolog)
Distal cohesin
and Aurora B

Chiasma
Paired sister
AIR-2 chromatids
(paternal
Arrows point to chiasmata REC-8 REC-8 homolog)

FIGURE 45.10  BIVALENTS (PAIRED HOMOLOGOUS CHROMOSOMES) ARE HELD TOGETHER BY CHIASMATA AFTER DISAS-
SEMBLY OF THE SYNAPTONEMAL COMPLEX. A, Three diplotene bivalents from the grasshopper species Chorthippus jucundus are held
together by three (left), one (middle), and four (right) chiasmata. The middle cross-shaped bivalent is telocentric; the other two longer bivalents
are submetacentric. (For an explanation of the terminology, see Fig. 7.2.) Lactopropionic orcein staining. B, Caenorhabditis elegans chromosomes
at metaphase I. Aurora B kinase AIR-2 (red) is located distal to chiasmata. Cohesin subunit REC-8 (green) is all along the chromosomes.
C, Explanatory diagram. (A, Courtesy José A. Suja and Julio S. Rufas, Universidad Autónoma de Madrid, Spain. B, Courtesy Josef Loidl, Max
Perutz Labs, Vienna Biocenter, Austria.)
790 SECTION X  n  Cell Cycle
A. Normal
SB
FIGURE 45.11  ABNORMAL PACHYTENE CHROMOSOMES IN AN INFERTILE MALE. A, Normal pachytene chromosome spread from
a testis biopsy showing synaptonemal complexes (red), MLH1 foci (recombination sites [green]), and centromeres (blue). B, Abnormal pachytene
spread from an infertile patient containing one synaptonemal complex with an area of asynapsis and one synaptonemal complex with a gap
(arrows). SB, sex body (the paired X and Y chromosomes). (Courtesy Renée H. Martin, University of Calgary, Alberta, Canada.)
on the 23 pairs of homologous chromosomes, if one
excludes the five acrocentric short arms, which do not
normally undergo crossovers. Remarkably, there is typi-
cally only one chiasma produced for most arms; human
males typically have 46 to 53 chiasmata (Fig. 45.11).
Even more remarkably, that single chiasma can hold
homologous stably paired for over 40 years in human
females, yet still be released on schedule when the
oocyte matures into an egg.
Only a small fraction of DNA breaks formed by Spo11
mature into full crossovers, because a mechanism called
crossover interference decreases the likelihood that
DNA breaks near a crossover-designated recombination
event will also become crossovers. This interference
tends to spread crossovers apart across the genome. If
all breaks had an equal probability of forming crossovers,
small chromosomes might be left without a crossover in
a significant fraction of meiotic nuclei if large chromo-
somes used all of the structural components necessary
to form crossovers and chiasmata.
Crossover interference has been defined genetically
for almost 100 years, but its mechanism is not certain.
Interference may be mediated by the synaptonemal
complex. Organisms such as the fission yeast Schizosac-
charomyces pombe and the mold Aspergillus nidulans
that naturally lack synaptonemal complex also lack
interference. Furthermore, the frequency of meiotic
recombination is directly proportional to the length of
the synapsed chromosome axis (ie, the length of the
synaptonemal complex), rather than the actual length
of DNA in the chromosome. For example, in human
females, the synaptonemal complex is roughly 50%
longer than in males, and females undergo recombina-
tion about twice as frequently as males. However, other
observations suggest that interference may be established
before the synaptonemal complex forms.
B. Infertile male
SB
Cohesion and Chromosomal Movements
During Meiosis I
Chromosomes in mitosis achieve a dynamic alignment at
metaphase as a result of a balance of forces in the spindle.
In mitosis, the two kinetochores of the sister chromatids
are attached to microtubules emanating from opposite
spindle poles, and each chromatid is pulled toward the
pole that its kinetochore faces (Fig. 45.12A).
In meiosis I, homologs linked by chiasmata (called
bivalents) are balanced at the metaphase plate, but the
organization differs in three important ways from mitosis.
First, the two kinetochores of the sister chromatids are
fused and act as a single unit oriented toward one spindle
pole. The structure of the meiosis I kinetochore is most
easily explained if the two kinetochores are each rotated
90 degrees toward one another relative to their position
on mitotic chromosomes and then fused (Fig. 45.12A).
In yeast, this coorientation of sister kinetochores requires
the presence of a meiosis-specific kinetochore protein—
spo13 (meikin in vertebrates)—that associates with
sister kinetochores from pachytene until anaphase of
meiosis I. Spo13/meikin recruits polo kinase to kineto-
chores, but the critical kinase substrates are not known.
Second, the physical connection between the fused
kinetochores is not broken in anaphase I. As a result, at
anaphase the two homologs with their paired sister
chromatids move in opposite directions.
A third major difference between bivalents in meiosis
I and mitotic chromosomes is that cohesion of homolo-
gous chromosome arms distal to chiasmata (Figs. 45.2
and 45.10) rather than cohesion between sister chroma-
tids at centromeres resists the poleward pulling of
the kinetochores at the spindle midzone at metaphase I
(Fig. 45.12B). This reflects specialized behavior of the
meiotic cohesin complex in which the meiosis-specific
 CHAPTER 45  n  Meiosis 791

A. Kinetochore movement
Kinetochore

90° rotation
Microtubules of kinetochores

Sister chromatids
Mitosis Meiosis Meiosis

B. Centromere behavior in meiosis

Paired sister
kinetochores
Microtubules
Chiasmata
Recombination
2×
Arms of sisters Homolog
separate pairing
Paired Paired sister
homologs chromatids
Anaphase I Metaphase I Leptotene
Chiasmata released
Homologs separate

Sister Sisters
centromeres segregate
separate
Paired
sisters
Anaphase I Metaphase II Anaphase II
FIGURE 45.12  CHROMOSOMAL BEHAVIOR DURING MEIOSIS I AND II. During meiosis I, sister chromatids are tightly paired along their
lengths, sister kinetochores are fused, and homologs are held together at the metaphase plate by chiasmata. During anaphase I, loss of cohesion
between the arms of sister chromatids releases the chiasmata and allows homologous chromosomes to segregate to opposite spindle poles.
During metaphase of meiosis II, sister chromatids are held together only at their centromeres. Release of centromeric cohesion at meiosis II allows
the sister chromatids to segregate to opposite spindle poles.

Rec8 and Rad21L proteins replace Scc1 (see Figs. 8.18
and 44.16). After premeiotic DNA replication, the
meiotic cohesion complex keeps sister chromatids
together all along the arms. The cohesin complex plus
synaptonemal complex proteins SYCP3 and SYCP2 are
required for assembly of the dense axial elements that
extend along the length of the chromosome during
synapsis.
In mitosis, cohesion is released between sister chro-
matid arms during prometaphase, but in meiosis I it is
retained distal to chiasmata until the onset of anaphase
(Figs. 45.7 and 45.12), when Rec8 and Rad21L along
the chromosome arms are cleaved by a protease called
separase (see Fig. 44.16). Separation of sister chromatid
arms allows the chiasmata to resolve (untangle), and
the homologous chromosomes segregate to opposite
spindle poles.
In the meantime, the Rec8 and Rad21L at centromeres
are protected from cleavage and continue to hold the
sister chromatid centromeres tightly paired until ana-
phase of meiosis II. This protection requires a class of
proteins called Shugoshins (from the Japanese, meaning
“guardian spirit”), which recruit the protein phosphatase
2A (PP2A). Rec8 must be phosphorylated for separase to
cleave it efficiently, so the localized phosphatase can
block the cleavage reaction. Cleavage of centromeric
Rec8 releases sister chromatid cohesion at the onset of
anaphase II similar to the release of cohesion during
mitosis (see Fig. 44.16).
Behavior of the Sex Chromosomes
in Meiosis
Of the 46 human chromosomes, two sex chromosomes
carry genes that define the sex of the individual. The
other 22 pairs of chromosomes are called autosomes.
If genetic recombination is required to stabilize
homologous chromosomes at the metaphase plate in
792 SECTION X  n  Cell Cycle
A C
X 2
Y
9 has been called the pachytene checkpoint, as cells arrest
B niscent of the pachytene stage. Apoptosis eliminates
Synapsed pseudoautosomal region
Y Meiosis I and Meiosis II
X Meiosis is unique in that it involves two M phases with
Condensed unpaired portion
of X and Y chromosomes
FIGURE 45.13  SEX CHROMOSOMES OF A CHINESE
HAMSTER AT PACHYTENE. A–B, The X and Y chromosomes are
synapsed at the pseudoautosomal region. Elsewhere, the unpaired
chromatin adopts a highly condensed morphology. C, In the same
preparation, autosomes are completely synapsed and show a lesser
degree of condensation. (From Dresser ME, Moses MJ. Synaptonemal
complex karyotyping in spermatocytes of the Chinese hamster (Crice-
tulus griseus). IV. Light and electron microscopy of synapsis and
nucleolar development by silver staining. Chromosoma. 1980;76:
1–22.)
meiosis I, how is this accomplished for the X and Y
chromosomes? The answer in most mammals is that the
X and Y chromosomes have a short region of homolo-
gous sequence (approximately 2.6 million base pairs in
humans) that does pair and undergo genetic recombina-
tion during meiosis. This pseudoautosomal region
must undergo genetic recombination in every meiosis I
cell for the X and Y chromosomes to be partitioned
correctly. Thus, the X and Y chromosomes act like short
homologous chromosomes with large regions of unre-
lated DNA attached (Fig. 45.13). Unpaired regions of
the X and Y chromosomes acquire a distinct chromatin
structure during late pachytene.
Cell-Cycle Regulation of Meiotic Events
Meiosis employs the full set of functions that regulate
the division of somatic cells (see Chapters 40 to 43).
However, the peculiarities of the meiotic cell cycle
require additional regulation. One major difference from
somatic cells is that meiotic chromosomes must undergo
recombination and form chiasmata to segregate properly
at the first meiotic division. Like somatic cells, meiotic
cells have a DNA damage response that arrests cell-cycle
progression in the presence of DNA breaks (see Fig.
43.11). In addition, they have a “crossover assurance”
checkpoint that can detect the presence of stalled or
abnormal recombination intermediates. Such intermedi-
ates accumulate if there are problems with the core
recombination enzymes or if the assembly of the syn-
aptonemal complex (required for the completion of
recombination) is defective. When such problems are
detected, cells arrest in meiotic prophase I. In yeast, this
late in meiotic prophase with nuclear morphology remi-
mammalian germ cells that arrest owing to defects in
recombination.
Suppression of DNA Replication Between
no intervening S phase. On exit from meiosis I, Cdk1
kinase is reactivated immediately. This blocks assembly
of prereplication complexes (see Fig. 42.8), thereby
blocking DNA replication. At least two pathways contrib-
ute to reactivation of Cdk1.
The first involves downregulation of translation of
Wee1 protein kinase in meiosis. Wee1 is a mitotic inhibi-
tor (see Fig. 43.3) that inactivates Cdk1 by phosphoryla-
tion at Tyr15. The absence of Wee1 in meiosis I was first
observed in Xenopus laevis but this seems to be a uni-
versally conserved way of reactivating Cdk1 without an
S phase. Ectopic expression of Wee1 in mature X. laevis
oocytes prevents reactivation of Cdk1 immediately after
the meiosis I division. As a result, the oocytes reenter
interphase and replicate their DNA. Meiotic cells also
express a specialized isoform of Cdc25, the phosphatase
that counteracts Wee1 (see Fig. 43.1).
Metaphase II Arrest and
the Mitogen-Activated Protein
Kinase Pathway
Following their activation and release from the ovary
(ovulation), oocytes of many vertebrates arrest in meta-
phase II of meiosis until they are fertilized. The activity
that is responsible for this arrest was discovered in
X. laevis eggs arrested in metaphase of meiosis II and is
called cytostatic factor (CSF). Injection of cytoplasm
containing CSF into one blastomere of a two-cell frog
embryo blocks the next cell cycle at metaphase, just
like the egg (Fig. 45.14). Therefore, CSF can even
block somatic cells indefinitely in mitotic metaphase.
CSF activity appears in meiosis II and disappears after
fertilization.
One active component of CSF is the X. laevis homolog
of a well-known viral oncogene, v-mos, the transforming
gene of the Moloney murine sarcoma virus, which causes
solid tumors in mice. The v-mos gene is a mutated
form of the cellular c-mos gene. Vertebrates express
c-mos (a mitogen-activated protein [MAP] kinase kinase
kinase; see Fig. 27.5) exclusively in oocytes and eggs.
Injection of either v-Mos or c-Mos proteins into dividing

A. Two-cell embryo B then also phosphorylates Emi2. Emi2 phosphorylated by
Inject egg cytosol,
with activated
c-Mos, into one
blastomere
C The fate of cells undergoing meiosis, as well as the timing
c-Mos removed
by antibody
before injection
D. The MAP kinase cascade that arrests eggs
in metaphase of meiosis II:
c-Mos MEK MAPK p90Rsk [Emi2] CSF
FIGURE 45.14  ROLE OF C-MOS IN THE CYTOSTATIC
FACTOR PATHWAY. A, Diagram of the experiment that identified
c-Mos as an essential component of cytostatic factor (CSF) required
for arrest of eggs in meiotic metaphase. One blastomere of an Xenopus
laevis embryo at the two-cell stage was injected with cytoplasm from
a metaphase-arrested egg containing CSF activity. B, This blastomere
(right half of the embryo) remained blocked in metaphase while the left
blastomere divided many times. C, The same experiment was per-
formed, but prior to injection, the c-Mos was removed from the egg
cytoplasm by absorption with a specific antibody. Both the injected
and uninjected blastomeres continued to divide normally. D, The
mitogen-activated protein (MAP) kinase (MAPK) pathway leading to
metaphase II arrest in vertebrate eggs. (B–C, Micrographs courtesy
George Vande Woude, NCI, Frederick, MD. Modified from Sagata N,
Watanabe N, Vande Woude GF, et al. The c-Mos proto-oncogene
product is a cytostatic factor responsible for meiotic arrest in vertebrate
eggs. Nature. 1989;342:512–518.)
blastomeres of early frog embryos arrests the cells at
metaphase (Fig. 45.14). These experiments led to a
proposal that c-Mos is CSF.
CSF arrest requires the MAP kinase (MAPK) signal
transduction pathway (see Fig. 27.5). Mos activates the
pathway by phosphorylating mitogen activated protein/
extracellular signal-related kinase kinase (MEK), which
then activates MAPK. MAPK then activates a downstream
kinase called p90Rsk (Fig. 45.14D). Introduction of
constitutively active c-Mos or p90Rsk into X. laevis eggs
induces metaphase arrest like CSF. However, this is not
the whole story, because metaphase arrest is maintained
in extracts depleted of p90Rsk. Thus the pathway must
include at least one unidentified step beyond p90Rsk.
The extra component of CSF is an anaphase-promoting
complex/cyclosome (APC/C) inhibitor called Emi2. A
burst of cytoplasmic Ca2+ released at fertilization (see Fig.
26.15) activates Ca2+/calmodulin-dependent protein
kinase II (CaMKII), which phosphorylates Emi2. This
CHAPTER 45  n  Meiosis 793
modification creates a binding site for polo kinase, which
polo kinase is recognized by SCFβTrCP, which ubiquitylates
it, marking it for destruction. This results in activation of
the APC/C (see Fig. 40.15), termination of the CSF meta-
phase arrest, and completion of meiosis II.
Timing of Meiosis in Humans
of meiotic events, differs significantly between human
males and females.
Males produce approximately 100 million sperm a day
in a process called spermatogenesis. This process
continues throughout adult life. Spermatogenesis starts
with the division of stem cells called spermatogonia
and involves eight divisions prior to meiosis. These
divisions are unusual in that cytokinesis is incomplete,
and the cells remain connected by intercellular bridges.
The process could produce up to 256 cells, but usually
some cells die and others fail to divide, so a more typical
number is around 200 cells arising from the initial
stem cell division. When these cells pass through
meiosis (they are then referred to as spermatocytes) the
final result is approximately 800 postmeiotic sperma-
tids. Spermatids then undergo a complex program of
differentiation, resulting in the production of highly
specialized spermatozoa. The entire process of sper-
matogenesis takes approximately 64 days, the bulk of
which is spent in meiosis I. Approximately 16 days are
spent in pachytene, the longest stage of the meiosis I
prophase. In contrast, only about 8 hours are spent in
meiosis II.
By the twentieth week of fetal life each ovary of a
human female contains approximately 3 × 106 primordial
follicles, each with an oocyte arrested in the diplotene
stage of meiosis. This lengthy arrested stage is referred
to as dictyate. Thereafter, arrested primordial follicles
are recruited continuously to mature into growing
primary follicles. However, successful follicular growth
depends on follicle-stimulating hormone (FSH), so before
puberty—when the hypothalamus-pituitary-ovarian axis
matures—all activated oocytes undergo programmed
cell death and degenerate in a process known as atresia.
At birth, the ovary contains approximately 1,000,000
germ cells of which approximately 300,000 to 400,000
remain at puberty. Following puberty and in response to
high levels of FSH each month, a cohort of follicles with
their dictyate-arrested oocytes is activated to complete
meiosis and grow. Only one of these activated oocytes
matures fully and is shed from the ovary in response to
a surge of luteinizing hormone. The other follicles degen-
erate by atresia. As the successful oocyte is shed from
the ovary, it completes meiosis I and is arrested at meta-
phase of meiosis II by CSF. It remains arrested at this
stage until fertilization occurs. By the time of menopause
794 SECTION X  n  Cell Cycle
First polar body
Metaphase II oocyte
50 µm
FIGURE 45.15  POLAR BODY FORMATION. Asymmetric cleav-
age during mouse oogenesis produces a large oocyte and small polar
body. (Modified from Jiao ZX, Xu M, Woodruff TK. Age-associated
alteration of oocyte-specific gene expression in polar bodies: potential
markers of oocyte competence. Fertil Steril. 2012;98:480–486.)
the ovarian reserve is almost depleted, leaving approxi-
mately 1000 primordial follicles in the ovary.
In human females, meiosis produces only one mature
egg. Both meiotic cell divisions are asymmetrical, pro-
ducing one large and one very small and short-lived cell.
The small cells are called polar bodies (Fig. 45.15).
Meiotic Defects and Human Disease
Abnormalities in meiosis are surprisingly common but are
not widely observed in human populations because their
consequences are extremely severe. In fact, meiotic
abnormalities are a leading cause of fetal death, particu-
larly during the first trimester of pregnancy in humans.
The two major causes of problems are chromosome
nondisjunction during the meiotic divisions and the gen-
eration of unbalanced chromosomal rearrangements via
faulty recombination.
When chromosomes fail to segregate properly in one
or both meiotic divisions (nondisjunction), the daughter
cells lack the normal haploid complement of chromo-
somes. Embryos that have gained an entire set of chro-
mosomes are referred to as polyploid. In human
embryos, polyploidy is a common type of chromosomal
abnormality. It is estimated that 1% to 3% of all concep-
tions are triploid (69 chromosomes; 23 from one parent
and 46 from the other parent). Two-thirds of these arise
from two sperm fertilizing one egg (nothing wrong with
meiosis there). In other cases, they come from a diploid
gamete, the result of a defective meiotic segregation.
Very few triploid embryos survive to birth.
Most chromosomal abnormalities in human embryos
result from the loss or gain of one or more chromosomes
during meiosis. This condition is called aneuploidy.
Most zygotes that arise from aneuploid gametes die
during fetal development. (Any fetal death is a spontane-
ous abortion, commonly called a miscarriage.) It is now
thought that at least 30% of all conceptions result in
spontaneous abortions. Furthermore, more than 60% of
those spontaneous abortions are aneuploid. These figures
probably underestimate the frequency of meiotic abnor-
malities. Many spontaneous abortions occur very early
during pregnancy and many are never detected at all.
Few fetuses lost in the first 4 to 6 weeks of gestation are
tested in a laboratory, so their karyotypes are unknown.
Meiotic errors involving certain autosomes can
produce fetuses that survive to birth. Females with three
or more copies of the gene-rich X chromosome survive,
because all but one X chromosome are inactivated
(silenced) in somatic cells (see Fig. 8.6). Rare individuals
with three copies of chromosome 13 or chromosome18
survive to birth; but those who do typically die shortly
thereafter. The exception is individuals trisomic for
chromosome 21 (a condition that is commonly known
as Down syndrome). These individuals have intellec-
tual disability as well as other characteristic phenotypic
features, including decreased life expectancy. Why do
individuals with Down syndrome survive whereas others
affected by aneuploidy do not? Perhaps the very small
number (233) of coding genes on chromosome 21
includes none whose dosage is critical for survival.
The frequency of certain types of aneuploidy, such as
trisomy for chromosome 21, increases with the ages of
the mother and father. Only 0.04% of children of 20-year-
old mothers old have trisomy 21. This number rises
dramatically with maternal age; nearly 5% of the concep-
tions in mothers 45 years old have trisomy 21 (Fig.
45.16). This maternal age effect is a leading cause of
human genetic disease. Some believe that during the
many years of arrest of oocytes in meiosis I dictyate,
chiasmata joining homologous chromosomes gradually
dissociate. A mechanism to explain this might be the
progressive loss of cohesion between sister chromatids
as the mother ages. There appears to be no mechanism
to replace cohesin complexes that are gradually lost in
dictyate oocytes. Mice with a mutation in a key subunit
of the cohesin complex (Fig. 45.7; see also Fig. 8.18)
exhibit a pattern of chromosome nondisjunction with
increasing maternal age that looks much like that seen
in aging human mothers. Another potential source of
errors lies in the mechanism of spindle assembly in
oocytes, which does not involve centrosomes and
appears to be more prone to errors than in somatic cells
(see Chapter 44).
Not all cases of human aneuploidy originate from the
mother. One of the most common aneuploidies, 45,X
(see Table 45.1 for an explanation of nomenclature)
involves the loss of the paternal X or Y chromosome 70%
to 80% of the time. This aneuploidy accounts for nearly
10% of spontaneous abortions. In addition, about 7% of
 CHAPTER 45  n  Meiosis 795

TABLE 45.1  Aneuploidies Involving the Sex Chromosomes in Newborn Humans

Karyotype Frequency Sex Comments
47,XXY* 1/1000 M Klinefelter syndrome. Increased height, sterile, a proportion may
have some learning difficulties.
47,XYY 1/1000 M Increased height, generally fertile, typically with chromosomally
normal offspring. A proportion may have some learning difficulties.
Other X or Y aneuploidy 1/1350
Total: 1 in 360 male births
47,XXX 1/900 F Increased height, generally fertile, typically with chromosomally
normal offspring. A proportion have serious learning difficulties.
45,X 1/4000 F Turner syndrome. Reduced height, infertile, normal intelligence.
Of 45,X embryos, 99% terminate as spontaneous abortions.
Other X or Y aneuploidy 1/2700
Total: 1 in 580 female births
*This number gives the total number of chromosomes, followed by the complement of sex chromosomes.
Modified from Nussbaum RL, McInnes RR, Willard HF. Genetics in Medicine. 6th ed. Philadelphia, PA: WB Saunders; 2001:150.

[Table 45.1]) reveal that spontaneous abortion is a highly

efficient protective mechanism for the elimination of
chromosomal imbalances that arise from errors in
meiosis.
Chromosome number
3.0

ACKNOWLEDGMENTS
16
We thank Abby Dernburg, Jim Haber, Scott Hawley, Amy
% Trisomies

MacQueen, Adele Marston, and Alberto Pendas for their

2.0 suggestions on revisions to this chapter.

21
SELECTED READINGS

1.0 de Massy B. Initiation of meiotic recombination: how and where?

18
0 synaptonemal complex: protein components, assembly and role in
<19 20–24 25–29 30–34 35–39
Maternal age [years]
FIGURE 45.16  RELATIONSHIP BETWEEN MATERNAL AGE
AND THE INCIDENCE OF HUMAN TRISOMIES. (Modified from
Nagaoka SI, Hassold TJ, Hunt PA. Human aneuploidy: mechanisms
and new insights into an age-old problem. Nat Rev Genet. 2012;
13:493–504.)
instances of trisomy 21 are of paternal origin. Clearly,
more than one mechanism is responsible for the genera-
tion of aneuploid offspring.
These rather sobering statistics reveal two important
facts about human reproduction. First, the production
of gametes is error prone. This has been confirmed by
direct studies, in which 20% of eggs and 3% to 4% of
sperm were found to have chromosomal abnormalities.
Second, the much lower rates of chromosomal abnor-
malities seen in live births (approximately 0.3% overall
Conservation and specificities among eukaryotes. Annu Rev Genet.
2013;47:563-599.
Duro E, Marston AL. From equator to pole: splitting chromosomes in
mitosis and meiosis. Genes Dev. 2015;29:109-122.
Fraune J, Schramm S, Alsheimer M, Benavente R. The mammalian
40
meiotic recombination. Exp Cell Res. 2012;318:1340-1346.
Gutiérrez-Caballero C, Cebollero LR, Pendás AM. Shugoshins: from
protectors of cohesion to versatile adaptors at the centromere.
Trends Genet. 2012;28:351-360.
Hiraoka Y, Dernburg AF. The SUN rises on meiotic chromosome
dynamics. Dev Cell. 2009;17:598-605.
Lam I, Keeney S. Mechanism and regulation of meiotic recombination
initiation. Cold Spring Harb Perspect Biol. 2014;7:a016634.
Nagaoka SI, Hassold TJ, Hunt PA. Human aneuploidy: mechanisms and
new insights into an age-old problem. Nat Rev Genet. 2012;13:
493-504.
Scherthan H. A bouquet makes ends meet. Nat Rev Mol Cell Biol.
2001;2:621-627.
Schmidt A, Rauh NR, Nigg EA, Mayer TU. Cytostatic factor: an activity
that puts the cell cycle on hold. J Cell Sci. 2006;119:1213-1218.
Subramanian VV. Hochwagen A: The meiotic checkpoint network:
step-by-step through meiotic prophase. Cold Spring Harb Perspect
Biol. 2014;6:a016675.
Zickler D, Kleckner N. Recombination, Pairing, and Synapsis of Homo-
logs during Meiosis. Cold Spring Harb Perspect Biol. 2015;7(6).
This page intentionally left blank
 CHAPTER 46 

Programmed Cell Death

Necessity for Cell Death in stimuli, particularly when induction of apoptosis is

Multicellular Organisms
The ability to undergo programmed cell death (Box
46.1) is a built-in latent capacity in most cells of multi-
cellular organisms. Cell death is important for embryonic
development, maintenance of tissue homeostasis, estab-
lishment of immune self-tolerance, killing by immune
effector cells, and regulation of cell viability by hormones
and growth factors. Both extrinsic signals and internal
imbalances can lead cells to kill themselves. Furthermore,
many metazoan cells will die if they fail to receive sur-
vival signals from other cells. Abnormalities of the cell
death program contribute to several important diseases,
including cancer, Alzheimer disease, and AIDS. Cell
death programs are ancient: much of the current network
was present in the last eumetazoan common ancestor
(see Fig. 2.8).
inhibited. Programmed necrosis looks morphologically
like accidental cell death. A third pathway leading to cell
death involves autophagy (see Fig. 23.7). Although
usually regarded as a protective response to starvation,
autophagy has been implicated in certain examples of
cell death, particularly during development.
Necrosis corresponds to what most of us naively
imagine cell death would be like. Owing to lack of cel-
lular homeostasis, water rushes into the dying cell,
causing it to swell until the plasma and organelle mem-
branes burst. As a result, the cell undergoes a generalized
NEIGHBORING CELL
Fas ligand
TNF
Fas receptor
TNF
receptor

Programmed Cell Death Versus Accidental Extrinsic

DISC
Cell Death: Apoptosis Versus Necrosis pathway
Complex II Complex I
Active
Although cells die in many ways, it is useful to focus on Bcl2 caspases
killer
two poles of this spectrum: apoptosis and necrosis. Fig. Necroptosis
Survival

46.1 summarizes the major pathways of programmed

Apoptosis Inflammation
cell death. (Details are filled in as we progress through
the chapter.) Apoptosis, the most widely studied MITOCHONDRION Pyroptosis
pathway for programmed cell death, is cellular suicide Apoptosome
resulting from activation of a dedicated intracellular Bcl2
killer
Signaling
Inflammasome
program. Fig. 46.2 shows a detailed description of the Intrinsic platforms
pathway
events of apoptosis. At the other end of the spectrum Pathogen
DNA damage, etc
necrosis, also called accidental cell death, occurs when attack NUCLEUS
cells sustain a structural or chemical insult that causes
FIGURE 46.1  OVERVIEW OF PROGRAMMED CELL DEATH
the cells to swell and undergo membrane lysis (Fig.
DISCUSSED IN THIS CHAPTER. All these types of death hinge on
46.3). Examples of such insults include extremes of the assembly of a signaling platform (boxed) that is often involved in
temperature and physical trauma. Cells can also initiate activating proteases called caspases. These pathways are all dis-
active programmed necrosis in response to certain cussed in greater detail in the text.

797
798 SECTION X  n  Cell Cycle

BOX 46.1  Key Terms Apoptosis

Apoptosis: Type of programmed cell death that was

identified due to a particular pattern of morphologic Junctions
changes but now is defined by the action of molecular Mitochondria
pathways involving cell surface receptors or mitochon-
dria and resulting in the activation of specialized pro- Nucleus
teases. The name comes from the Greek, referring to
shedding of the petals from flowers or leaves from
Microvilli contract
trees. Apoptotic death occurs in two phases. During Intercellular junctions break
the latent phase, the cell looks morphologically normal Chromatin begins to condense
but is committed to death. The execution phase is
characterized by a series of dramatic structural and
biochemical changes that culminate in fragmentation
of the cell into membrane-enclosed apoptotic bodies.
Activities that cause cells to undergo apoptosis are said
to be proapoptotic. Activities that protect cells from
apoptosis are said to be antiapoptotic.
Autophagic Cell Death: It is still debated how widely Cell shrinks
autophagy is used as a pathway for cell death, although Chromatin condenses around
it is accepted that the pathway (which is widely nuclear periphery
assumed to be primarily a survival pathway when cells
are starved for nutrients) can promote cell death during
development. Autophagy may also either promote or
inhibit apoptosis under specialized circumstances.
Necroptosis: Programmed necrosis that occurs when
tumor necrosis factor (TNF) and certain other cell-
surface receptors are activated. Activation of these
receptors normally leads to a proinflammatory response
and cell survival, but can lead to apoptosis. If certain Cell blebs violently
Chromatin condensation
components of the apoptotic pathway are missing, continues
cells instead undergo necroptosis, apparently as a
backup pathway.
Necrosis (Accidental Cell Death): Death that results
from irreversible injury to the cell. Cell membranes
swell and become permeable. Lytic enzymes destroy
the cellular contents, which then leak out into the inter-
cellular space, leading to an inflammatory response.
Programmed Cell Death: Any active cellular process
that culminates in cell death. This may occur in Cell fragments into membrane-
response to developmental or environmental cues or enclosed apoptotic bodies
as a response to physiological damage detected by the
cell’s internal surveillance networks.
Pyroptosis: Often in response to intracellular pathogens,
this involves activation of caspase 1. The infected
cells secrete interleukin-1β and interleukin-18, which
promote an inflammatory response, and also undergo
a form of cell death that resembles necrosis.

Apoptotic bodies phagocytosed

by neighboring cells and
roving macrophages

FIGURE 46.2  APOPTOSIS—ACTIVE SUICIDE—TYPICALLY

AFFECTS SINGLE CELLS. Neighboring cells remain healthy. Apop-
totic cell death usually does not lead to an inflammatory response.
 CHAPTER 46  n  Programmed Cell Death 799

Necrosis
Trauma Dissolution of
cellular structures

Junctions

Mitochondria

Nucleus

Cells and organelles swell Cell lysis

Chromatin condenses Invasion of phagocytic cells
Membranes compromised: Inflammation
H 2O fluid rushes in H2O

FIGURE 46.3  NECROSIS USUALLY RESULTS FROM IRREVERSIBLE INJURY TO CELLS. Typically, groups of cells are affected. In most
cases, necrotic cell death leads to an inflammatory response (red “activated” macrophages).

Point of Reprieve
Insult no return (very rare)

Latent Execution
Condemned Committed
Rescue by Rescue usually Morphologic
survival factors impossible changes
FIGURE 46.4  TWO PHASES OF APOPTOSIS. The latent phase
can be subdivided into two stages: a condemned stage, during which
the cell is proceeding on a pathway toward death but can still be
rescued if it is exposed to antiapoptotic activities, and a committed
stage, beyond which rescue is usually impossible.

process of autodigestion and dissolution, until it spills its

cytoplasmic contents out into the surroundings (Fig.
46.3). This produces local inflammation as phagocytic
cells are activated, flock to the site, and ingest the debris
(see Figs. 22.3 and 30.13). Because agents that damage
cells act over areas that are large in comparison to the
size of a single cell, necrosis often involves large groups
of neighboring cells.
In contrast to necrosis, apoptotic cells shrink rather
than swelling and they typically do not lyse (Fig. 46.2).
Apoptosis is a two-stage process. On receipt of the
proapoptotic signal that triggers the pathway to death,
cells enter a latent phase of apoptosis (Fig. 46.4). The
duration of the latent phase of apoptosis, during which
cells appear healthy, can be extremely variable, ranging
from a few hours to several days.
FIGURE 46.5  SCANNING ELECTRON MICROGRAPH OF
INTACT AND APOPTOTIC MOUSE SARCOMA CELLS. Intact cells
are covered with microvilli, whereas apoptotic cells have numerous
smooth blebs. These cells were stimulated to undergo apoptosis as a
result of interference with RNA metabolism. (From Wyllie AH, Kerr JFR,
Currie AR. Cell death: the significance of apoptosis. Int Rev Cytol.
1980;68:251–306.)
Ultimately, the cells enter the execution phase
of apoptosis, lasting about an hour, during which
they undergo dramatic morphologic and physiological
changes. These include:
• Loss of microvilli and intercellular junctions (Fig. 46.5)
• Shrinkage of the cytoplasm
800 SECTION X  n  Cell Cycle

• Dramatic changes in cytoplasmic motility with activa-

tion of violent blebbing (Fig. 46.6)
• Loss of plasma membrane lipid asymmetry (see Fig.
13.7), so the distribution of phosphatidylserine is
randomized and it appears in the outer leaflet of the
membrane
• Hypercondensation of the chromatin and its collapse
against the nuclear periphery
• The “explosive” fragmentation of the cell into
membrane-enclosed apoptotic bodies that contain
remnants of the nucleus, mitochondria, and other 0 min 114 116
organelles.
All these changes are instigated by the action of a
specific set of death-inducing proteases and are discussed
at length later.
In tissues, surrounding cells rapidly phagocytose
apoptotic bodies in response to the phosphatidylserine
and other markers on their surfaces (Fig. 46.7). Apoptosis
can thus be considered to be the disassembly of the cell
into “bite-sized” membrane-bound vesicles, so the cel-
lular contents are not usually released into the environ- 118 120 148
ment unless phagocytosis is delayed. Surface markers on
apoptotic bodies cause cells that ingest them to secrete FIGURE 46.6  APOPTOSIS OF A TRANSFORMED PIG KIDNEY
antiinflammatory cytokines. As a result, apoptotic death CELL FOLLOWING EXPOSURE TO ETOPOSIDE, A DRUG USED
usually does not lead to an inflammatory response and IN CANCER CHEMOTHERAPY. The dramatic cytoplasmic blebbing
results in the disassembly of the cell into membrane-enclosed vesicles.
apoptotic cells seem to vanish from tissues without (Courtesy L.M. Martins and K. Samejima, Wellcome Trust Institute for
a trace. Cell Biology, University of Edinburgh, United Kingdom.)

A. Attraction of phagocytes via D

soluble “find me” signals
n
Mi
gration sign

Phagocyte Apoptotic
cell
al
s

B. Recognition and phagocytosis via

displayed “eat me” signals and
lack of “don’t eat me” signals

C. Production of antiinflammatory
cytokines

IL-10
TGF-β

FIGURE 46.7  PHAGOCYTOSIS OF APOPTOTIC CELLS. A–C, Phagocytosis that occurs when cells express “eat me” signals results in the
production of antiinflammatory cytokines. D, Electron micrograph of a phagocytosed apoptotic body containing a nuclear fragment. The nucleus
(n) of the epithelial cell that engulfed this apoptotic body is shown at top. In this case, apoptosis occurred during allograft rejection in a pig.
(A, Modified from Lauber K, Blumenthal SG, Waibel M, et al. Clearance of apoptotic cells: getting rid of the corpses. Mol Cell. 2004;12:277–287.
B, From Wyllie AH, Kerr JFR, Currie AR. Cell death: the significance of apoptosis. Int Rev Cytol. 1980;68:251–306.)
 CHAPTER 46  n  Programmed Cell Death 801

Examples of Cells That Undergo
Programmed Cell Death
The following are six common causes of programmed
cell death (Fig. 46.8).
Developmentally Defective Cells
During molecular maturation of T-lymphocyte antigen
receptors (see Figs. 27.8 and 28.9), immature T cells in
the thymus (known as thymocytes) rearrange the genes
encoding the receptor α and β chains. To function
properly, the T-cell receptor must recognize major his-
tocompatibility complex (MHC) glycoproteins on other
thymic cells during antigen presentation (see Fig. 27.8).
T lymphocytes whose T-cell receptors cannot interact
with the spectrum of MHC glycoproteins expressed in a
given individual are ineffective in the immune response.
These cells die by apoptosis in a process known as posi-
tive selection (Fig. 46.9).
Many of the newly created receptors bind to foreign
antigens, but others interact with self-antigens. The
latter are potentially harmful and are eliminated through

A B C D
Up to 80% of
Epithelial cells must die neurons die in
to allow fusion of palate some ganglia

Mammary epithelium Over 95% of

cells die when deprived immature
of hormones at end of T cells die
lactation in thymus

Cells of Müllerian
ducts die in males E12.5 E13.5 E14.5

Prostate cells die Cells of

interdigital E
when deprived of
hormone webbing die Dying cells
(yellow)

FIGURE 46.8  PROGRAMMED CELL DEATH DURING DEVELOPMENT. A, Examples of cells that undergo programmed cell death.
B–D, Programmed cell death in the embryonic mouse paw. At day 12.5 of development, the digits are fully connected by webbing. By day 13.5,
the webbing has started to die, and by day 14.5, all the webbing cells are gone. E, Cells undergoing programmed cell death take up acridine
orange, whereas cells of the surrounding healthy tissue do not. (Micrographs courtesy William Wood and Paul Martin, Department of Anatomy
and Developmental Biology, University College of London, United Kingdom.)

THYMUS PERIPHERY, SPLEEN, AND LYMPH NODES

TCR-β gene Pre-TCR TCR-α gene TCR T-cell

rearrangement rearrangement stimulation

Common T-cell Pro-T4 cell Immature Mature Mature T-cell T-cell blast
precursor thymocyte thymocyte

No IL-7R signal No productive No TCR signal (no Autoreactive Lack of TCR No growth-
TCR-β gene positive selection) TCR (negative signal factor signals
rearrangement No productive TCR-α selection) Strong TCR
gene rearrangement restimulation

APOPTOSIS

FIGURE 46.9  EXAMPLES OF SIGNALS THAT PROMOTE DIFFERENTIATION OR PROGRAMMED CELL DEATH OF IMMATURE
THYMOCYTES IN THE THYMUS AND MATURE T CELLS IN THE PERIPHERY. Thymocytes that make functional T-cell receptors (TCRs)
and do not recognize self-antigens mature, provided that they receive survival signals, such as interleukin-7. Pro-T cells are also known as
double-negative thymocytes (referring to their lack of the two cell-surface markers CD4 and CD8). Immature thymocytes express CD4 and CD8
and are known as double-positive thymocytes. Thymocytes undergo apoptosis if they produce defective T-cell receptor, recognize self-antigens,
suffer DNA damage, or receive a death stimulus (glucocorticoid hormone). More than 98% of immature thymocytes die without leaving the thymus.
(Modified from Strasser A. The role of BH3-only proteins in the immune system. Nat Rev Immunol. 2005;5:189–200.)
802 SECTION X  n  Cell Cycle
apoptosis in a process known as negative selection
(Fig. 46.9). The drug cyclosporine, which inhibits apop-
tosis in thymocytes, can cause autoimmune disease.
Overall, defects in T-cell receptor assembly are extremely
common, and up to 98% of immature T cells die by
apoptosis without leaving the thymus.
Similar positive and negative selection steps occur
during the maturation of B lymphocytes (see Fig. 28.10),
which is accomplished by a combination of gene rear-
rangements and facilitated mutagenesis. B lymphocytes
expressing antibodies directed against self-antigens or
producing antibodies whose affinity for antigen is below
a critical threshold are eliminated through apoptosis.
Excess Cells
Programmed cell death is also widely used for quality
control during development. For example, in the brain,
embryonic ganglia often have many more neurons than
are required to enervate their target muscles. Production
of excess cells is part of a Darwinian strategy to ensure
that a sufficient number of axons reach their targets.
Programmed cell death eliminates excess neurons that
fail to make appropriate connections. Up to 80% of
neurons in certain developing ganglia die in this way.
Because of the importance of apoptosis during its devel-
opment, the brain is often seriously affected in mice
engineered to lack components of the apoptotic pathway.
Cells That Serve No Function
The elimination of obsolete cells whose function has
been completed is most evident in organisms, such as
insects and amphibians that undergo metamorphosis
during development. For example, a burst of thyroid
hormone initiates programmed cell death for resorption
of the tadpole tail.
Mammals also use programmed cell death to eliminate
obsolete tissues during development. For example,
during human embryonic development, the digits of
hands and feet are connected by a tissue webbing that
is eliminated by programmed cell death (Fig. 46.8).
During craniofacial development, the hard palate
develops from two lateral precursors, each covered in a
protective layer of epithelial cells. As the two halves
grow together at the midline of the nasopharynx, they
remain separated by this epithelial covering until, in
response to a developmental cue, the epithelial cells at
the midline undergo programmed cell death. Then the
two halves of the palate can fuse. Failure of the epithelial
cells to die at the appropriate time can interfere with the
fusion of the bone, causing cleft palate.
Populations of cells that are fully functional may
become obsolete as a result of physiological changes
in the status of an organism. For example, in male
mammals, the prostate and other accessory glands of the
reproductive system are regulated by the levels of circu-
lating male hormone. If hormone levels fall below a
critical threshold, these organs virtually disappear in a
relatively brief time as their constituent cells undergo
massive apoptotic death. Should levels of circulating
androgens rise again, the remaining prostatic stem cells
proliferate and reconstruct the gland. A similar cycle of
growth and involution is seen in the mammary gland of
female mammals, which exhibits substantial differences
in size and cellular composition in the lactating and
nonlactating states. Interference with survival signaling
by sex hormones is one important strategy that is com-
monly used in the treatment of breast and prostate cancer.
Programmed cell death also eliminates certain popula-
tions of cells that never serve any function. The müllerian
ducts develop into the female oviduct. Male embryos
also develop progenitors of these ducts, which serve no
purpose and are eliminated by apoptosis.
Cells With Perturbed Cell Cycles
Chapters 40 to 43 describe how biochemical circuits
called checkpoints regulate the cell cycle. Cells respond
to DNA damage by blocking cell-cycle progression while
attempting to repair the DNA. The p53 transcription
factor, an important downstream checkpoint EFFECTOR
induces the expression of genes encoding proteins that
arrest the cell cycle. p53 also turns on genes encoding
proteins that induce cell death. It is generally thought
that if the damage cannot be repaired quickly, the pro-
death factors win out, and the outcome is apoptosis.
Double-strand DNA breaks induced by ionizing radiation
and DNA damage induced by chemotherapeutic agents
frequently result in cell death rather than repair.
A second important cell-cycle checkpoint regulates
the transition from the G1 phase to S phase. Passing the
restriction point (see Fig. 41.8) represents the commit-
ment of the cell to undergo another cycle of DNA repli-
cation and division. Restriction point control centers on
the regulation of the E2F family of transcription factors.
E2F regulates genes that promote cell-cycle progression.
E2F also regulates the expression of genes that promote
apoptosis. If activated too strongly, E2F can initiate
apoptosis as, for example, after DNA damage (see Fig.
41.14) or when restriction point control has broken
down. Cells that die in response to inappropriate
signals to proliferate include those infected by certain
viruses or overexpressing genes that drive cell prolifera-
tion (such as c-myc and c-fos [see Fig. 46.15]). Apoptotic
death of cells with an inappropriate stimulus to prolifer-
ate is an important defense against cancer.
Virus-Infected Cells
Cytotoxic T lymphocytes eliminate virus-infected cells
by causing them to undergo programmed cell death
either by apoptosis or by a second related pathway. For
example, programmed cell death accounts for at least
part of the loss of mature CD4+ T-helper cells (see Fig.
28.9) in people infected with HIV-1. When exposed to

agents that normally stimulate cell proliferation, these
cells instead undergo apoptosis. Paradoxically, it appears
that many of the dying cells are not themselves infected
with HIV.
Chemotherapeutic Killing of Cells
Exposure of cancer cells to many of the agents used in
chemotherapy does not kill the cells outright. Instead,
they die because the drugs cause intracellular damage
that acts as a signal for the induction of apoptotic cell
death. Thus, the treated cancer cells kill themselves.
Genetic Analysis of Apoptosis
Several key components that are involved in the apop-
totic execution of mammalian cells were discovered by
genetic analysis of the nematode worm Caenorhabditis
elegans. Because C. elegans is optically clear, it is pos-
sible to see every cell in a developing worm using dif-
ferential interference contrast optics (see Fig. 6.2). This
enabled investigators to trace the lineage of every cell in
an adult worm back to the fertilized egg. These studies
led to the surprising discovery that programmed cell
death is one of the most common fates for newborn C.
elegans cells. Of the 1090 somatic cells that are produced
during embryogenesis of the C. elegans hermaphrodite,
131 undergo programmed cell death at reproducible
locations and times.
Mutations in at least 14 C. elegans genes affect pro-
grammed cell death (Fig. 46.10). These are divided into
three classes: (a) genes that mark cells for subsequent
programmed death; (b) genes that are involved in cell
killing and its regulation; and (c) genes that are involved
egl-1 Determination
ced-9 ced-3
ced-4 Killing
Dead cell
ced-1, -6, -7
ced-2, -5, -10, -12 Engulfment
nuc-1 Degradation
Mammalian homologs
ced-9 protein = Bcl-2 family (antiapoptotic)
egl-1 protein = Bcl-2 family (BH3-only proapoptotic)
ced-4 protein = Apaf-1
ced-3 protein = initiator caspases
FIGURE 46.10  GENETIC DISSECTION OF PROGRAMMED
CELL DEATH. The ced (cell death abnormal) mutants of the nematode
worm Caenorhabditis elegans affect the killing, engulfment, and deg-
radation stages of programmed cell death.
CHAPTER 46  n  Programmed Cell Death 803
in the phagocytosis and subsequent processing of the
cell corpses. These mutants are collectively known as
“cell death abnormal” (ced) mutants. Interestingly, this
repertoire of nematode genes is vastly simplified from
the apoptosis genes in the last eumetazoan com­mon
ancestor (see Fig. 2.8). By contrast, humans have
approximately 300 genes involved in apoptosis.
The three best-known worm cell death genes are ced-
3, ced-4, and ced-9. If either ced-3 or ced-4 is inactivated,
all cells throughout the organism that should die by
apoptosis are reprieved. These cells remain alive and are
apparently functional. Interestingly, these worms have
normal life spans. This suggests that programmed cell
death is not involved in the normal aging process, at least
not in C. elegans. Ced-9 negatively regulates ced-3 and
ced-4. In worms with ced-9 loss-of-function mutations
many cells die that normally stay alive. This is deleterious
for the organism, so ced-9 mutants die. The key gene
triggering apoptosis is egl-1. Its regulation appears to
govern apoptosis in the worm. All these genes have
mammalian counterparts (discussed more fully later).
Ced-3 is a member of a specialized family of cell
death proteases called caspases. Ced-4 is a scaffolding/
adapter protein that plays an essential role in the activa-
tion of Ced-3 from its zymogen precursor. The mamma-
lian counterpart of Ced-4 is apoptotic protease-activating
factor-1 (Apaf-1). Ced-9 is a member of the Bcl-2 family
of cell death regulators (Fig. 46.14). Some Bcl-2 family
members protect against cell death, but others actively
promote cell death. Egl-1 is a BH3-only proapoptotic
regulator.
Phagocytosis turned out to be surprisingly complex,
with eight C. elegans genes encoding proteins that
are involved in the engulfment and processing of cell
corpses. Several are signaling proteins that reorganize
the cytoskeleton to permit the cell to move toward and
engulf its target. Another, the nuc-1 gene, encodes one of
several nucleases that digest the DNA of the dead cell in
lysosomes of cells that ingest the corpse. In mammals, this
digestion typically is initiated within the dying cell itself.
Signals and Pathways of Apoptosis
Two principal pathways lead to cell death by apoptosis.
These are introduced only briefly here and expanded
upon later. The intrinsic pathway (see Fig. 46.17) is
activated by internal surveillance mechanisms or signals
sent (or not sent) by other cells. Signals that induce this
pathway include DNA damage, exposure to chemicals
that interfere with a variety of cellular pathways and
excessive activation of factors that promote cell-cycle
progression. Withdrawal of nutrients or survival signals
from the environment also activates the intrinsic
pathway. Those survival signals include lymphokines,
such as interleukin-2 and interleukin-3, which are essen-
tial for survival of thymocytes; nerve growth factor,
which is required for survival of many neurons; and
804 SECTION X  n  Cell Cycle
extracellular matrix, which is required for survival of
epithelial cells. Signals that activate the intrinsic pathway
converge on mitochondria, which release key factors
that drive the apoptotic response.
Signals from other cells are the primary triggers of the
extrinsic pathway (see Fig. 46.18). Direct contact of a
killer cell with the target cell activates specific receptors
that initiate this pathway, starting at the plasma mem-
brane. Activation of the extrinsic pathway is one strategy
that cytotoxic T lymphocytes use to kill cells that are
recognized as foreign (or as harboring foreign patho-
gens). This pathway is also widely used to control cell
populations in the immune system. In many cells, the
extrinsic pathway activates the intrinsic pathway, which
is then responsible for killing the cell.
Protein Regulators and Effectors of Apoptosis
Because the penalty for misregulation of apoptosis is
death, it is not surprising that the process is carefully
regulated. This is essential for cells but complicates
matters for students. This section first lays out the
over­all strategy in generic terms and then fills in some
important details.
A cascade of proteases called caspases drives apop-
tosis. Each caspase is harmless until activated (usually by
dimerization for initiator caspases and proteolytic cleav-
age for effector caspases). Activation of a small number
of initiator caspases triggers a cascade that amplifies the
response and results in activation of numerous effector
caspases. The ability of effector caspases to activate
further initiators and effectors further amplifies the
response.
This strategy of employing amplification and positive
feedback has two powerful advantages. First, it can
provide a very rapid change in the state of the cytoplasm,
from pro-life to pro-death within seconds. Second, the
relatively small number of initiator caspases that trigger
the cascade constitutes a feasible target for negative regu-
lators that can rapidly quell responses that are initiated
under borderline conditions or by mistake. This is benefi-
cial but also complicates the overall system. If initiator
caspases start apoptosis and are then inactivated by
suppressers, how does the response ever take hold? The
answer is at least one more level of regulation: inhibitors
of the inhibitors. This additional layer of regulation is
thought to enable the rapid burst of caspase activation
that triggers the onset of apoptotic execution.
The following sections discuss the workhorses of
apoptosis—the caspases—followed by regulation of the
response.
Caspases
Caspases (cysteine aspartases) are specialized proteases
with a cysteine in their active site that cleave their targets
on the C-terminal side of aspartate residues. Caspases
generally inactivate cellular survival pathways and
specifically activate other factors that promote cell death.
In certain specialized instances caspases can participate
in pathways leading to activation of nuclear factor κB
(NF-κB; see Figs. 10.21 and 27.8) and the immune
response and also in terminal differentiation. These
nonlethal roles of caspases are not discussed further here.
C. elegans has three caspases, one of which (Ced-3)
is essential for cell death. In contrast, mammals have as
many as 17 caspase-related genes (Fig. 46.11A). Analysis
based on sequence comparisons divides caspases into
two major subfamilies. The caspase 1 subfamily encodes
enzymes that process pro–interleukin-1β to yield mature
interleukin-1β and pro–interleukin-18 to yield mature
interleukin-18. Macrophages secrete these cytokines,
which cause fever and inflammation. The second caspase
3 subfamily of enzymes participates almost exclusively
in apoptotic cell death.
Like many proteases, caspases are synthesized as
inactive zymogens, known as procaspases. All living
vertebrate cells synthesize procaspases constitutively.
Procaspases typically consist of three domains: an N-
terminal prodomain followed by the large and small
subunits of the mature enzyme (Fig. 46.11A–B). Aspar-
tate residues between these three domains are targets
for cleavage by caspases. Procaspases are activated either
by multimerization or by cleavage and release of the
prodomains. Following procaspase cleavage, the two
large and two small subunits associate in a compact,
block-like heterotetrameric molecule (Fig. 46.11B–D).
Depending on the enzyme, multimerization or cleavage
of the procaspase permits a major conformational change
in the polypeptide, creating two stable active site pockets
between the large and small subunits.
Two classes of caspases are involved in cell death.
Initiator caspases have long prodomains (Fig. 46.11A).
They exist as monomers in cells and become autoactivated
when scaffolding cofactors promote their aggregation.
Activation is induced by dimerization and does not require
proteolytic cleavage (although cleavage typically follows
activation). Sequences within the extended prodomains
are involved in targeting the initiator procaspases to the
appropriate cellular locations and in interactions with
scaffolding factors.
Effector caspase zymogens are monomers with
short prodomains. These inactive enzymes are typically
incapable of autoactivation. Instead, they are activated
through cleavage by initiator caspases or by other active
effector caspases.
Scaffolding proteins and adapters play an essential
role in activating initiator caspases by forming multi-
protein activation platforms. For the intrinsic pathway
of cell death, factors released from mitochondria (dis-
cussed later) activate the scaffold protein Apaf-1, which
is related to AAA ATPases (adenosine triphosphatases)
(see Box 36.1). Active Apaf-1 forms a ring-like structure
with seven spokes called the apoptosome (Fig. 46.17).
 CHAPTER 46  n  Programmed Cell Death 805

~p20 ~p10
A B. Caspase maturation
Effector caspases 0 28 175 277
Casp-3 LS SS Procaspases (zymogens) Active enzyme
303 Pro
Casp-7 LS SS Prodomain
DED DED LS SS
293
Casp-6 LS SS Caspase 8 (an initiator caspase)

Initiator caspases 479

Casp-8 DED DED LS SS LS SS

521 Caspase 3 (an effector caspase)

Casp-10 DED DED LS SS
416
Casp-9 Card LS SS
435 C. Caspase 1 D. Caspase 3
Casp-2 Card LS SS

Proinflammatory caspases 377

Casp-4 Card LS SS
418
Casp-5 Card LS SS
373
Casp-11 Card
419
Casp-12 Card
404
Casp-1 (ICE) Card LS SS
242
Casp-14

FIGURE 46.11  INTRODUCTION TO CASPASES. A, The 13 enzymatically active mammalian caspases fall into three groups. Where it has
been determined, the portions of the zymogens that give rise to the large (blue) and small (yellow) subunits are shown. B, Initiator caspases have
large prodomains that participate in subcellular targeting. Two initiator procaspases come together to form the active enzyme. C–D, Ribbon
diagrams of crystal structures of caspases 1 and 3. The catalytic residues come primarily from the large subunit (blue). The space-filling structure
(red) represents a peptide inhibitor covalently bound in the active site of the enzyme.

Binding of procaspase 9 zymogen to this platform pro-
motes dimerization and activation of the enzyme.
The scaffold proteins for the extrinsic death pathway
are cytoplasmic domains of cell-surface receptors. When
these receptors bind their ligands on the surface of other
cells, they form stable trimeric complexes that recruit
adapter proteins to form a signaling complex called the
“death-inducing signaling complex (DISC).” Interac-
tions involving multiple protein–protein interaction
motifs link the procaspase 8 zymogen to the DISC (Box
46.2), resulting in dimerization, activation, self-cleavage,
and release of active caspase 8.
Caspases cleave as many as 1000 cellular proteins
(Fig. 46.12). Some targets are structural proteins, but
many are involved in cellular signaling. For example,
caspases cleave several protein kinases. Many kinases
have pseudosubstrate motifs that autoinhibit the kinase
and can be moved out of the way in response to physi-
ological stimuli (see Fig. 25.4). Caspase cleavage often
neatly clips off these regulatory domains, thereby pro-
ducing constitutively active enzymes. These unregulated
kinases may alter signaling pathways to promote cell
death. Caspases also cleave and inactivate several pro-
teins that normally function in the detection and repair
of DNA damage.
Caspases can also create factors that directly promote
cell death. The most obvious example is caspase activa-
tion of other caspases in the death cascade. Caspases
also act indirectly to cause mitochondria to release
factors that promote cell death through the intrinsic
pathway. Caspase cleavage of an inhibitory chaperone is
responsible for activation of the nuclease that ultimately
destroys the chromosomal DNA of most cells undergoing
apoptosis (see section on CAD nuclease).
Natural Caspase Inhibitors
Because most healthy cells express initiator procaspases
with the potential to oligomerize and kill the cell, it is
important to have a mechanism that dampens any poten-
tial “noise” in the pro-apoptotic pathway. The inhibitor
of apoptosis protein (IAP) family is defined by the pres-
ence of a motif of approximately 80 amino acids known
as a baculovirus IAP repeat (BIR) domain. This is a
type of Zn2+ finger (see Fig. 10.14) that mediates protein–
protein interactions. IAP proteins inhibit caspases in two
ways. First, they bind the caspase and invade the active
site, thereby blocking its access to substrates. Second,
several IAPs are E3 ubiquitin ligases (see Fig. 23.3) that
ubiquitylate caspases and tag them for destruction by
proteasomes.
If IAP proteins inactivate caspases, then how is the
apoptotic response ever initiated? Cells also express
several molecules that can bind and inactivate IAPs. The
best characterized of these, is the second mitochondrial
activator of caspases (Smac or DIABLO). Smac is nor-
mally sequestered in mitochondria, but is released into
806 SECTION X  n  Cell Cycle

BOX 46.2  Matchmaking for Cell Death: the Key Is in the Domains

There are so many different proteins involved in apoptosis
that even experts have a difficult time keeping them all
straight. However, an understanding of the general principles
is much simplified if we recognize that most proteins
involved in apoptosis regulation are built from a relatively
limited number of modules, which act as sites for protein–
protein interactions. The following are the most important
modules for apoptosis.
Bcl-2 family members are defined by the presence of
short regions of conserved sequence, referred to as BH
domains (Bcl-2 homology). One of these, the approxi-
mately 20-residue BH3 domain, found in all Bcl-2 family
members, promotes complex formation between Bcl-2
family members by docking into a so-called BH3 binding
groove.
Four domains regulate caspase targeting and activation.
Although the amino acid sequences of these domains have
diverged extensively, all are regions of approximately 80 to
90 residues that form a characteristic bundle of six α-helices.
These domains all were present in the last eumetazoan
common ancestor.
1. The death domain (DD) is found in many proteins that
are involved in signaling pathways related to cell death.
These include cell death receptors, such as Fas (and
others), and adapter molecules, such as FADD (Fas-
associated death domain).
2. The death effector domain (DED) is found in adapters
such as FADD, the prodomains of caspases 8 and 10, and
certain inhibitors of apoptosis.
3. The Pyrin domain (PYD) is found in proteins involved
in inflammation (such as microbial sensors) and the
activation of caspase 1.
4. The caspase recruitment domain (CARD) is found in
several adapter proteins involved in cell death, including
CED-4 and Apaf-1, and in caspases 1, 2, and 9.
The domains have similar folds, but different distributions
of surface charge. As a result, they prefer to interact with
themselves (ie, DD–DD, DED–DED, PYD–PYD, and CARD–
CARD). Such interactions are said to be homophilic. When
a new apoptosis effector protein is identified, it is possible
to predict from an analysis of its amino acid sequence which
of the known proteins it is most likely to interact with.

A. Inactivation of protective factors and activation B. Pathway of nuclear apoptosis CYTOPLASM

of proapoptotic factors by caspases
Initiator Effector Cytoplasmic
caspases caspases targets
(8, 9, etc.) (3, 6, 7, etc.)
p53 Focal
pRb Surface Ras
mdm2 receptors contact RasGAP
Raf FAK Paxillin
Actin
Cdk/cyclins Gas2
active MEK PP2A Cb1/1b CAD / Caspase-6
p27 ICAD PARP
p21 Wee1 Lamins
APC PI3K
A A
Cdk/cyclin ERK ICAD/ I CA D CAD P RP L M I NS
inactive NF-κB DFF45 Akt Domain NUCLEUS
STAT1 nuclease
CAD Death Bad
Cell-cycle ? Bcl-2 Fragmentation
arrest ? nuclease
PKCθ Bcl-XL
Survival PKCδ JNK-p38 Bid

MEKK1 PAK2
Mst1/Krs

FIGURE 46.12  SOME WAYS THAT CASPASES PROMOTE CELL DEATH. A, A few of the many proteins cleaved by caspases in apoptotic
cell death. Proteins shown in green normally have a role in keeping the cell alive and are inactivated by caspases. Proteins shown in red are
turned into active death-promoting factors as a result of caspase cleavage. Proteins shown in black are not cleaved and are included to show
the pathways that are affected by cleavage. Caspases inactivate a number of pathways that promote cell survival, thereby strongly reinforcing
the decision of the cell to die. B, Some of the roles of caspases in disassembly of the nucleus. CAD, caspase-activated deoxyribonuclease; ERK,
extracellular signal-regulated kinase; ICAD, inhibitor of caspase-activated deoxyribonuclease; JNK, c-Jun N-terminal kinase; MEK, mitogen
activated protein/extracellular signal-related kinase kinase; NF-κB, nuclear factor κB; PI3K, phosphatidylinositol 3′-kinase; PKC, protein kinase
C; STAT, signal transducer and activator of transcription.

the cytoplasm when the intrinsic pathway of apoptosis
is initiated as a result of permeabilization of the mito-
chondrial outer membrane. It then binds to the BIR
domain, inactivating the IAPs. Mathematical modeling
shows that inactivation of IAPs by Smac is needed to
make the proapoptotic signal act like a switch rather
than a graded response.
IAPs were discovered in studies of the mechanisms
viruses use to avoid being eliminated by cell death.
When viruses infect cells and disassemble their capsids,
they become vulnerable to suicide defense mechanisms:
If cells can kill themselves before the virus has time to
complete its life cycle, they will take the virus with them,
and the organism will survive. To defend against this,
 CHAPTER 46  n  Programmed Cell Death 807

viruses pilfer genes encoding cellular proteins and adapt
them for their own means. For example, insect baculo-
viruses make two proteins that inhibit apoptosis, keeping
the cell alive long enough for the virus to reproduce.
One of these, IAP, was derived from a cellular gene. The
origin of the second IAP inhibitor, p35, is less clear. p35
is a broad-spectrum caspase inhibitor that is thought to
work by a serpin-like mechanism. Serpins are special
protease substrates that, after cleavage, form a tight
complex with the enzyme and inactivate it. Several
mammalian pox viruses also make a serpin-like inhibitor
of certain caspases called CrmA.
CAD Nuclease and Its Chaperone ICAD
The chromosomal DNA is destroyed during apoptosis.
The nucleases involved in cleaving the cellular DNA
during (and after) apoptotic cell death fall into two
classes. Cell autonomous nucleases degrade the DNA
within the dying cell (Fig. 46.13A). The best known is

A. DNA cleavage during apoptosis B. Active CAD nuclease

DNA

Proapoptotic Execution Engulfment Clearance

stimulus
Large Nucleosomal Further digestion
fragments fragments within phagocytic cell

Cell autonomous Waste management

nucleases nucleases

Necrosis Lysis Extracellular digestion

C. ICAD regulates CAD function

D. Drug-induced DNA E. Effect of purified CAD on
Apoptotic stimuli cleavage in vivo isolated nuclei in vitro
Caspase activation
23
CAD / Inactive 9.4
ICAD ICAD 6.6

Active
CAD 2.0
ICAD Dimerized CAD
protein cleaves DNA

0.56
CAD mRNA
CYTOPLASM NUCLEUS 3 ICAD/CAD
0 1 2 3 4 6h 1 2 3 4 + Casp-3

FIGURE 46.13  NUCLEASES DIGEST THE CELLULAR DNA DURING APOPTOSIS. A, In apoptosis, the DNA is digested first to large
fragments and later to nucleosome-sized pieces (see Fig. 13.1) by cell autonomous nucleases expressed within the dying cell. Waste management
nucleases made by other cells also have an essential role in cleaning up apoptotic and necrotic debris. B, The predominant cell autonomous
nuclease (CAD) has a scissors-like structure. C, ICAD is an inhibitory chaperone for CAD, promoting its folding on the ribosome and continuing
as an inhibitor when CAD is stored in the nucleus. ICAD cleavage leads to CAD activation. D, Cleavage of the chromosomal DNA by CAD during
chemotherapy-induced apoptosis of a leukemia cell line. DNA separated according to size by electrophoresis on an agarose gel was stained with
ethidium bromide. The ladder of bands reflects cleavage between adjacent nucleosomes. E, Activated CAD causes chromatin condensation and
appearance of an apoptotic morphology in isolated cell nuclei. Cloned CAD and ICAD were expressed together in Escherichia coli and incubated
with nuclei. ICAD cleavage with caspase 3 released active CAD, which degrades the nuclear DNA (Lane 3). Other lanes: DNA gel size markers
(left); nuclei incubated with buffer or caspase 3 alone (Lanes 1 and 2, respectively); same experiment as in Lane 3 but performed by using a
mutant ICAD that could not be cleaved by caspase 3 (Lane 4). To the right is an electron micrograph of a thin section of one nucleus with
condensed chromatin at the nuclear periphery. CAD, caspase-activated deoxyribonuclease; ICAD, inhibitor of caspase-activated deoxyribonucle-
ase; mRNA, messenger RNA. (A, Modified from Samejima K, Earnshaw WC. Trashing the genome: The role of nucleases during apoptosis. Nat
Rev Mol Cell Biol. 2005;6:677–688. B, For more information, see Protein Data Bank [PDB; www.rcsb.org] file 1V0D from Woo EJ, Kim YG, Kim
MS, et al. Structural mechanism for inactivation and activation of CAD/DFF40 in the apoptotic pathway. Mol Cell. 2004;14:531–539. D, From
Kaufmann SH. Induction of endonucleolytic DNA cleavage in human acute myelogenous leukemia cells by etoposide, camptothecin, and other
cytotoxic anticancer drugs: a cautionary note. Cancer Res. 1989;49:5870–5878. E, Courtesy K. Samejima, Wellcome Trust Institute for Cell
Biology, University of Edinburgh, United Kingdom.)
808 SECTION X  n  Cell Cycle

the caspase-activated DNase (CAD; see later discussion). Bcl-2 family

A mitochondrial nuclease known as endonuclease G may
also degrade DNA in some cell types. Cell autonomous Protectors Killers Regulators
nucleases are dispensable for cell death and for the life Bcl-2 Bax Bad Bik / Nbk
Bcl-xL Bak Bid HRK
of the organism. They might have evolved to eliminate Bcl-W Bok / Mtd Bim Puma
viral DNA as part of the suicide defense response Mcl-1 Bcl-xS BmF Noxa
described in the previous section. A1 Hrk
Boo/Diva C. elegans Egl-1
Waste management nucleases clean up the debris C. elegans ced-9
after cells die. They either function within lysosomes of Adenovirus E1B19K
cells that have ingested apoptotic cell fragments or are Epstein-Barr virus BHRF1
secreted into the extracellular space. DNase II, one of
the most important waste management nucleases, is
essential for life. Mouse embryos that lack DNase II BH4 BH3 BH1 BH2 Protectors
become overwhelmed with undegraded DNA and die.
Cell autonomous nucleases act in two stages. After BH3 BH1 BH2 Killers
an initial cleavage of the chromosomes into fragments
of roughly 50,000 base pairs, DNA is usually cleaved BH3 Regulators
between nucleosomes, producing a characteristic
“ladder” of DNA fragments with a periodicity of approxi-
mately 200 base pairs. This ladder is seen when DNA FIGURE 46.14  THE BCL-2 FAMILY OF APOPTOTIC REGULA-
TORS. C. elegans, Caenorhabditis elegans.
isolated from apoptotic cells is subjected to gel electro-
phoresis. The responsible nuclease is CAD. CAD is nor-
mally present in a complex with ICAD (inhibitor of CAD normally survive into the adulthood. A Ced-9 mutation
[Fig. 46.13C]). The complex of CAD and ICAD is also kills the worm. Human Bcl-2 is functionally and structur-
known as DNA fragmentation factor (DFF). ICAD is a ally homologous to C. elegans Ced-9 and can substitute
chaperone required to fold CAD into an active conforma- for it in living worms. This ability of a human gene to
tion during translation on the ribosome. However, ICAD protect nematodes reveals that the fundamental machin-
also inhibits the nuclease activity of CAD. This dual func- ery of apoptotic cell death has been conserved over great
tion of ICAD guarantees that only inactive CAD can be evolutionary distances.
synthesized in healthy cells. During apoptosis, caspase 3 Bcl-2 family members are defined by the presence of
cleaves ICAD and releases active CAD nuclease. one to four short blocks of conserved protein sequence
called BH (Bcl-2 homology) domains. Antiapoptotic
Bcl-2 Proteins and the Intrinsic Pathway Bcl-2 protectors typically have four of the domains.
Proapoptotic Bcl-2 killers typically have three of these
of Apoptotic Cell Death
domains, while the Bcl-2 regulators have only the BH3
As mentioned previously, mitochondria are key players in domain. The BH3 domain is a short helix that fits into
a pathway to cell death that is triggered by a variety of a groove on the surface of both Bcl-2 protectors and
toxic insults (Fig. 46.17). The Bcl-2 family of proteins killers, forming complexes that regulate their activity. It is
regulates these mitochondrial events. The following sec- believed that the Bcl-2 protectors regulate the behavior of
tions describe this important protein family and the regu- Bcl-2 killers by a similar interaction. For example, the Bcl-2
lation of the intrinsic pathway of apoptosis. protein forms a complex with a proapoptotic Bcl-2 killer
called Bax, thereby interfering with the ability of Bax to
Bcl-2 Proteins kill cells. Binding of BH3-only proteins to Bcl-2 protectors
Bcl-2 proteins can be grouped into three subfamilies (Fig. can inactivate their antiapoptotic functions. Egl-1 is a
46.14). Bcl-2 protectors protect cells against apoptosis. BH3-only protein and this is how it triggers apoptosis. A
Bcl-2 killers (eg, Bax and Bak) are proapoptotic proteins new generation of BH3 mimetic drugs induces apoptosis
that actively kill cells. Bcl-2 regulators (widely known as of cancer cells by mimicking this second mechanism.
BH3-only proteins) promote cell killing by either interfer- Genetic experiments in mice revealed several differ-
ing with the protectors or activating the killers. Bcl-2 ent functions for Bcl-2 family members. Mice born
proteins primarily regulate the release of death-promoting without Bcl-2 have deficiencies of the immune system
factors from mitochondria when cells receive signals that are best understood if one role of this protein in vivo
that activate the intrinsic pathway. is to render lymphocytes resistant to proapoptotic signals
C. elegans genetics identified a gene, ced-9, that pro- during immune system maturation. Mice lacking another
tects cells against apoptosis and another gene, egl-1, pro-life family member, Bcl-xL, die during embryogenesis,
that inactivates ced-9 protein and triggers apoptosis. In apparently as a result of widespread death of neurons
ced-9 mutants, many cells die during development that in the central and peripheral nervous systems and

hematopoietic cells in the liver. In contrast, loss of the
killers Bax plus Bak makes cells highly resistant to apop-
tosis by a wide variety of intrinsic pathway stimuli.
Bcl-2 Family Members and Cancer
A gene that prevents cells from dying poses a potential
danger in multicellular organisms, in which rates of cell
proliferation and death must be balanced carefully. In
fact, the name Bcl-2 comes from the discovery that this
gene is the culprit responsible for certain types of B-cell
lymphoma. These particular lymphomas arise when a
chromosome translocation, involving chromosomes 14
and 18, moves the Bcl-2 gene into the immunoglobulin
heavy-chain gene cluster, a site of very active gene
transcription in B lymphocytes. Elevated transcription of
Bcl-2 is thought to be directly responsible for the cancer-
ous phenotype in these patients, making Bcl-2 a cancer-
promoting oncogene (see Chapter 41).
Unlike many other oncogenes, Bcl-2 overexpression
does not cause cell proliferation. Instead, it disrupts
the balance of regulation between life and death. Cells
that overexpress Bcl-2 protein actually grow somewhat
slower than normal but are highly resistant to many
stimuli that normally promote cell death. The net result
is an accumulation of B cells: a lymphoma. Other anti-
apoptotic Bcl-2 family members are amplified in a variety
of solid tumors and loss of microRNAs directed at Bcl-1
is thought to play a major role in development of chronic
lymphocytic leukemia. Fig. 46.15 shows an example of
Bcl-2 conferring resistance to death when the cell cycle
is perturbed by expression of an oncogene.
Intrinsic Pathway of Apoptotic Death
In addition to their role in energy production, mitochon-
dria have an essential role as sensors of the health of the
cell. If cells sense insults from which they cannot recover,
mitochondria trigger the intrinsic pathway of cell
death (Fig. 46.17). Bcl-2 family members regulate this
pathway. The regulation seems straightforward in C.
elegans, in which the protector CED-9 (Bcl-2–like) binds
to the CED-4 scaffolding protein (Apaf-1-like) and inter-
feres with its activation of the CED-3 caspase. Apoptosis
is induced when the regulator BH3-only protein EGL-1
binds to CED-9 and blocks it from inactivating CED-4.
In mammals, the situation is more complex, partly
because Bcl-2 family members are more numerous and
partly because they do not interact in such a straightfor-
ward fashion. In mammals, two of the killer proteins,
Bax and Bak, are essential for activation of the intrinsic
pathway. In healthy cells, Bak is loosely associated
with the mitochondrial outer membrane, and Bax is in
the cytoplasm (Fig. 46.17). On receipt of a proapoptotic
stimulus, Bax and Bak insert deeply into the mitochon-
drial outer membrane and form oligomeric membrane
CHAPTER 46  n  Programmed Cell Death 809
100
Cell viability (% control)
80
60 Normal levels of c-myc
40
Overexpressed c-myc
and overexpressed Bcl-2
20
Overexpressed c-myc
0
0 30 60 90 120
c-myc induction (min)
FIGURE 46.15  BCL-2 PROTECTS CELLS FROM ONCOGENE-
INDUCED CELL DEATH. Chinese hamster ovary cells die when they
are induced to express abnormally high levels of the c-myc protein,
but simultaneous expression of the Bcl-2 protein rescues them from
this effect. These cells contain many copies of the c-myc gene under
control of a promoter that is activated when the cells are briefly
exposed to high temperature (43°C for 90 minutes). The curves show
the percentage of viable cells remaining at various times following the
induction of c-myc expression. The Bcl-2 gene was introduced into
these cells on a plasmid under the control of a promoter that is always
active. The blue line represents the parental cell line lacking either the
cloned c-myc or Bcl-2 genes. (Note that approximately 40% of these
cells die following the heat treatment used to induce c-myc expres-
sion.) The yellow line shows that the cells that produce high levels of
c-myc protein alone rapidly die (by apoptosis). The red line shows that
cells expressing both the c-myc and Bcl-2 proteins survive the treat-
ment almost as well as the parental cells. (From Bissonnette RP,
Echeverri F, Mahboubi A, et al. Apoptotic cell death induced by c-myc
is inhibited by bcl-2. Nature. 1992;359:552–554.)
pores that allow the release of pro-apoptotic factors from
the mitochondrial intermembrane space. Binding of
antiapoptotic Bcl-2 family members to Bax/Bak somehow
prevents mitochondrial outer membrane permeabiliza-
tion. BH3-only family members can either directly
promote death by facilitating Bax/Bak oligomerization or
indirectly promote it by binding and neutralizing anti-
apoptotic Bcl-2 family members.
The proapoptotic factors released from the mitochon-
drial intermembrane space by Bax and Bak include the
electron transport protein cytochrome c (see Fig. 19.5),
Smac, and endonuclease G (Fig. 46.16). These mitochon-
drial proteins actively promote apoptotic cell death. In
the cytoplasm, cytochrome c binds to the scaffolding
protein Apaf-1, a mammalian homolog of C. elegans
CED-4 protein, causing it to undergo a conformational
change that exposes a hidden bound adenosine diphos-
phate (ADP) to solvent. Exchange of this ADP for deoxy-
adenosine triphosphate (dATP) allows Apaf-1 to form a
wheel-like structure with seven spokes called the apopto-
some (Fig. 46.17). Apaf-1 in the apoptosome binds caspase
9 through an N-terminal caspase recruitment domain.
Binding to the apoptosome elevates the catalytic
activity of procaspase 9 approximately 2000-fold without
the need for its cleavage. Thus, the active form of caspase
9 is an oligomeric complex of the procaspase with
the apoptosome. Activated caspase 9 then cleaves
810 SECTION X  n  Cell Cycle

multiple procaspase 3 zymogens, amplifying the cell

A. Life death cascade. It also cleaves itself, triggering its release
Cytochrome c
Smac, AIF from the apoptosome with a resulting loss of activity.
Intermembranous
space
Endonuclease G This autocleavage acts like a timer limiting apopto-
Others?
some activity.
Mitochondrial
outer membrane This cascade can be further amplified in at least two
permeabilization ways. First, caspase 3 cleaves other effector caspases,
directly amplifying the cascade. In addition, active cas-
B. Death pases cleave the BH3-only protein Bid, which then acti-
Bid vates more Bax and Bak in a feedback loop, thereby
Caspases
Bax promoting the release of more cytochrome c and Smac,
Other Bak
Cytochrome c
and enhancing caspase 9 activation.
Activation of signals
downstream Smac It was extremely surprising to find that an essential
caspases AIF metabolic protein such as cytochrome c has a second
Caspase 9 Endonuclease G function that is essential for death. Among the studies
activation
Other Others
supporting the Jekyll-and-Hyde–like nature of this protein
targets in life and death was the engineering of mice whose
To nucleus
Nuclear Other cytochrome c can function in electron transport but
disassembly targets cannot bind Apaf-1. These mice die as a result of brain
abnormalities caused by insufficient cell death.
FIGURE 46.16  MITOCHONDRIA INTEGRATE A CELL’S LIFE
AND DEATH DECISIONS. A, In healthy cells, a number of factors
that promote apoptosis are stored in the intermembrane space of the
mitochondria. B, In cells undergoing apoptosis, BH3-only proteins
Extrinsic Pathway of Apoptotic Death
trigger the proapoptotic Bcl-2 family members to induce the release Cells express at least six different cell surface molecules,
of these death-promoting factors; this initiates an amplifying cycle that
collectively termed death receptors, that can trigger
ultimately leads to cell death. AIF, apoptosis-inducing factor; Smac,
second mitochondrial activator of caspases. apoptotic death. Protein ligands on the surface of other

A. Life Metabolites up C. Apoptosome

to 10,000 D 3 (Apaf-1 scaffold)
9

Bak Activation of
Bid downstream
3 caspases
Autoactivation
VDAC
of caspase 9 (stays
3
6 bound to apoptosome)
MOMP
Caspases
cleave Bid
Bax B. Death
tBid 9
Proteins not to scale

Procaspase 9 9

Procaspase CARD domain

Apaf-1
CARD
domain Cytochrome c
dADP
Aggregation
Apaf-1 of Apaf-1 scaffold
* Conformational (apoptosome)
* *AIF
Smac, (ATP)
change in Apaf-1
endonuclease G (exchanges dADP for ATP) 27nm

FIGURE 46.17  INTRINSIC CELL DEATH PATHWAY. A–B, Following activation by the BH3-only protein Bid, Bax and Bak form a pore that
releases cytochrome c. Released cytochrome c binds to Apaf-1, inducing a conformational change leading to formation of the apoptosome,
which binds and activates procaspase 9 via scaffold-induced oligomerization. Activated caspase 9 subsequently activates downstream effector
caspases, leading to cell death. C, Molecular organization of the apoptosome. (C, Modified from PDB file 3JBT from Zhou M, Li Y, Hu Q, et al.
Atomic structure of the apoptosome: mechanism of cytochrome c- and dATP-mediated activation of Apaf-1. Genes Dev. 2015;29:2349–2361.)
 CHAPTER 46  n  Programmed Cell Death 811

cells bind and activate these receptors, turning on path-
ways that can lead to apoptotic death.
One well-characterized death receptor called Fas (also
known as Apo1 or CD95) is a member of the tumor
necrosis factor receptor family (see Fig. 24.9). Fas is a
type I membrane protein whose extracellular domain
consists of three cysteine-rich domains (Fig. 24.9 shows
the atomic structure of the related trimeric tumor necro-
sis factor receptor with bound ligand). The cytoplasmic
domain of Fas contains a death domain of approxi-
mately 80 residues, which is shared by all the death
receptors (see Box 46.2).
The Fas ligand is a trimeric 40-kD intrinsic membrane
protein found on the surface of cells. Cytotoxic T lym-
phocytes use Fas ligand to rid the body of virally infected
cells. When a cytotoxic T lymphocyte contacts a target
cell, the Fas ligand on the lymphocyte surface binds to
Fas on the target cell and initiates the extrinsic pathway
of apoptotic death (Fig. 46.18). Ligand binding activates
signaling from the intracellular death domain of Fas,
possibly by stabilizing Fas trimers or by altering their
conformation. Activated Fas binds an adapter protein
called FADD (Fas-associated protein with a death
domain), assembling the DISC. The DISC is an activation
platform that binds procaspase 8 through interactions
involving another type of motif called the death effec-
tor domain, which is present on both FADD and
the prodomain of procaspase 8. Procaspase 8 monomers
dimerize on the DISC and acquire catalytic activity.
These dimers can cleave neighboring dimers, creating
and releasing heterotetrameric active caspase 8, which
can initiate the caspase cascade by activating downstream
effector caspases. Frequently, caspase 8 cleaves the BH3-
only protein Bid, thereby activating the intrinsic death
pathway (Fig. 46.17).
The extrinsic pathway poses considerable risk for the
cell. Fas is constitutively present in the cell membrane
and can form at least transient trimers in the absence of
binding by its ligand. How do cells avoid the accidental
activation of apoptosis caused by chance binding of
procaspase 8 to naturally occurring transient Fas trimers?
Cells express a caspase-related protein called cFLIP
(FLICE-like inhibitory protein; FLICE is another name for
caspase 8) that looks very much like a catalytically dead

Unfolded FasR
binding domains

TARGET
FasR CD95 APO-1 CELL
8

8 8
A. Preligation
Monomers C. Release of active caspase 8
KILLER 8
CELL

Uncleaved caspase 8
Fas active on DISC
FADD
ligand Death domain 8

Death effector 8
Released active
domain caspase 8
Trimers DISC 8
(signaling
platform)
8

Procaspase 8

B. Ligand docked on a trimerized receptor Caspase 8

Molecular cleaves Bid Procaspases 3,6,7
scaffold forms 8 7
Bid 3 6

8
E. Activation of the D. Activation
intrinsic death of effector
8
pathway (see caspases 7
Clustering of Fig. 46.17) Active caspases 3,6,7
8
caspase 8
6 3
F. Death

FIGURE 46.18  EXTRINSIC CELL DEATH PATHWAY. The pathways shown are downstream of the Fas cell death receptor. A, Preligation.
B, Ligand docked on a trimerized receptor. C, Release of active caspase. D, Activation of effector caspases. E, Activation of the intrinsic death
pathway (Fig. 46.17). F. Death (see the text for a detailed description). DISC, death-inducing signaling complex; FADD, Fas-associated death
domain.
812 SECTION X  n  Cell Cycle
version of procaspase 8. When expressed at high levels,
cFLIP coassembles with procaspase 8 monomers creat-
ing an enzyme with altered activity that does not trigger
cell death. This role of cFLIP may be to dampen the Fas
response locally to ensure that the cascade does not get
activated by mistake. Another role of FLIP is to combine
with caspase 8 to ensure that TNFα (tumor necrosis
factor α) signaling, which occurs in many immune cells,
promotes inflammation rather than killing cells by
necroptosis (see later).
Role of the Fas Death Receptor in Normal
and Diseased Cells
Fas is important for regulation of the immune system,
but also has a very unexpected role in cancer cells. Mice
with mutated Fas (the lpr mutation) or Fas ligand (the
gld mutation) accumulate excessive lymphocytes. In the
appropriate genetic background, these mice tend to
develop autoimmune disorders.
Fas is important in regulating the life span of activated
tissue T and B lymphocytes. Normally, T cells die within
a few days of their activation during an immune response.
Activation initiates the expression of Fas ligand on the T
cells themselves. This new Fas ligand interacts by an
unknown mechanism with Fas already on the cell surface,
causing the cell to commit apoptotic suicide. T-cell
activation also downregulates the expression of cFLIP,
thus permitting activated caspase 8 to more effectively
kill the cell. A similar mechanism (export of Fas and
Fas ligand to the surface of the same cell) is responsible
for some examples of p53-induced cell death and some
instances of cell death following exposure to chemo-
therapeutic agents.
These features of Fas might seem to make this system
useful in the treatment of cancer. Unfortunately this is
not the case, because Fas does more than signal to
promote cell death. It can also signal to several pathways
to promote cell survival and migration. In fact, Fas signal-
ing in cancer cells can actually promote tumor growth
and metastasis. This serves as an example of the com-
plexity of cell signaling networks.
Some tissues, like the lens of the eye and the testis,
avoid immune and inflammatory responses by express-
ing Fas ligand. Immune effector cells (which express
Fas on their surface) that enter these tissues encounter
Fas ligand and die by apoptosis. These tissues are known
as immune-privileged. Not surprisingly, certain tumor
cells subvert this strategy as protection against the
immune system. Melanoma cells expressing Fas ligand
establish tumors particularly efficiently. Some tumor
cells, especially those from colon and lung cancers, also
defend themselves against immune surveillance with
so-called decoy receptors. A secreted Fas decoy
receptor blocks Fas ligand on cytotoxic T cells so that
it cannot engage Fas on the surface of the tumor
cells. Other decoy receptors remain membrane bound
but do not signal cell death when they bind ligand
because their intracellular domains lack functional death
domains.
Linking Apoptosis to the Cell Cycle
by p53
No obligate link exists between particular cell-cycle
phases and apoptosis. Noncycling G0 cells can undergo
apoptosis, and cycling cells appear able to do so from
any cell-cycle phase. However, one link between apop-
tosis and the cell-cycle machinery is firmly established.
This involves the p53 tumor suppresser and DNA
damage.
The p53 transcription factor is one of the downstream
EFFECTORS of the DNA damage response pathway (see
Fig. 43.11). When cells sense DNA damage induced by
agents such as ionizing radiation, p53 levels rise dramati-
cally. When stabilized and activated by phosphorylation
(see Fig. 41.14) p53 upregulates the expression of a
number of genes, including the Cdk inhibitor p21, which
blocks the entry into the S and M phases. p53 also can
trigger an apoptotic response in instances in which the
DNA damage is too severe to repair (Fig. 46.19). This
tumor-suppressor protein is critical in the body’s defense
against cancer. Mutations in the p53 gene/protein are
found in approximately 50% of all human cancers.
A direct connection between p53 and apoptosis
was revealed by overexpressing the cloned p53 gene
in different cell types. In most cells, overexpression of
p53 arrests the cell cycle at the G1/S boundary.
However, ectopic expression of cloned p53 in certain
cancer-derived cell lines causes the cells to undergo
apoptosis.
The role of p53 in apoptosis was confirmed in trans-
genic mice lacking a functional p53 gene (p53 knockout
mice). These mice develop normally but are extremely
prone to cancer at a very young age. Thymocytes isolated
from p53 knockout mice are highly resistant to the
induction of apoptosis by ionizing radiation and other
agents that cause DNA breaks (Fig. 46.19B). However,
p53 is not involved in all types of apoptosis. For example,
the same thymocytes isolated from p53 knockout mice
show normal induction of apoptosis following exposure
to glucocorticoid hormone (Fig. 46.19).
p53 promotes apoptosis by functioning as a transcrip-
tional activator. It controls, among others, the well-
studied death-promoting genes Bax, Fas (CD95/APO-1),
and APAF-1. However, the key target gene is PUMA (p53
modulated upregulator of apoptosis), a BH3-only protein
that promotes apoptotic cell death by activating Bax and
Bak. PUMA knockout mice show defects in cell death
pathways that are essentially identical to those seen in
p53 knockout mice and not seen in mice that lack Bax,
Fas, or Apaf-1.

A. Glucocorticoid
hormone–treated
100
Cell viability (%)
Cell viability (%)
80
60
40
20
0 0
0 5 10 15 20 25
Time (h)
C. Control
FIGURE 46.19  LINK BETWEEN p53 AND DNA DAMAGE-
INDUCED APOPTOTIC DEATH. A–B, Survival of thymocytes from
three strains of mice after exposure to glucocorticoids or irradiation.
Cell death was from apoptosis. The strains were as follows: wild-type
mice (yellow), heterozygous mice having one good copy of the p53
gene and one defective copy (red), and mice lacking any functional
copy of the p53 gene (blue). Thymocytes that lack p53 are resistant
to radiation-induced apoptosis but show normal induction of apopto-
sis following exposure to glucocorticoid hormone. C–D, Induction of
p53 accumulation following radiation of the small intestine. Black
arrows indicate cells with increased levels of p53. Red arrows indicate
apoptotic cells. (A–B, From Lowe SW, Schmitt EM, Smith SW, et al.
p53 is required for radiation-induced apoptosis in mouse thymocytes.
Nature. 1993;362:847–849. C–D, Courtesy John Hickman, Molecular
and Cellular Pharmacology Group, University of Manchester, United
Kingdom.)
Other Types of Programmed Cell Death
The terms apoptosis and programmed cell death are
sometimes viewed as synonymous. However, a number
of specific examples of programmed cell death have
been described that lack the features that classically
define apoptosis.
Inflammatory Cell Death: Pyroptosis
and Necroptosis
One of the defining features of apoptosis is that dying
cells disappear without causing an inflammatory
response. However, in certain instances, it is protective
for the body to have cell death be accompanied by
CHAPTER 46  n  Programmed Cell Death 813
B. Irradiated inflammation as this can trigger an immune response in
the early phases of infection.
p53 Pyroptosis (proinflammatory programmed cell
100 –/– death) occurs when signaling by a variety of cytoplasmic
80
receptors in response to pathogen infection leads to the
60 formation of the inflammasome, a cytosolic complex
40
p53 that contains numerous caspase 1 activation sites. Pyrin
+/–
20 +/+
domains (PYDs) in these receptors are linked to caspase
recruitment domains (CARDs), which in turn recruit
0 5 10 15 20 25 caspases 1, 4, and 5 (1 and 11 in mice). Despite being
Time (h) the first caspase to be described, caspase 1 was the last
to be shown to be involved in a programmed cell death
D. Irradiated response.
The function of caspase 1 is to produce interleukin-1β
(IL-1β) and interleukin-18 (IL-18) by proteolytic process-
ing of their precursors. These proteins are secreted
together with caspase 1 by an unconventional pathway
that is still being investigated and may involve cell lysis.
IL-1β is a proinflammatory cytokine that triggers the
fever response by acting on the hypothalamus. It also
promotes the proliferation of immune cells, among other
activities. IL-18 promotes increased immune cell recruit-
ment and activation, as well as modifying local cells to
facilitate adaptive immune responses. This proinflamma-
tory signaling facilitates a ramping up of the immune
response to the pathogen.
How pyroptosis leads to cell death is still being inves-
tigated, but the morphology of the dying cells resembles
that seen in necrosis. As a result, the internal components
of the cell are released into the extracellular space,
provoking a strong inflammatory response (as in necro-
sis, above, and necroptosis, below).
Necroptosis (programmed necrosis) is a cell death
pathway that may function as a backup when the extrinsic
pathway of apoptosis is not functioning. Activation of the
CD95 (Fas) and TNFR1 (tumor necrosis factor receptor
1) cell-surface receptors can have three possible out-
comes. TNFR1 can signal via complex I (which contains
protective IAP proteins) to activate the canonical NF-κB
pathway to promote cell survival (Fig. 46.1). Alternatively
TNFR1 can activate receptor interacting protein kinases 1
and 3 (RIPK1/RIPK3) and signal to form complex II (con-
taining FADD, TRADD [tumor necrosis factor receptor-
associated death domain], caspase 8, and cFLIP). When
caspase 8 is associated with cFLIP, it inactivates RIPK3
and cells survive. Complex II activates necroptosis if
either caspase 8 or cFLIP is missing or inactivated and
if RIPK1/RIPK3 are active. RIPK1/RIPK3 activity leads
to permeabilization of the cell membrane, cell lysis and
death. As in other forms of necrosis, the released cellular
contents cause a local inflammatory response.
Necroptosis is thought to be a backup pathway for
apoptosis during viral infection. Mouse kidney cells
infected with a virus that encodes a caspase 8 inhibitor
proceed down the necroptosis death pathway. Removal
of the gene from the virus restores apoptosis. Mice
814 SECTION X  n  Cell Cycle

lacking RIPK3 show increased susceptibility to viral

infection. Murine cytomegalovirus has proteins that help
it to evade both apoptosis and necroptosis, supporting Primary
lesion
this argument.

Autophagic Death
Autophagy is a catabolic, energy producing process that
allows cells to survive in adverse conditions by recycling
amino acids liberated by degradation of cellular proteins
and organelles (see Fig. 23.7). Autophagy can be acti-
vated by a wide range of stimuli, including nutrient and
oxidative stress, accumulation of unfolded proteins,
intracellular bacterial infection, and oncogenic stress. In
addition to being a response to starvation, autophagy can
also participate in antiviral responses by participating in
antigen presentation by immune cells.
Connections between autophagy and cell death are
complex and debated. In certain cases during develop-
ment, autophagic pathways can lead directly to cell
death. These dying cells lack chromatin condensation
and exhibit extreme vacuolization of the cytoplasm. In
some cells, autophagy can regulate apoptosis. For
example, degradation of proapoptotic BH3-only proteins
by autophagy provides a mechanism protecting cells
against apoptosis. Alternatively, degradation of protec-
tive proteins can cause autophagy to actively promote
apoptotic cell death.
Connections between autophagy and cancer are
complex. It has been argued that by promoting cell
survival under adverse conditions, autophagy might help
promote the establishment of cancers particularly in
avascular areas and by helping cancer cells to survive
chemotherapy. However, mice heterozygous for Beclin1
(a scaffolding protein that functions early in assembly of
the phagophore membrane; see Fig. 23.7) develop
cancers, suggesting that Beclin-1 is a tumor suppressor
and that autophagy may protect against tumorigenesis.
Interestingly, Beclin-1 has a BH3 domain, and although
it is not a canonical BH3-only proapoptotic protein, its
function is negatively regulated by Bcl-2. Also, autophagy
is activated in oncogene-induced senescence and inhibit-
ing autophagy delays senescence. Thus autophagy might
prevent cancer formation by helping damaged cells to
become senescent.
Importance of Programmed Cell Death in
Human Disease
Why have studies of programmed cell death so caught
the scientific eye? One likely answer is that cell death is
a point of intersection between cell signaling pathways,
cell structure, the cell cycle, and, of course, human
disease. This chapter has mentioned the roles that aber-
rations in programmed cell death play in the etiology
of autoimmunity, AIDS, and cancer. Cell death is firmly
established as a key factor in neurodegenerative diseases,
Penumbra: zone of apoptotic Primary focus
death starting within 24 hours of necrotic death
of the initial lesion
FIGURE 46.20  BRAIN DAMAGE IN STROKE. Secondary pro-
grammed cell death caused by oxygen deprivation in the penumbra
greatly increases the size of the affected area of the brain in stroke.
such as Huntington disease and Alzheimer disease, as
well as in myocardial infarction and stroke (Fig. 46.20).
At a practical level, the realization that many successful
chemotherapeutic agents act by inducing cancer cells
to undergo apoptosis motivated searches for newer
and better drugs that elicit this response. The generally
disappointing outcome of those studies may largely be
because other pathways (eg, necroptosis) kick in when
apoptosis is inhibited. Alternatively, as more is learned
about mechanisms of necroptosis and pyroptosis, these
alternative types of programmed death become attractive
therapeutic targets, as their induction may not only kill
the target cells, but also provoke a localized immune
response that might attack cancer cells or pathogens.
With such important practical problems to be solved, pro-
grammed cell death will continue to occupy a prominent
position in cell biology research over the coming years.
ACKNOWLEDGMENTS
We thank Charles Earnshaw, Scott Kaufmann, Luis
Miguel Martins, and Andrew Thorburn for their sugges-
tions on revisions to this chapter.
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Czabotar PE, Lessene G, Strasser A, Adams JM. Control of apoptosis by
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