CHAPTER

The Eukaryotic
Cell Cycle

A two-cell C. elegans embryo stained with ant ibodies against tubulin

(red) and CeBUB-1, a spindle checkpoint protein (green). DNA is stained
with DAPI (blue). CeBUB-1 is localized on the chromosomes and
kinetochore-attached spindle microtubules during metaphase in the
smaller, posterior cell (right). It is presumed to monitor chromosome
attachment and tension. The larger, anterior cell (left) has already entered
anaphase, and CeBUB-1 is no longer detectable on the chromosomes or
spindle microtubules. Thus asynchrony of this second cell cycle in the C.
e/egans embryo allows the observation of both the presence of a
functional spindle checkpoint protein during metaphase and its absence
after initiation of anaphase. [Encanada et al., 2005, Mol. Bioi. Ce/1 16:1 056.]

roper control of cell division is vital to all organisms. In molecular mechanisms regulating eukaryotic cell division dis-

P unicellular organisms, cell division must be balanced

with cell growth so that cell size is properly maintained.
If several divisions occur before parental cells have reached
the proper size, daughter cells eventually become roo small to
be viable. If cells grow too large before cell division, the cells
function improperly and the number of cells increases slowly.
In developing mu lticellular organisms, the replication of each
cell must be precisely controlled and timed to faithfully and
reproducibly complete the developmental program in every
individual. Each type of cell in every tissue must control its
replication precisely for normal development of complex or-
gans such as the brain or the kidney. In a normal adult, cells
divide only when and wliere they are needed. However, loss
of normal controls on cell replication is the fundamenta l de-
fect in cancer, an all-too-familiar disease that kills one in
every six people in the developed world (see Chapter 24 ). The
cussed in this chapter have gone a long way in explaining
how replication control goes awry in cancer cells. Appropri-
ately, Leland Hartwell, Tim Hunt, and Paul Nurse were
awarded the Nobel Prize in Physiology or Medicine in 2001
for the initial experiments that elucidated the master regula-
tors of cell division in all eukaryotes.
The term cell cycle refers to the ordered series of events
that lead to cell division and the production of two daughter
cells, each containing chromosomes identical to those of the
parental cell. Two main molecular processes take place dur-
ing the cell cycle, with resting intervals in between: during
the S phase of the cycle, each parental chromosome is dupli-
cated to form two identical sister chromatids; in mitosis (M
phase), the resulting sister chromatids are distributed to each
daughter cell (Figure 19-1). Chromosome replication and
segregation to daughter cells must occur in the proper order

OUTLINE

19.1 Overview of the Cell Cycle and Its Control 875 19.5 Entry into Mitosis 897

19.2 Model Organisms and Methods to Study the 19.6 Completion of Mitosis: Chromosome Segregation
Cell Cycle 877 and Exit from Mitosis 903

19.3 Regulation of CDK Activity 883 19.7 Surveillance Mechanisms in Cell Cycle
Regulation 906
19.4 Commitment to the Cell Cycle and DNA
Replication 890 19.8 Meiosis: A Special Type of Cell Division 913
(;} OVERVIEW ANIMATION: Cell Cycle Control
FIGURE 19-1 The fate of a single parental chromosome through-
out the eukaryotic cell cycle. Following mitosis (M), daughter cells
contain 2n chromosomes in diploid organisms and ln chromosomes in
haploid organisms. In proliferating cells, G 1 is the period between the
"bi rth" of a cell following mitosis and the initiation of DNA synthesis,
which marks the beginning of the 5 phase. At the end of the 5 phase,
cells enter G2 containing twice the number of chromosomes as G1 cells
(4n in diploid organisms, 2n m haploid organisms). The end of G2 is
marked by the onset of mitosis, during which numerous events leading
to cell division occur. The G~< 5, and G2 phases are collectively referred
to as interphase, the period between one mitosis and the next. Most
nonproliferating cells in vertebrates leave the cell cycle in G~< entering
the G0 state. Although chromosomes condense only during mitosis,
here they are shown in condensed form throughout the cell cycle to
emphasize the number of chromosomes at each stage. For simplicity,
the nuclear envelope is not depicted.
in every cell division. If a cell undergoes chromosome segrega-
tion before the replication of all chromosomes has been com-
pleted, at least one daughter cell will lose generic information.
Likewise, if a second round of replication occurs in one region
of a chromosome before cell division occurs, the genes encoded
in that region are increased in number out of proportion to
other genes, a phenomenon that often leads to an imbalance of
gene expression that is incompatible with viability.
High acc uracy a nd fidelity are required to ensure that
DNA replication is carried out correctly and that each
daughter cell inherits the correct number of each chromo-
some. To achieve this, cell division is controlled by surveil -
lance mechanisms known as checkpoint pathways that
prevent initiation of each step in cell division until earlier
steps on which it depends have been completed and mistakes
that occurred during the process have been corrected. Muta-
tions that inactivate or alter the no rmal operation of these
checkpoint pathways contribute to the generation of ca ncer
cells because they result in chromosomal rearrangements
and abnormal numbers of chromosomes, which lead to fur-
ther mutations and changes in gene expression level that
cause uncontrolled cell growth (see Chapter 24 ).
In the late 1980s, it became clear that the molecular
processes regulating the two key events in the cell cycle-
chromosome replication and segregation-are fundamen-
tally similar in all eukaryotic cells. Initially, it was surprising
to many researchers that cells as diverse as budding yeast
and developing human neurons use nearly identical pro-
teins to regulate their division. However, like transcription
874 CHAPTER 19 • The Eukaryotic Cell Cycle
and protein synthesis, control of cell division appears to be
a fundamental cellular process that evolved and was largely
optimized early in eukaryotic evolu tion. Because of this
similarity, research with diverse organisms, each with its
own particular experimental advantages, has contributed
to a growing understanding of how cell cycle events are
coordinated and controlled. Biochemical, genetic, imaging,
and micromanipulation techniques a ll have been employed
in studying various aspects of t he eukaryotic cell cycle.
These studies have revea led that cel l division is controlled
primarily by regulating the timing of entry into the cell di -
vision cycle, nuclear DNA replication, and mitosis.
The master controllers of the cell cycle are a small number
of heterodimeric protein kinases that contain a regulatory sub-
unit (cyclin) and a cata lytic subunit (cyclin-dependent kinase,
or CDK). These heterodimeric kinases regulate the activities of
multiple proteins involved in entry into the cell cycle, DNA
replication, and mitosis by p hosphorylating them at specific
regulatory sites, activating some and inhibiting others to coor-
dinate their activities. Regulated degradation of proteins also
plays a prominent role in important cell cycle transitions. Since
protein degradation is irreversible, this ensures that the pro-
cesses move in only one di rection through the cell cycle.
In this chapter, we first present an overview of th e cel l
cycle and then describe the various experimental systems that
contributed to our current understanding of it. We will then
discuss cyclin-dependent kinases (CDKs) and the many dif-
ferent ways these key cell cycle controllers can be regulated.
Next, we' ll examine each cell cycle phase in greater detail
with an emphasis on how control of CDK activity governs
the events that take place in each phase. We will then discuss
the system of checkpoint pathways that establish the order of
the cell cycle and ensure that each cell cycle phase occurs with
accuracy. In our discussion we will emphasize the general
principles governing cell cycle progression and will use a spe-
cies-spanning nomenclature when discussing the factors con-
trolling each cell cycle phase. The chapter concludes with a
discussion of meiosis, a special type of cell division that gen-
erates haploid germ cells (egg and sperm), and the molecular
mechanisms that distinguish it from mitosis.
19.1 Overview of the Cell Cycle
and Its Control
We begin our discussion by reviewing the stages of the eu-
karyotic cell cycle, presenting a summary of the current
model of how the cycle is regulated. We will see how DNA
replication leads to the creation of two identical DNA mol-
ecules during the DNA synthesis phase and how these DNA
molecules are compacted and structured for their segrega-
tion into daughter cells. We will then introduce the master
regulators of the cell cycle, the cyclin-dependent kinases, be-
fore concluding with an overview of the principles that gov-
ern the cell cycle to ensure that the process occurs in the
correct temporal fashion and without mistakes.
~ FOCUS ANIMATION: Mitosis
VIDEO: Visualizing Mitosis with Fluorescent Probes
FIGURE 19- 2 The stages of mitosis.
During prophase, the nuclear envelope Spindle
poles
breaks down, microtubules form the mitotic
spindle apparatus, and chromosomes
condense. At metaphase, attachment of
chromosomes to inicrotubules via their
kinetochores is complete. During anaphase,
microtubule motors and shortening of
spindle microtubules pull the sister chroma- Prophase
tids toward opposite spindle poles. After
chromosome movement to the spindle poles,
chromosomes decondense, and cells
reassemble nuclear membra~es around the
daughter-cell nuclei and undergo cytokinesis.
The Cell Cycle Is an Ordered Series of Events
leading to Cell Replication
As illustrated in Figure 19-1, the cell cycle is divided into four
major phases. Cycling (replicating) mammalian somatic cells
grow in size and synthesize RNAs and proteins required for
DNA synthesis during the G 1 (first gap) phase. When cells
have reached the appropriate size and have synthesized the
required proteins, they enter the cell cycle by traversing a
point in G 1 known as START. Once this point has been
crossed, cells are committed to cell division. The first step to-
ward successful cell division is entry into the S (synthesis)
phase, the period in which cells actively replicate their chro-
mosomes. After progressing through a second gap phase, the
G 2 phase, cells begin the complicated process of mitosis, also
called the M (mitotic) phase, which is divided into several
stages (Figure 19-2).
In discussing mitosis, we commonly use the term chro-
mosome for the replicated structures that condense and be-
come visible in the light microscope during the early stages
of mitosis. Thus each chromosome is composed of two iden-
tical DNA molecules resulting from DNA replication, plus
the histones and other chromosome-associated proteins (see
Figure 6-39). The two identical DNA molecules and associ-
ated chromosomal proteins that form one chromosome are
called sister chromatids. Sister chromatids are attached to
each other by protein cross-links along their lengths.
Sister
Met aphase
G,
19.1 Overview of the Cell Cycle and Its Cont rol 875
 During interphase, the part of the cell cycle between the
end of one M phase and the beginning of the next, the outer
nuclear membrane is continuous with the endoplasmic reticu-
lum. With the onset of mitosis in prophase, the nuclear enve-
lope retracts into the endoplasmic reticulum in most cells
from higher eukaryotes, and Golgi membranes break down
into vesicles. This is necessary so that the microtubules, nu-
cleated by the centrosomes, can interact with the chromo-
somes to form the mitotic spindle, consisting of a
football-shaped bundle of microtubules with a star-shaped
cluster of microtubules radiating from each end, or spindle
pole. A multiprotein complex, the kinetochore, assembles at
each centromere. After nuclear envelope breakdown, the ki-
netochores of sister chromatids associate with microtubules
coming from opposite spindle poles (see Figure 18-37), and
chromosomes align in a plane in the center of the cell at meta-
phase. During the anaphase period of mitosis, sister chroma-
tids separate. They initially are pulled by microtubules
toward the spindle poles and then are further separated as the
spindle poles move away from each other (see Figure 19-2).
Once chromosome separation is complete, the mitotic
spindle disassembles and chromosomes decondense during
telophase. The nuclear envelope re-forms around the segre-
gated chromosomes as they decondense. The physical divi-
sion of the cytoplasm, called cytokinesis, yields two daughter
cells. Following mitosis, cycling cells enter the G 1 phase, em-
barking on another turn of the cycle.
The progression of cell cycle stages is the same for all
eukaryotes, though the time it takes to complete one turn of
the cycle varies considerably between organisms. Rapidly
replicating human cells progress through the full cell cycle in
about 24 hours: G 1 takes 9 hours; the S phase, 10 hours; G 2,
4.5 hours; and mitosis, 30 minutes. In contrast, the full cycle
takes only 90 minutes in rapidly growing yeast cells. The cell
divisions that take place during early embryonic develop-
ment of the fruit fly Drosophila melanogaster are completed
in as little as 8 minutes!
In multicellular organisms, most differentiated cells
'·exit" the cell cycle and survive for days, weeks, or in some
cases (e.g., nerve cells and cells of the eye lens) even the life-
time of the organism without dividing again. Such postmi-
totic cells generally exit the cell cycle in Gh entering a phase
called G 0 (see Figure 19-1 ). Some G 0 cells can return to the
cell cycle and resume replicating; this re-entry is regulated,
thereby providing control of cell proliferation.
Cyclin-Dependent Kinases Control
the Eukaryotic Cell Cycle
As mentioned in the chapter introduction, passage through the
cell cycle is controlled by heterodimeric protein kinases that
comprise a catalytic subunit and a regulatory subunit. The
concentrations of the catalytic subunits, the cyclin-dependent
kinases (CDKs), are constant throughout the cell cycle. How-
ever, they have no kinase activity unless they arc associated
with a regulatory cyclin subunit. Each CDK can associate with
a small number of different cyclins that determine the substrate
876 CHAPTER 19 • The Eukaryotic Cell Cycle
specificity of the complex, that is, which proteins it phosphory-
lates. Each cyclin is only present and active during the cell cycle
stage it promotes and hence restricts the kinase activity of the
CDKs it binds to just that cell cycle stage. Cyclin-CDK com-
plexes activate or inhibit hundreds of proteins involved in cell
cycle progression by phosphorylating them at specific regula-
tory sites. Thus proper progression through the cell cycle is
governed by activation of the appropriate cyclin-CDK com-
plex at the appropriate time. As we will see, restricting cyclin
expression to the appropriate cell cycle stage is one of the
many mechanisms cells employ to regulate the activities of
each cyclin-CDK heterodimer.
Several Key Principles Govern the Cell Cycle
The goal of each cell division is to generate two daughter cells
of identical genetic makeup. To achieve this, cell cycle events
must occur in the proper order. DNA replication must always
precede chromosome segregation. Today we know that the ac-
tivity of the key proteins that promote cell cycle progression,
the CDKs, fluctuates during the cell cycle. For example, CDKs
that promoteS phase are active during S phase but are inactive
during mitosis. CDKs that promote mitosis are only active dur-
ing mitosis. These oscillations in CDK activity "are a fundamen-
tal aspect of eukaryotic cell cycle control, and we have gained
some understanding over the last few years as to how these
oscillations are generated. Oscillations are generated by posi-
tive feedback mechanisms, where specific CDKs promote their
own activation. These positive feedback loops are coupled to
subsequent negative feedback mechanisms where, indirectly or
with a built-in delay, CDKs promote their own inactivation.
Oscillators not only propel the cell cycle forward but also cre-
ate abrupt transitions between different cell cycle states, which
is essential to bring about distinct cell cycle states.
Laid on the cell cycle oscillator machinery is a system of
surveillance mechanisms that further ensures that the next
cell cycle event is not activated before the preceding one has
been completed or before errors that occurred during the
preceding step are corrected. These surveillance mechanisms
are called checkpoint pathways, and their job is it to ensure
accuracy of the chromosome replication and segregation
processes. The system that ensures that chromosomes are
segregated accurately is so efficient, a mis-segregation event
occurs only once in 10 4-10 1 divisions! These multiple layers
of control put on the cell cycle control machinery ensure that
the cell cycle is robust and error free.
KEY CONCEPTS of Section 19.1
Overview of the Cell Cycle and Its Control
• The eukaryotic cell cycle is divided into four phases: G 1
(the period between mitosis and the initiation of nuclear
DNA replication), S (the period of nuclear DNA replica-
tion), G 2 (the period between the completion of nuclear
DNA replication and mitosis), and M (mitosis).
·.

• Cells commit to a new cell division at a specific point in G 1
.. · known as START.
• Cyclin-CDK complexes, composed of a regulatory cyclin
subunit and a catalytic cyclin-dependent kinase (CDK) sub-
unit, drive progression of a cell through the cell cycle.
Cyclins activate CDKs and are present only in the cell cy-
cle stage that they promote.
CDK activities oscillate during the cell cycle. Positive and
negative feedback loops drive these oscillations.
• Surveillance mechanisms, called checkpoint pathways,
guarantee that each cell cycle step is completed correctly be-
fore the next one is initiated.
.· 19. 2 Model Organisms and Methods
to Study the Cell Cycle
The unraveling of the molecular mechanisms governing cell
cycle progression in eukaryotes was remarkably speedy and
fueled by the powerful combination of genetic and biochem-
ical approaches. In this section we will discuss several model
systems and their contribution to the discovery of the mo-
lecular mechanisms of cell division. The three most impor-
tant systems employed to study the cell cycle are the
single-celled yeasts Saccharomyces cerevisiae (budding yeast)
and Schizosaccharomyces pombe (fission yeast) and oocytes
and early embryos of the frog Xenopus laevis. We will also
discuss the fruit fly Drosophila melanogaster, which proved
extremely powerful in the study of the interplay between cell
division and development as well as how the study of mam-
malian tissue culture cells led to the characterization of cell
cycle control in mammals.
The studies of the cell divisiOn cycle in many different
experimental systems also led to two remarkable discoveries
about the general control of the cell cycle. First, complex
molecular processes such as initiation of DNA replication
and entry into mitosis are all regulated and coordinated by a
small number of master cell cycle regulatory proteins. Sec-
ond, these master regulators and the proteins that control
them are highly conserved, so that cell cycle studies in fungi,
sea urchins, insects, frogs, and other species are directly ap-
plicable to all eukaryotic cells, including human cells.
Budding and Fission Yeast Are Powerful Systems
for Genetic Analysis of the Cell Cycle
Budding and fission yeast have proved to be valuable sys-
tems for the study of the cell cycle. Although they both be-
long to the kingdom of fungi, they are only distantly related.
Both organisms can exist in the haploid state, carrying only
one copy of each chromosome. The fact that these yeasts can
exist as haploid cells makes them powerful genetic systems.
It is easy to generate mutations that inactivate genes in hap-
loids because there is only one copy of each gene (a diploid
system would require an inactivating mutation in each of the
two copies of the gene to render its activity nonfunctional).
Haploid yeast can be easily employed to screen or select for
mutants with specific defects, such as defects in cell prolif-
eration. Additional advantages of the two systems are the
relative ease with which one can manipulate the expression
of individual genes, their fully sequenced genomes, and the
ease with which they can be cultivated and manipulated so
that cultures of yeast cells progress through the cell cycle in
a synchronous manner.
Budding yeast cells are ovoid in shape and divide by bud-
ding (Figure 19-3a). The bud is the future daughter cell and
begins to form concomitant with the initiation of D A repli-
cation and continues to grow throughout the cell cycle (Fig-
ure 19-3b). Cell cycle stage can therefore be inferred from the
size of the bud, which makes S. cerevisiae a useful system for
identifying mutants that are blocked at specific steps in the
cell cycle. Indeed, it was in this organism that Lee Hartwell
and colleagues first identified mutants that were defective in
progressing through specific cell cycle stages. Like mamma-
lian cells, the budding yeast cell cycle has a long G 1 phase,
and the study of the budding yeast cell cycle shaped our un-
derstanding of how the G 1-S phase transition is controlled.
Fission yeast cells are rod shaped and grow entirely by
elongation at their ends (Figure 19-4a). After the completion
of mitosis, cytokinesis occurs by the formation of a septum
(Figure 19-4b). The molecular mechanisms governing G 2 and
entry into mitosis are very similar between fission yeast and
metazoan cells, and studies with this organism revealed the
molecular events surrounding the GrM phase transition.
Budding and fission yeast are both useful for the isola-
tion of mutants that are blocked at specific steps in the cell
cycle or that exhibit altered regulation of the cycle. Since cell
cycle progression is essential for viability, scientists isolated
conditional mutants that encode proteins that are functional
at one temperature but become inactive at a different, often
elevated, temperature (e.g., due to protein misfolding at the
non-permissive temperature). Mutants arrested at a particu-
lar cell cycle stage are easily distinguished from normal di-
viding cells by microscopic examination. Thus, in both of
these yeasts, cells with temperature-sensitive mutations caus-
ing defects in specific proteins required to progress through
the cell cycle were readily isolated (see Figure 5-6). Such cells
are called cdc (cell division cycle) mutants.
How can one identify which gene is defective in a given
cdc mutant? The wild-type alleles of recessive temperature-
sensitive cdc mutant alleles can be isolated readily by trans-
forming haploid mutant cells with a plasmid library prepared
from wild-type cells and then plating the transformed cells at
the non-permissive temperature (Figure 19-5). Haploid mu-
tant cells cannot form colonies at the non-permissive tem-
perature. However, a transformed mutant cell can grow into
a colony if it also contains a plasmid that carries the wild-
type allele that complements the recessive mutation; the plas-
m ids bearing the wild-type allele can then be recovered from
those cells, allowing the identification of the complementing
gene. Because many of the proteins that regulate the cell
19. 2 Model Organisms and Methods to Study the Cell Cycle 877
G) VIDEO: Mitosis and Budding in 5. cerevisiae
(a) (b)
Chromosome
segregation;
nuclear
division
Spindle
formation;
nuclear
migration
FIGURE 19-3 The budding yeastS. cerevisiae. (a) Scanning
electron micrograph of 5. cerevisiae cells at va rious stages of the cell
cycle. The larger the bud, which emerges at the end of the G1 phase,
the farther along in the cycle the cell is. (b) Main events in the 5.
cerevisiae cell cycle: Daughter cells are born smaller than mother cells
and must grow to a greater extent in G1 before they are large enough
to enter the S phase. START is the point in the cell cycle after which
cycle are highly conserved, human cDNAs cloned into yeast
expression vectors often can complement yeast cell cycle mu-
tants, leading to the rapid isolation of human genes encoding
cell cycle control proteins. In fact, it was the ability of the
human gene encoding CDKJ to complement the growth de-
fects caused by inactivation of fission yeast CDKl that led to
rhe appreciation of the high degree of conservation that ex-
ists among eukaryotic cell cycle regulators.
Frog Oocytes and Early Embryos Facilitate
Biochemical Characterization
of the Cell Cycle Engine
Biochemical studies require the preparation of cell extracts
from many cells. For biochemical studies of the cell cycle, the
eggs and early embryos of amphibians and marine
invertebrates are particularly suitable. These organisms typi-
878 CHAPTER 19 • The Eukaryotic Cell Cycle
( ' ; ; \ Mother
D cell
Cytokinesis
Daughter
cell
Growth
START
® Spindle pole
body
duplication
Bud
emergence
DNA
replication
cells are irreversibly committed to undergoing a cell cycle. G2 is not
well defined in budd ing yeast and is therefore denoted in parentheses.
Note that the nuclear envelope does not disassemble during mitosis
in 5. cerevisiae and other yeasts. The small 5. cerevisiae chromosomes
do not condense sufficiently to be visible by light microscopy.
[Part (a) courtesy of E. Schachtbach and I. Herskowitz.]
cally have large eggs, and fertilization is followed by multiple
synchronous cell cycles. By isolating large numbers of eggs
from fema les and fertilizing them simultaneously by add ition
of sperm (or treating them in ways that mimic fertilization ),
researchers can obtain extracts from cells at specific points in
the cell cycle for analysis of proteins and enzymatic activities.
To understand how X. laevis oocytes and eggs can be
used for the analysis of cell cycle progression, we must first
lay out the events of oocyte maturation, w hich can be reca-
pitulated in vitro. So far, we discussed mitotic division. Oo-
cyres, however, undergo a meiotic division (see Figure 19-38
fo r an overview of meiosis). As oocytes develop in the frog
ovary, they replicate their DNA and become arrested in G 2
for 8 months, during which time they grow in size to a diam-
eter of 1 mm, stockpiling all the materials needed for the
mu ltiple cell divisions of the early embryo. When stimulated
by a male, an adu lt fema le's ovarian cells secrete the steroid
g VIDEO: Mitosis and Cell Division in 5. pombe

(a) (b) Cytokinesis

Nuclear division
~
Chromosome
segregation
0 START

DNA
replication
Spindle
formation

Chromosome
condensation

~
Spindle pole
body duplication

FIGURE 19-4 The fission yeastS. pombe. (a) Scanning electron
micrograph of 5. pombe cells at various stages of the cell cycle. Long
cells are about to enter mitosis; short cells have just passed through
cytokinesis. (b) Main events in the 5. pombe cell cycle. START is the
Cell growth
point in the cell cycle after which cells are irreversibly committed to
undergoing a cell cycle. As in 5. cerevisiae, the nuclear envelope does
not break down during mitosis. [Part (a) courtesy of N. Hajibagheri.)

Transform with
plasmid library EXPERIMENTAL FIGURE 19-5 Wild-type cell division
15
of wi ld-type cycle (COO genes can be isolated from aS. cerevisiae
cdc28 S. cerevisiae DNA
cells grown Transformed genomic library by functional complementation of cdc
at 25 °C form Gene X cdc28 15 cells mutants. Mutant cells with a temperature-sensitive mutation in a
colonies grown at 37 C

©J?@\§) 0 @
CDC gene are transformed with a genomic library prepared from
wild-type cells and plated on nutrient agar at the non-permissive
temperature (37 °(). Each transformed cell takes up a single
Gene Y No colony plasmid containing one genomic DNA fragment. Most such

0 formation fragments include genes (e.g., X and Y) that do not encode the

@~
defective Cdc protein; transformed cells that take up such
@ fragments do not form colonies at the non-permissive temperature.
CDC28 The rare cell that takes up a plasmid containing the wild-type

0 Q@@ version of the mutant gene (in this case CDC28, a cyclin-dependent
kin asP) is complemented, allowing the cell to replicate and form a

@~~
Isolate CDC2B
---------+ ~ plasmid
(?520
<=@
0 colony at the non-permissive temperature. Plasmid DNA isolated
from this colony carries the wild-type CDC gene corresponding to
the gene that is defective in the mutant cells. The same procedure
is used to isolate wild-type cdc • genes in 5. pombe. See Figures
Cells in colony at
various cell cycle 5-17 and 5-18 for more detailed illustrations of the construction
stages and screening of a yeast genomic library.

19. 2 Model Organisms and Methods to Study the Cell Cycle 879
(a) First polar body
Progesterone
l
--....
D
Oocyte Meiosis I
-.
arrested in G 2
(b)
~·
• """' •
, ....
:
,;,.,:::.;~;:.~t,;., ---~
FIGURE 19-6 Progesterone sti mulates meiotic maturation of
Xenopus oocytes. (a) Step D : Progesterone treatment of G2-arrested
Xenopus oocytes surgically removed from the ovary of an adult female
causes the oocytes to enter meiosis I. Two pairs of synapsed homolo-
gous chromosomes (blue) connected to meiotic spindle microtubules
(green) are shown schematically to represent cells in metaphase of
meiosis I. Step fJ : Segregation of homologous chromosomes and a
highly asymmetrical cell division expels half the chromosomes into
a small cell called the first polar body. The oocyte immediately com-
mences meiosis II and arrests in metaphase II to yield an egg. Two
chromosomes connected to spindle microtubules are shown
hormone progesterone, which induces the G 2-arrested oo-
cytes to enter meiosis. As we will see in Section 19.8, meiosis
consists of two consecutive chromosome segregation phases
known as meiosis I and meiosis II. Progesterone triggers oo-
cytes to undergo meiosis I and progress to the second meiotic
metaphase, where they arrest and await fertilization (Figure
19-6). At this stage the cells are called eggs. When fertilized
by sperm, the egg nucleus is released from its metaphase II
arrest and completes meiosis. The resulting haploid egg nu-
cleus then fuses with the haploid sperm nucleus, producing a
diploid zygote nucleus. DNA replication follows, and the
first mitotic division of embryogenesis begins. The resulting
embryonic cells then proceed through 11 more rapid, syn-
chronous cell cycles, generating a hollow sphere of cells
called the blastula. Cell division then slows, and subsequent
divisions are non-synchronous, with cells at different posi-
tions in the blastula dividing at different times.
The advantage of using X. laevis to study factors in-
volved in mitosis is that large numbers of oocytes and eggs
\:an be prepared that are all proceed ing synchronously
through the cell cycle events that follow progesterone treat-
ment and fertilization. This makes it possible to prepare suf-
ficient amounts of extract for biochemical experiments from
cells that were all at the same point in the cell cycle. It was in
this system that the cyclin-CDK complexes that trigger mito-
sis and the oscillatory nature of their activity was first dis-
880 CHAPTER 19 • The Eukaryotic Cell Cycle
Second polar body 11 synchronous
divisions
!
Egg arrested Male Female First cleavage Blastula
in metaphase pronucleus pronucleus
of meiosis II
schematically to represent egg cells arrested in metaphase of meiosis II.
Step 11: Fertilization by sperm releases eggs from their metaphase
arrest, allowing them to proceed through anaphase o.f meiosis II and
undergo a second highly asymmetrical cell division that eliminates one
chromatid of each chromosome in a second polar body. The resulting
haploid female pronucleus fuses with the haploid sperm pronucleus
to produce a diploid zygote. Step B :The zygote undergoes DNA
replication and the first mitosis. Step 111: The first mitosis is followed by
11 more synchronous divisions to form a blastula. (b) Micrograph of
Xenopus eggs. [Part (b) copyright e ISM/Phototake.]
covered. This activity was called maturatio n-promoting
factor (MPF) because of its abi lity to induce entry into meio-
sis when injected into Grresting oocytes.
Fruit Flies Reveal the Interplay Between
Development and the Cell Cycle
The development of complex tissues often requires specific
modifications to the cell cycle. Understanding the interplay
between development and cell division is thus crucial if we .·.'
want to understand how complex organisms are built. Dro-
sophila melanogaster has established itself as the premier
model system for studying the interplay between develop-
ment and the cell cycle. Not only does the development of
this organism involve several highly unusual cell cycles, the
powerful genetic techniques that can be applied to fruit flies
facilitated the discovery of genes involved in the developmen-
tal control of the cell cycle. The fi rst 13 nuclear divisions of
the fertilized Drosophila embryo all occur in ::t common cyto-
plasm and are rapid cycles of DNA replication and mitosis
(with no gap phases), fueled by key cell cycle regulators that
were stockpiled in the egg cytoplasm as it matured. These
divisions are called the syncytial divisions and occur in uni-
son (Figure 19-7). As maternal stockpiles run out, gap phases
are introduced, first G 2 , followed by G 1• Most cells in the
embryo cease to divide at this point, form plasma membranes,
 0 VIDEO: Syncytial Divisions of the Drosophila Embryo
Mitotic divisions
in the syncytium
Endocycles in Endocycles in
differentiating larval tissues
larval tissues
Increase in
Mitotic divisions cel l size
in the
nervous system
1st 2nd 3rd
in star instar instar
~~
Embryo Larval stages
FIGURE 19-7 Cell division patterns during the life cycle of
Drosophila melanogaster. After fertilization, nuclei in the embryo
undergo 13 rapidS phase-M phase cycles. These are followed by three
divisions that include a G2 phase. All these nuclear divisions occur
·. within a common cytoplasm and are therefore called the syncytial
divisions. During late stages of embryogenesis and t hroughout larval
development (with the exception of the nervous system), cells undergo
endocycles. This leads to an increase in cellular ploidy and size and
and utilize a specialized cell cycle known as the endocycle. In
the endocycle, cells replicate their DNA but do not undergo
mitosis. This leads to an increase in gene dosage and fuels
increased macromolecule biosynthesis, which allows individ-
ual cells to grow in size. Thus the embryo, which has now
developed into a crawling larva, grows simply by an increase
in cell size and not through cell multiplication. A select num-
ber of cells do not share this fate. These cells are in the ima-
ginal disks, the organs that will give rise to the adu lt fly
tissues during metamorphosis. Metamorphosis occurs during
the pupa stage and transforms larvae into adult flies. The di-
visions that give rise to the adult fly are canonical cell cycles
leading to the adult fly being a diploid organism.
The Study of Tissue Culture Cells Uncovers
Cell Cycle Regulation in Mammals
Cell cycle regulation in hllman cells is more complex than in
other non-mammalian systems. To understand this increased
level of complexity and to understand the cell cycle alterations
that are the cause of cancer, it is important to study the cell
cycle not only in model organisms but also in human cells.
Researchers use normal or tumor cells grown in plastic dishes
to study the properties of the human cell cycle, a method
called tissue culture or cell culture. It is, however, important
to note that many of the cell types used to study the human
<.:dl cycle themselves have altered cell cycle properties due to
genetic alterati ons that occurred during their culturing o r be-
cause they were isolated from human tumors. Furthermore, in
vitro culture conditions do not resemble those found in the
organism and could lead to altered behavior of cells. Although
some aspects of mammalian cell division are not recapitulated
in cell culture conditions-such as the importance of tissue
Mitosis
(stem cells)
Meiosis
(egg and sperm)
Division and
d1fferent1ation Endocycles
of imaginal disks (ovary)
~ :
Pupa
T_~
Adult
hence larval growth. In the pupa, during a process called metamorpho-
sis, imaginal disks, the tissues that give rise to the adult organs,
undergo mitotic divisions and then differentiate to form adult
structures. Several types of divisions are seen in the adult fly. Stem cells
undergo mitotic divisions, meiosis gives rise to sperm and egg, and
endocycles create polyploid cells in the ovary. [Adapted from Lee and
Orr-Weaver, 2003, Ann. Rev. Genet. 37:545- 578.)
organization and developmental signals governing cell cycle
control--<:ell culture systems nevertheless provide critical in-
sights into the mammalian cell's intrinsic mechanisms govern-
ing cell division. Researchers also work toward establishing
culture systems that more closely resemble the cell architec-
ture in tissues. For example, polymers are currently being de-
veloped that allow scientists to grow cells in 3-D culture.
As we wi ll see in Chapter 21, primary human cells and
other mammalian cells have a finite life span when cultured
in vitro. Normal human cells, for example, divide 25-50
times, but thereafter proliferation slows and eventually
stops. This process is called replicative senescence. Cells can
escape this process and become immortalized, allowing re-
searchers to establish cell lines. Although these cell lines har-
bor genetic alterations that affect some aspects of cell
proliferation, they are nevertheless a useful tool to study cell
cycle progression in human cells. These cell lines provide an
inexhaustible supply of cells that, as we will see next, can be
manipulated to progress through the cell cycle in a synchro-
nous manner, allowing for the analysis of protein levels and
enzymatic activity at different stages of the cell cycle.
Researchers Use Multiple Tools
to Study the Cell Cycle
The experimental analysis of cell cycle properties requires that
we are able to determine the cell cycle stage of individual cells.
Light microscopy provides some estimate of cell cycle progres-
sion. For example, light microscopy allows a researcher to
determine whether cultured mammalian cells are in interphase
(Gh S phase, and G2 ) or in mitosis. Mammalian tissue culture
cells are flat and adhere to the plastic dish during interphase
but round up and form spherical structures as they undergo
19. 2 Model Organisms and Methods to Study the Cell Cycle 881
FIGURE 19-8 Human cells undergoing
mitosis. He La Kyoto cells were filmed as they
underwent mitosis. The images shown were
taken every 20 minutes. Cells are flat during
interphase, but as cells undergo mitosis, they
round up and divide. Subsequently, they flatten
out again. [Courtesy of Sejal Vyas and Paul Chang, MIT.]

mitOSIS (Figure 19-8). Fluorescence microscopy of cellular
structures or the analysis of specific cell cycle markers, that is,
proteins that are only present in certain cell cycle stages, al-
lows for a more accurate determination of cell cycle stage.
In addition to microscopic tools, cell cycle researchers
use flow cytometry to determine the D~A content of a cell
population (Figure 19-9; see also Figure 9-2). Cells are
treated with a DNA-binding fluorescent dye and the amount
of dye that incorporates into the DNA of cells can then be
quantitatively assessed using a flow cytometer. Cells are then
sorted by their DNA content, and the percentage of cells in
GI> S phase, and G 2 or mitosis can be assessed in this man-
ner. Cells in G 1 will have half as much DNA as cells in G2 or
mitosis. Cells undergoing DNA synthesis in S phase will
have an intermediate amount of DNA.
To characterize different cell cycle events, it is essential to
examine cell populations that progress through the cell cycle in
unison. Researchers achieve this by reversibly arresting cells in
1200
900
Unreplicated
.!!!.
Qi
a particular cell cycle stage. This cell cycle arrest is usually ac-
complished by restricting nutrients or adding anti-growth fac-
tors, which cause cells to arrest in G 1• In budding yeast, for
example, cells treated with a mating pheromone arrest in G 1•
When the pheromone is removed from cells (usually by wash-
ing them extensively), cells exit the G 1 arrest and progress
through the cell cycle in a synchronous manner. In mammalian
cells, removal of growth factors by removing serum from the
culture medium (serum starvation) arrests cells in G 0 • Re-
add ition of serum allows cells to re-enter the cell cycle. Other
methods involve blocking a certain cell cycle.step with chemi-
cals. Hydroxyurea inhibits DNA replication, leading to an S
phase arrest. On removal of the drug, cells will resume DNA
synthesis in unison. Nocodazole disrupts the mitotic spindle
and halts the cell cycle in mitosis. Once the drug is washed
away, cells will resume progression through mitosis in a syn-
chronous manner. In budding and fission yeast, the conditional
cell division cycle (cdc) mutants introduced ea rlier have proved
a powerful tool for creating synchronous cultures. Tempera-
ture-sensitive cdc mutants, when incubated at the non-permis-
sive temperature, arrest in a particular cell cycle stage because
they are defective in a certain key cell cycle protein. Returning
cells to the permissive temperature allows cells to continue with
the cell division cycle in a synchronous fas hion.

-(.)

0
Cii 600
.0
E KEY CONCEPTS of Section 19.2
:::;)
z Model Organisms and Methods to Study the Cell Cycle
Replicated
300
The ability to isolate mutants and the powerful genetic
tools of budding and fission yeast allowed for the isolation
of key factors important for cell cycle regulation.

1C 2C
DNA content
: XPE. MEN AL FIGURE 19 ·9 Analysis of DNA content by
flow cytometry. Haploid yeast cells were grown in <.ulture and stained
with propidium iodide, a fluorescent dye that incorporates into DNA.
The x axis shows DNA content, they axis the number of cells. The DNA
content ana lysis shows two predominant populations of cells: cells
with unreplicated DNA (lC) and with replicated DNA (2C). The cells
between the two peaks represent cells that are in the process of
undergoing DNA replication. [Courtesy of Heid1 Blank.]
Frog eggs and early embryos from synchronously fertil-
ized eggs provide sources of extracts fo r biochemical studies
of cell cycle events and identified the oscillatory nature of
cyclin-CDK complexes.
Fruit flies are a powerful system to investigate the inter-
play between cell division and the developmental programs
responsible for building multicellular organisms.
Human tissue culture cells are used to study the properties
of the mammalian cell cycle.

882 CHAPTER 19 • The Eukaryotic Cell Cycle


• The generation of synchronized cell populations through
reversibly arresting cells in a particular cell cycle stage allows
the researcher to examine the behavior of proteins and cel-
lular processes during the cell cycle.
19.3 Regulation of CDK Activity
In the following seuiuns we describe the current model of eu-
karyotic cell cycle regulation summarized in Figure 19-10 and
present some of the experiments that led to this understanding.
As we will see, results obtained with different experimental
systems and approaches have provided insights into each of the
transition points in the cell cycle. A key discovery in cell cycle
studies was that cyclin-dependent kinases govern progression
through the cell cycle. Three key features about these kinases
are important to keep in mind throughout this chapter:
• Cyclin-dependent kinases (CDKs) are only active when
bound to a regulatory cyclin subunit.
• Different types of cyclin-CDK complexes initiate different
event~. G 1 CDKs and G 1 /S phase CDKs promote entry into
the cell cycle, S phase CDKs trigger S phase, and mitotic
CDKs initiate the events of mitosis (Figure 19-11 ).
• Multiple mechanisms are in place to ensure that the different
CDKs are only active in the stages of the cell cycle they trigger.

(i) VIDEO: Dynamic Behavior of Mitotic Cyclin in Hela Cells

APC/C and phosphatases

induce late steps in mitosis

APC/C ubiquitin-protein
ligase induces
anaphase

Prophase Metaphase

M
Mitotic CDKs G 1 CDKs and
induce mitosis G 1/S phase CDKs
prepare cells
for S phase

SCF
ubiquitin-protein
ligase induces
S phase
s
S phase CDKs activate
DNA replication
FIGURE 19-1 0 Regulation of cell cycle transitions. Cell cycle down and chromosomes align on the mitotic spindle but they cannot
transitions are regulated by cyclin-CDK protein kinases, protein separate until the anaphase-promoting complex (APC/C), a ubiquitin-
phosphatases, and u biquitin-protein ligases. Here the cell cycle is protein ligase, ubiquitinylates the anaphase inhibitor protein securin,
diagrammed, with the major stages of mitosis shown at the top. In early marking it for degradation by proteasomes. This results in degradat ion
G1, no cyclin-CDKs are active.ln mid-G1, G1/S phase CDKs activate of protein complexes linking the sister chromatids and the onset of
transcription of genes required for DNA replication. S ph11se is initiated anaphase as sister chromatids separate. After chromosome movement
by the SCF ubiquitin-protein ligase that ubiquitinylates inhibitors of S to the spindle poles, the APC/C ubiquitinylates mitotic cyclins, causing
phase CDKs, marking them for deg radation by proteasomes. The S phase their degradation by proteasomes. The resulting drop in mitotic CDK
CDKs then activate DNA replication and DNA synthesis commences. activity, along with the action of protein phosphatases, results in
Once DNA replication is complete, cells enter G2• In late G2, mitotic CDKs chromosome decondensation, reassembly of nuclear membranes
trigger entry into mitosis. During prophase, the nuclear envelope b reaks around the daughter-cell nuclei, and cytokinesis.

19.3 Regulation of CDK Act ivity 883

FIGURE 19-11 An overview of how CDKs regulate cell cycle G 1/S phase S phase Mitotic APC/C
progression. Cells harbor different types of CDKs that initiate different CDKs CDKs CDKs activity
events of the cell cycle. Importantly, the CDKs are only active in the
stages of the cell cycle they trigger. G/S phase CDKs are active at the
G1-5 phase transition to trigger entry into the cell cycle. 5 phase CDKs
-~
are active during 5 phase and trigger 5 phase. Mitotic CDKs are active
·~
u
during mitosis and trigger mitosis. A ubiquitin ligase known as the <(
anaphase-promoting complex or cyclosome (APC/C) catalyzes two key
cell cycle transitions by ubiquitinylating proteins, hence targeting them
for degradation. The APC/C initiates anaphase and exit from mitosis.

Metaphase-anaphase
transition

In thts section we will first discuss the properties of CDKs
and investigate the structural basis of their activation and
regulation. We will then see how cyclins activate CDKs and
investigate the multiple regulatory mechanisms that restrict
the different cyclins to the appropriate cell cycle stage. We
will see that protein degradation plays an essential part in
this process. In addition, we will discuss how post-transla-
tional modifications to CDKs an d inhibitory proteins that
directly bind to cyclin-CDK complexes are essential addi-
tional control mechanisms in restricting different cycl in-
CDK activities to the appropriate cell cycle stage.
Cyclin-Dependent Kinases Are Small Protein
Kinases That Requ ire a Regulatory Cyclin
Subunit for Their Activity
Cyclin-dependent kinases are a family of small (30-40 kD)
serine/threonine kinases. They are not active in the monomeric
form but, as mentioned previously, require an activating
subunit to be active as a protein kinase. In budding and fis-
sion yeast, a single CDK controls progression through the
cell cycle. Its activity is specified by cell-cycle-stage-specific
cyclin subunits. Mammalian cells contain as many as nine
CDKs, wtth four of them, CDKl, CDK2, CDK4, and CDK6,
having clearly been shown to regulate cell cycle progression.
They bind to different types of cycljns and together promote
different cell cycle transitions. CDK4 a nd CDK6 are G 1
CDKs and promote entry into the cell cycle, CDK2 functions
as a G/S phase and S phase CDK, and CDK1 is the mitotic
CDK. For historical reasons, the names of various cyclin-
dependent kinases from yeasts and vertebrates differ. When-
ever possible, we will use the general terms G 1, G /S phase,
S phase, and mitotic CDKs ro describe CDKs instead of the
species-specific terminology. Table 19-1 lists the different
names of the various CDKs and indicates when in the cell ·.
cycle they are active.
CDKs are not only regulated by cyclin binding but also
by both activating and inhibitory phosphorylation. To-
gether, these regulatory events ensure that CDKs are only
active at the appropriate cell cycle stage. The three-dimensional
structure of CDKs provides insight into how the activity of
these protein kinases is regulated. Unphosphorylated, inac-
tive CDK contains a flexible region, called the T loop, that
blocks access of protein substrates to the active site where
ATP is bound (Figure 19-12a). Steric blocking by the T loop
largely explains why free CDK, unbound to cyclin, has little
protein kinase activity. Unphosphorylatecl CDK bound to
one of its cyclin partners has minimal but detectable protein
kinase activity in vitro, although it may be essentially inac-
tive in vivo. Extensive interactions between the cyclin and

TABLE 19-1 Cyclins and CDKs: Nomenclature and Their Roles in the Mammalian Cell Cycle

CDK Cyclin Function General Name

- ~

- -- - ~ ~

-- ~ - - - - - -
CDKl Cyclin A, cyclin B Mitosis Mitotic CDKs

CDK2 Cyclin E, cyclin A Entry mto the cell cycle G/S phase CDKs
S phase S phase CDKs

CDK4 Cyclin D c. G, CDKs

Entry mto the cell cycle

CDK6 Cycli n D G, G 1 CDKs

Entry into the cell cycle

884 CHAPTER 19 • The Eukaryotic Cell Cycle

 (a) Free CDK2 (b) Low-activity cyclin A-CDK2
FIGURE 19-12 Structural models of human CDK2. (a) Free,
inactive CDK2 unbound to its cyclin subunit, cyclin A. In free CDK2, the
T loop blocks access of protein substrates to the 'Y phosphate of the
bound ATP, shown as a ball-and-stick model. The conformations of the
regions highlighted in yellow are altered when CDK is bound to cyclin
A. (b) Unphosphorylated, low-activity cyclin A-CDK2 complex.
Conformational changes induced by binding of a domain of cyclin A
(blue) cause the T loop to pull away from the active site of CDK2 so that
substrate proteins can bind. The cxl helix in CDK2, which interacts
the T loop cause a dramatic shift in the position of the T
loop, thereby exposing the CDK active site (Figure 19-llb).
As we will see shortly, high activity of the cyclin-CDK com-
plex requires phosphorylation of the activating threonine, in
the T loop, causing additional conformational changes in the
cyclin-CDK complex that greatly increase irs affinity for pro-
tein substrates (Figure 19-12c). As a result, the kinase activ-
ity of the phosphorylated complex is a hundredfold greater
than that of the unphosphorylated complex.
.. · Cyclins Determine the Activity of CDKs
Cyclins are so named because their levels change during the
cell cycle. They form a family of proteins that is defined by
three key features:
• Cyclins bind to and activate CDKs. The activity and sub-
strate specificity of any given CDK is primarily defined by
the particular cyclin to which it is bound.
' state. In molecular terms, this means that cells initiate DNA
• Cyclins are only present during the cell cycle stage that
they trigger and are absent in other cell cycle stages.
• Cyclins not only regulate a particular cell cycle stage but
also set in motion a series of events in preparation for the
next cell cycle stage. In this way, they propel the cell cycle
forward.
Cyclins are divided into four classes defined by their
.· presence and activity during the cell cycle: G 1 cyclins, G 1/S
cyclins, S phase cyclins, and mitotic cyclins (see Table 19-1 ).
The different types of cyclins are quite distinct from each
other in protein sequence, but all of them contain a con-
served 100 amino acid region known as the cyclin box and
possess similar three-dimensional structures.
(c) High-activity cyclin A-CDK2
extensively with cyclin A, moves several angstroms into the catalytic
cleft, repositioning key catalytic side chains required for the phos-
photransfer reaction. The red ball marks the position of the threonine
(Thr-160) whose phosphorylation activates CDKs. (c) Phosphorylated,
high-activity cyclin A-CDK2 complex. The conformational changes
induced by phosphorylation of the activating threonine (red ball) alter
the shape of the substrate-binding surface, greatly increasing the
affinity for protein substrates. [Courtesy of P. D. Jeffrey. See A. A. Russo et al.,
1996, Nature Struct. Bioi. 3:696.]
The G 1 cyclins are the lynchpin in coordinating the cell
cycle with extracellular events. Their activity is subject to
regulation by signal transduction pathways that sense the
presence of growth factors or cell proliferation inhibitory
signals. In metazoans, G 1 cyclins are known as cyclin Ds,
and they bind to CDK4 and CDK6. G 1 cyclins are unusual in
that their levels do not fluctuate in a specific pattern during
the cell cycle. Instead, in response to macromolecule biosyn-
thesis and extracellular signals, their levels gradually increase
throughout the cell cycle.
The G 1/S cyclins accumulate during late Gh reach peak
levels when cells enter S phase, and decline during S phase
(see Figure 19-11). They are known as cyclin E in metazoans
and bind to CDK2. The main function for cyclin t-CDK2
complexes, together with cyclin D-CDK4/6, is to trigger the
G 1-S phase transition. This transition is known as START
and is defined as the point at which cells are irreversibly
committed to cell division and can no longer return to the G 1
replication as well as duplicate their centrosomes, which is
the first step in the formation of the mitotic spindle that will
be used during mitosis.
S phase cyclins are synthesized concomitantly with G 1 cy-
clins, but levels remain high throughout S phase and do not
decline until early mitosis. Two types of S phase cyclins trigger
S phase in metazoans: cyclin E, which can also promote entry
into the cell cycle and is therefore also a G 1/S cyclin, and cyclin
A. Both cyclins bind CDK2 (see Table 19-1) and are directly
responsible for DNA synthesis. As we will see in Section 19.4,
these protein kinases phosphorylate proteins that activate
DNA helicases and load polymerases onto DNA.
Mitotic cyclins bind CDK l to promote entry into and
progression through mitosis. The metazoan mitotic cyclins
19.3 Regulation of CDK Activity 885
are cyclin A and cyclin B (note cyclin A can also trigger S
phase when bound to CDK2). Mitotic cyclin-CDK com-
plexes are synthesized during S phase and G 2 , but as we will
see shortly, their activities are held in check until DNA syn-
thesis is completed. In Section 19.5, we will see that once
activated, mitotic CDKs promote entry into mitosis by phos-
phorylating and activating hundreds of proteins to promote
chromosome segregation and other aspects of mitosis. Their
inactivation dunng anaphase prompts cells to exit mitosis,
which involves the disassembly of the mitotic spindle, chro-
mosome decondensation, the re-formation of the nuclear
envelope, and eventually cytokinesis.
Mitotic cyclins were the first cyclins to be discovered,
and it was their characterization that led to the discovery of
the oscillatory nature of the activities that govern cell cycle
progression. The experiment that led to their discovery is
described at the end of this chapter as a classic experiment in
cell biology (see Classic Experiment 19.1 , Figure 1).
The fact that cyclins are the rate-limiting proteins in trigger-
ing cell cycle transitions was discovered using the embryonic
(a) Untreated extract
Addition of
j s~erm nuclei
Time~
(c) RNase-treated extract+ wild-type mitotic cyclin mRNA
Addition of
sperm nuclei
i ~
Time~
- =Mitotic CDK activity
- =Mitotic cyclin concentration
EXPERIMENTAL FIGURE 19-13 Mitotic cyclins are rate
limiting for mitosis. In all cases, mitotic CDK activity and mitotic cyclin
concentration were determined at various times after addition of
sperm nuclei to a Xenopus egg extract treated as indicated in each
panel. Microscopic observations determined the occurrence of early
mitotic events {blue shading), including chromosome condensation
886 CHAPTER 19 • The Eukaryotic Cell Cycle
extract system. Using egg extracts from frogs, researchers
showed that mitotic cyclins are sufficient to induce mitosis.
Cytoplasmic extracts prepared from unfertilized Xenopus
eggs contain all the materials (mRNAs and proteins) re-
quired for multiple cell cycles. When nuclei prepared from
Xenopus sperm (sperm nuclei are used in this experiment
because they are readily isolated in large numbers) are added
to such an egg extract, the nucleus and DNA inside it are
induced to behave a~ if progressing through the cell cycle;
that is, they replicate their DNA and then undergo mitosis in
the extract. Concomitant with the final stages of mitosis, cy-
clin levels decline (Figure 19-13a). Researchers wanted to
determine whether this protein with its fluctuating levels in
fact had the ability to induce mitosis. Because mitotic cyclins
appeared to be unstable at the end of mitosis, the researchers
reasoned that removing the mRNA from the egg extract
would prevent the new synthesis .of all unstable proteins in
the next cell cycle, including that of the mitotic cyclins (all
other stable proteins would still be present in the extract). To
do this, they digested all mRNAs with a low concentration of
(b) RNase-treated extract
Addition of
sperm nuclei
i ~
Time~
(d) RNase-treated extract + nondegradable mitotic cyclin mRNA
Addition of
sperm nuclei
i ~
"'1<'------ Mitotic arrest -----~3>1
Time~
=Early mitotic events
= Late mitotic events
and nuclear envelope disassembly, and of late events {orange shading),
including chromosome decondensation and nuclear envelope
reassembly. See text for discussion. [See A. W. Murray et al.. 1989, Nature
339:275; adapted from A. Murray and T. Hunt, 1993, The Cell Cycle: An Introduction,
W. H. Freeman and Company.)
RNase, which then was inactivated by addition of a specific
inhibitor. This tr eatment destroys mRNAs without affecting
the tRNAs and rRNAs required for protein synthesis. When
sperm nuclei were added to the RNase-treated extracts, the
nuclei replicated their DNA, but they did not undergo mito-
sis. Furthermore, mitotic cyclin protein was not detected
(Figure 19-13b). Addition of mitotic cyclin mRNA, pro-
duced in vitro from cloned mitotic cyclin eDNA, to the
RNase-treated egg extract induced mitosis as observed with
the untreated egg extract (Figure l9-13c). Since mitotic cy-
clin is the only newly synthesized protein under these condi-
tions, these results demonstrate that the protein is the only
one limiting for entering mitosis. Subsequent studies showed
that G 1/S phase cyclins have similar properties. Their ex-
pression is sufficient to promote entry into the cell cycle,
and therefore all the other proteins needed for cell cycle
entry are present in unl imited amounts. It is thus clear that
the regu lation of cyclin levels is an essential aspect of the
cukaryotic cell cycle. As we will see in the following section,
cells utilize mu ltiple mechanisms to restrict cyclins to the
appropriate cell cycle stage and to keep them at the right
concentration.
Cyclin Levels Are Primarily Regulated
by Protein Degradation
Multiple mechanisms ensure that CDKs are active in the right
stage of the cell cycle. Table 19-2 lists these key regulators of
CDK!>. In this section we will discuss how the regulation of
cyclin levels is brought about. The timely activation of CDKs
depends, in part, on the presence of the appropriate cyclins in
the cell cycle stage where they are needed. Transcriptional
control of the cyclin subunits is one mechanism that ensures
proper temporal expression of tbe cyclins. In somatic cells and
yeast, waves of transcription factor activities help establish
waves of cyclin activity. A general principle here is that an
earlier wave of transcriptional activity helps produce the fac-
tors essential to generate a subsequent transcriptional wave.
As we will see in Section 19.4, transcription of the G 1/S phase
cyclins is promoted by the E2F transcription factor complex.

TABLE 19-2 Regulators of Cyclin -CDK Activity

Type of Regulator Function

Kinases and Phosphatases

CAK kinase Activates CDKs

Weel kinase Inhibits CDKs

Cdc25 phosphatase Activates CDKs

Cdc14 phosphatase Activates Cdh l to degrade mitotic cyclms

Cdc25A phosphatase Activates vertebrate S phase CDKs

Cdc25C phosphatase Activates vertebrate mitotic CDKs

Inhibitory Proteins

Sicl Binds and inhibits S phase CDKs

CKis p27KIPI, p57KII'2 , and p21 Cll' Bind and inhibit CDKs

INK4 Binds and inhibits G 1 CDKs

Rb Binds E2Fs, preventing transcription of multiple cell cycle genes

Ubiquitin-Protein Ligases

SCF Degradation of phosphorylated ~1cl or p27"JP 1 to activateS phase CDKs

APC/C + Cdc20 Degradation of securin, initiating anaphase. Induces degradation of B-type cyclins

APC/C + Cdhl Degradation of B-type cyclins in G 1 and geminin in metazoans to allow loading of replicative
helicases on DNA replication origins

19.3 Regulation of CDK Activity 887

Among the many other genes that E2F transcribes are the
transcription factors that will promote the synthesis of mitotic
cyclins.
The most important regulatory control that restricts cyclins
to the appropriate cell cycle stage is ubiquitin-mediated, protea-
some-dependent protein degradation. Because protein degra-
dation is an irreversible process, in the sense that the protein
can only be replenished through de novo protein synthesis, this
regulatory mechani~m i~ ideal to ensure that the cell cycle en-
gine is driven forward and cells cannot "go backward" in the
cell cycle. In other words, once a particular cyclin is degraded,
the processes that it activated can no longer take place.
Recall that during ubiquitin-mediated protein degrada-
tion, ubiquitin-protein ligases polyubiquitinylate substrate
proteins, marking them for degradation by the proteasomc
(see Figure 3-29). Cyclins are degraded through the action of
two different ubiquitin-protein ligases, SCF (named after the
first letters of its constituents, Skp 1, Cullin, f-box proteins)
and the anaphase-promoting complex or cydosome (abbrevi-
ated as APC/C in this chapter). SCF controls the G 1-S phase
transition by degrading G/S phase cyclins and, as we will see
m detail shortly, CDK inhibitory proteins. The APC/C de-
grades S phase and mitotic cyclins, thereby promoting the exit
from mitosis. APC/C also controls the onset of chromosome
segregation at the metaphase-anaphase transition by degrad-
ing an anaphase inhibitory protein (discussed in Section 19.6).
The SCF and APC/C are multisubunit ubiquitin ligases
that belong to the RING finger family of ubiquitin ligases.
Despite the fact that SCF and APC/C belong to the same
ubiquitin ligase family, their regulation is quite different.
The SCF recognizes its substrates only when they are phos-
phorylated. The SCF is continuously active throughout the
cell cycle, and cell-cycle-regulated phosphorylation of its
substrates ensur~s that they are only degraded at certain
stages of the cell cycle. In the case of APC/C-dependent pro-
tein degradation, the regulation is reversed. Substrates are
recognizable throughout the cell cycle, but the activity of the
APC/C is cell cycle regulated. APC/C is activated by phos-
phorylation at the metaphase-anaphase transition, through
the action of mitotic CDKs and other protein kinases. The
APC/C is then active throughout the rest of mitosis and dur-
ing G 1 to promote the degradation of cyclins and other mi-
totic regulators (see Figure 19-11 ). Substrate specificity of
active, phosphorylated APC/C is determined in part by its
association with one of two related substrate-targeting fac-
tors called Cdc20 and Cdh I. During anaphase APC/C bound
to Cdc20 ubiquitinylates proteins that lead to chromosome
segregation, while during telophase and Gh APC/C-Cdh 1
targets different substrates for degradation. The substrates
of the APC/C conrain recognition motifs. The first to be
identified is the destruction box. It is found in most S phase
and mitotic cyclins. This destruction box is both necessary
and sufficient to target proteins for degradation.
Today we know that cyclin degradation at the appropriate
cell cycle transition is essential for cell cycle progression. The
importance of cyclin degradation was again first demonstrated
in frog egg extracts. Recall that the accumulation of mitotic
888 CHAPTER 19 • The Eukaryotic Cell Cycle
cyclins not only coincided with entry into mitosis, but its dis-
appearance occurred concomitantly with exit from mitosis.
This finding raised the possibility that mitotic cyclin degrada-
tion was necessary for cells to exit from mitosis. To test this,
researchers added an mRNA encoding a nondegradable mi-
totic cyclin (lacking the destruction box) to a mixture of
RNase-treated Xenopus egg extract and sperm nuclei. As
shown in Figure 19-13d, entry into mitosis occurred on sched-
ule, but exit trom mitosis did not. This experiment demon-
strates that mitotic exit requires the degradation of mitotic
cyclin. Later studies showed that inhibiting the degradation of
other cyclins also severely affected cell cycle progression, indi-
cating that ubiquitin-mediated degradation of cyclins is an es-
sential aspect of the eukaryotic cell cycle.
CDKs Are Regulated by Activ,ating
and Inhibitory Phosphorylation
Regulating the levels of cyclins is not the only mechanism
that controls CDK activity. Activating and inhibitory phos-
phorylation events on the CDK subunit itself are essential to
control cyclin-CDK activity. Phosphorylation of a threonine
residue near the active site of the enzyme is required for CDK
activity. This phosphorylation is mediated by the CDK-acti-
vating kinase (CAK). In some organisms cyclin binding is a
prerequisite for CAK phosphorylation, whereas in others this
phosphorylation event occurs prior to cyclin binding. Al-
though the sequence of assembling active CDKs differs be-
tween organisms, it is clear that CAK phosphorylation of
CDK is not a rate-limiting step in CDK activation. CAK ac-
tivity is constant throughout the cell cycle and phosphory-
lates the CDK as soon as a cyclin-CDK complex is formed.
Two inhibitory phosphorylations on CDK play a critical
role in controlling CDK activity. In contrast to the CAK-in-
duced activating phosphorylation, these inhibitory phos-
phorylations are regulated. A highly conserved tyrosine (Y15
in human CDKs) and an adjacent threonine (T14 in humans)
are subject to regulated phosphorylation. Both residues are
situated in the ATP binding pocket of the CDK, and their
phosphorylation most likely interferes with positioning of
ATP in the pocket. Changes in the phosphorylation of these
sites are essential for the regulation of mitotic CDKs and
have also been implicated in the control of G/S and S phase
CDKs. As we will see in Section 19.5, a highly conserved
kinase called Weel brings about this inhibitory phosphory-
lation, and a highly conserved phosphatase called Cdc25
mediates dephosphorylation.
CDK Inhibitors Control Cyclin-CDK Activity
So far, we have discussed the impvrtam:e of regulating cyclin
protein levels and of CDK phosphorylation in controlling
CDK activity. The final layer of control that is of critical
importance in the regulation of CDKs is a family of proteins
known as CDK inhibitors, or CKis, that directly bind to the
cyclin-CDK complex and inhibit its activity. As we will see
in Section 19.4, these proteins play an especially important
role in the regulation of the G 1-S phase transition and its
integration with extracellular signals. It thus comes as no
surprise that the genes encoding these CKls are often found
mutated in human cancers (discussed in Chapter 24).
All eukaryotes harbor CKls involved in regulating S
phase and mitotic CDKs. Although these inhibitors display
little sequence simi larity, they are all essential to prevent pre-
mature activation of S phase and M phase CDKs. Inhibitors
of(; 1 C.J1Ks play an essential role in mediating a G 1 arrest i11
response to proliferation inhibitory signals. A class of CKis
called INK4s (inhibitors of kinase 4 ) includes several small,
closely related proteins that interact only with the G 1 CDKs.
Binding of INK4s to CDK4 and CDK6 blocks their interac-
tion with cyclin D and hence their protein kinase activity. A
second class of CKis found in metazoan cells consists of
three proteins-p21 ur, p27K 1P\ and p57K 1P2 . These CKis in-
hibit G 1/S phase CDKs and S phase CDKs and must be de-
graded before DNA replication can begin. As we will discuss
in Section 19.7, p21 CIP plays an important role in the response
of metazoan cells to DNA damage. CKis regulating G 1 CDKs
play a critical role in preventing tumor formation. For exam-
ple, both copies of the INK4 gene, which encodes p16, are
found inactivated in a large fraction of human cancers.
Special CDK Alleles Led to the Discovery
of CDK Functions
Different CDKs initiate different cell cycle phases by phos-
phorylating specific proteins. It is now clear that rather than
phosphorylating a small number of proteins that in turn
(a)
FIGURE 19-14 ATP analog- dependent CDK mutant. (a) Represen-
tation of the ATP-binding and catalytic sites of wild-type 5. cerevisiae
CDKl (called Cdc28 in budding yeast). Bound ATP and a phenylalanine
side chain (purple) in the vicinity of the binding pocket are shown in
stick format. (b) Bulky ATP analogs such as those containing a benzyl
group bound to the N6 amino nitrogen are too large to fit into the
ATP-binding pocket of wild-type protein kinases and thus cannot be
initiate a certain cell cycle stage, CDKs phosphorylate a myr-
iad of substrates, thereby directly initiating all aspects of a
given cell cycle phase. Analysis of a small number of sub-
strates has provided examples that show how phosphoryla-
tion by mitotic CDKs mediates many of the early events of
mitosis: chromosome condensation, formation of the mitotic
spindle, and disassembly of the nuclear envelope. We will
discuss these events in detail in the sections that follow.
In recent years systematic efforts to identify all CDK sub-
strates have been initiated. The challenge in identifying sub-
strates of a particular kinase is how to distinguish that kinase's
phosphorylation events from those carried out by other ki-
nases. A breakthrough in understanding which proteins are
targets of CDKs was facilitated by the engineering of a CDK
mutant in budding yeast that can utilize an analog of ATP that
is not bound by other kinases (Figure 19-14). This ATP ana-
log has a bulky benzyl group attached to N 6 of the adenine,
which makes the analog too large to fit into the ATP-binding
pocket of wild-type protein kinases. However, the ATP-bind-
ing pocket of the mutant CDK was modified to accommodate
this large ATP analog. Consequently, only the mutant CDK
can utilize this ATP analog as a substrate for transferring its 'Y
phosphate to a protein side chain. When the N 6 -benzyl ATP
analog with a labeled 'Y phosphate was incubated with yeast
cell extracts containing a recombinant mitotic CDK contain-
ing the altered ATP-binding pocket, multiple proteins were
labeled. True yeast mitotic CDK substrates could be verified
among these potential substrates by treatment of cells express-
ing the mutant CDK with a similar derivative of another ATP
analog that binds the mutant CDK in the same way as the
(b)
uttltzed by them. In the 5. cerevisiae CDK mutant, the phenylalanine at
position 88 is changed to glycine, which lacks a large side chain. The
mutant exhibits high protein kinase activity using N6-(benzyi)ATP.
These models of 5. cerevisiae CDK are based on crystal structures of the
PKA kinase domain, which shares extensive homology with the kinase
domain of 5. cerevisiae CDK. [See J. A. Ubersax et al., 2003, Nature 425:859;
K. Shah et al., 1997, Proc. Nat'/. Acad. Sci. USA 94:3565.]
19.3 Regulation of CDK Activity 889
N 6 -benzyl ATP analog but that cannot be used for substrate
phosphorylation. Because of the bulky benzyl group, it is ste-
rically blocked from binding to all other kinases, consequently
inhibiting only the mutant CDK. When cells expressing the
mutant CDK were treated with this specific CDK inhibitor,
most of the putative mitotic CDK targets identified initially
were found in a dephosphorylaryed state, indicating that these
proteins are indeed phosphorylated by the CDK in vivo as
well as in vitro. In yeast, th1s procedure identitied most of the
known CDK substrates plus more than 150 additional yeast
proteins. Similar approaches have also been used in mamma-
lian cells to identify CDK substrates. For example, a hunt for
substrates of the S phase CDK cyclin A-CDK2 revealed 180
potential substrates. These are currently being analyzed for
their functions in cell cycle processes.
KEY CONCEPTS of Section 19.3
Regulation of CDK Activity
• Cyclin-dependent kinases are activated by cyclin subunits.
Their activity is controlled at multiple levels.
Different cyclin subunits activate CDKs at different cell
cycle stages. Cyclins are present only in the cell cycle stages
that they promote.
Protein degradation is the key mechanism responsible for
restricting cyclins to the appropriate cell cycle stage. This
degradation is mediated by the u biquitin-proteasome system
and the ubiquitin ligases APC/C and SCF.
Activating and inhibitory phosphorylation on the CDK
subunit contributes to the regulation of CDK activity.
CDK inhibit9rs (CKls) inhibit CDK activity by directly
binding to the cyclin-CDK complex.
• CDKs initiate every aspect of each cell cycle stage by phos-
phorylating many different target proteins. Systematic efforts
using protein kinases engineered to bind only modified forms of
ATP have led to the identification of many of these substrates.
19.4 Commitment to the Cell Cycle
and DNA Replication
The previous section described the multiple mechanisms that
control the different cyclin-CDK complexes. In this and the
following two sections we will now examine each cell cycle
stage carefully and discuss how a particular cell cycle stage is
induced and controlled. We will examine how cells initiate
DNA replication and mitosis and how chromosomes are seg-
regated. We will focus on how cyclin-CDK complexes and
other key cell cycle regulators impact each cell cycle phase
and examine the mechanisms that coordinate their activities.
This section investigates how cells decide whether or not
to undergo cell division and how DNA replication is initi-
ated. The process of cell cycle entry is well understood in
890 CHAPTER 19 • The Eukaryotic Cell Cycle
budding yeast, and it was in this organism that the molecular
mechanisms underlying this cell cycle transition were initially
elucidated. We will therefore first examine the molecular
events governing cell cycle entry in budding yeast. We will
then investigate the striking similarities between the pathways
that govern cell cycle entry in yeast and metazoan cells and
discuss the realization that many genes involved in this deci-
sion are frequently found mutated in cancer. Next we will see
that the decision to enter the cell cycle is influenced by extra-
cellular events and learn about the signaling mechanisms that
convey these environmental cues to the cell cycle machinery.
Finally, we will discuss the molecular mechanisms that gov-
ern initiation of DNA replication. We will describe how deg-
radation of an S phase CKI is essential for this process and
discover how CDKs ensure that DNA replication occurs only
during 5 phase and then only once.
,
Cells Are Irreversibly Committed to Cell Division
at a Cell Cycle Point Called START
In most eukaryotic cells, the key decision of whether or not
a cell will divide is made at the point of whether or not to
enter 5 phase. In most cases, once a cell has. become commit-
ted to entering the cell cycle, it must complete it. The bud-
ding yeast Saccharomyces cerevisiae regulates its proliferation
in this manner, and much of our current understanding of
the molecular mechanisms controlling entry into the cell
cycle originated with genetic studies of S. cerevisiae.
When S. cerevisiae cells in G 1 have grown sufficiently in
size, they begin a program of gene expression that leads to
entry into the cell cycle. If G 1 cells are shifted from a rich
medium to a medium low in nutrients before they reach a
critical size, they remain in G 1 and grow slowly until they are
large enough to enter the cell cycle. However, once G 1 cells
reach the critical size, they become committed to completing
the cell cycle, entering 5 phase and proceeding through G 2
and mitosis, even if they are shifted to a medium low in nu-
trients. The point in late G 1 when S. cerevisiae cells become
irrevocably committed to entering and traversing the entire
cell cycle is called START.
CDK activity is essential for entry into 5 phase. This was
first realized in budding yeast, where temperature-sensitive
mutants in the gene encoding CDKl arrest in Gl> failing to
form a bud and to initiate DNA replication (CDKl is the
only CDK in the budding yeast genome and is known as
CDC28). We now know that a CDK cascade triggers entry
into the cell cycle. G 1 CDKs stimulate the formation of GtfS
phase CDKs, which then initiate bud formation, centrosome
duplication, and DNA replication. In yeast, the G 1 cyclin
gene is called CLN3 (Figure 1 9-15a). Its mRNA is produced
at a nearly constant level throughout the cell cycle, but ItS
translation is regulated in response to nutrient levels and, as
we will see shortly, CLN3 is a lynchpin in coupling cell cycle
entry to nutrient signals. Once sufficient Cln3 is synthesized
from its mRNA, Cln3-CDK complexes phosphorylate and
inactivate the transcriptional repressor Whi5. Phosphoryla-
tion of Whi5 promotes its export out of the nucleus, allowing
·.
·,
(a) S. cerevisiae
...
- (Cin3-CDKs) - Nutrients
mRNA
CLN1 or CLN2 gene
START
Budding/
S phase
1~Spindle pole body
duplication
FIGURE 19-15 Control of the G,-S phase transition (a) In budding
yeast, Cln3-CDK activity rises during G, and is controlled by nutrient
availability. Once sufficiently active, the kinase phosphorylates the
transcriptional inhibitor WhiS, promoting its export from the nucleus.
This causes the transcription factor complex SBF to induce the transcrip-
tion of the G1/S phase cyclins CLN 1and CLN2 and of other genes whose
products are needed for DNA replication. G1/S phase CDKs further
phosphorylate WhiS, promoting further CLN1 and CLN2 transcription.
Once sufficiently high levels of G/S phase CDKs have been produced,
START is traversed. Cells enter the cell cycle: they initiate DNA replication,
the transcription factor complex SBF to induce transcription
of the G 1/S phase cyclin genes CLNl and CLN2 as well as
other genes important for DNA replication. Once produced,
Cln1/2-CDKs contribute to further Whi5 phosphorylation.
This positive feedback loop ensures the rapid accumulation
of G 1/S phase CDKs. Once a critical level of Clnl/2-CDKs is
reached, these G/S phase CDKs promote bud formation, entry
into S phase, and the duplication of the centrosome (also
known as the spindle pole body, which later in the cell cycle
will organize the mitotic spindle). This state of G /S phase
CDK activity, sufficient to initiateS phase, bud formation, and
centrosome duplication, is the molecular definition of START.
The E2F Transcription Factor and Its
Regulator Rb Control the G1-S Phase
Transition in Metazoans
The molecular events governing entry into S phase in mam-
malian-and in fact all metazoan--cells are remarkably simi-
lar to those of budding yeast (Figure l9-15b). G 1 cyclins arc
present throughout G 1 and are often found to be expressed at
increased levels in response to growth factors. In turn, the G 1
(b) Metazoans
- Growth factors
mRNA
Cycl in E or cycl in A g en e
START
S phase
/ Centrosome
duplication
bud formation, and spindle pole body duplication. (b) In vertebrates,
G1-CDK activity rises during G1 and is stimulated by the presence of
growth factors. When signaling from mitogens is sustained, the resulting
cyclin D-CDK4/6 complexes begin phosphorylating Rb, releasing some
E2F, which stimulates transcription of the genes encoding cyclin E, CDK2,
and E2F itself. The cyclin E-CDK2 complexes further phosphorylate Rb,
resulting in a positive feedback loop that leads to a rapid rise in the
expression and activity of both E2F and cyclin E-CDK2. Once G1/ S phase
CDK is sufficiently high, cells traverse START. They commence DNA
replication and centrosome duplication.
CDKs activate members of a small family of related tran-
scription factors, referred to collectively as E2F transcription
factors (E2Fs). During Gt. E2Fs are held mactive through
their association with the retinoblastoma protein (Rb), and
G 1 CDKs activate E2Fs by phosphorylating and inactivating
Rb. E2Fs then activate genes encoding many of the proteins
involved in DNA synthesis. They also stimulate transcription
of genes encoding the G 1/S phase cyclins and the S phase cy-
clins. Thus the E2Fs function in late G 1 similarly to the S.
cerevisiae transcription factor complex SBF.
Key to the regulation of E2F function is the Rb protein.
When E2Fs are bound to Rb, they function as transcriptional
repressors. This is because Rb recruits chromatin-modifying
enzymes that promote deacetylation and methylation of spe-
cific histone lysines, causing chromatin to assume a condensed,
transcriptionally in;~ctive form. Rb was initially identified as
the gene mutated in retinoblastoma, a childhood cancer of the
retina. Subsequent studies found RB to be inactivated in al-
most all cancer cells either by mutations in both alleles of RB
or by abnormal regulation of Rb phosphorylation.
Rb protein regulation by G 1 CDKs in mammalian cells is
analogous to that of Cln3-CDK regulation of Whi5 in yeast.
19.4 Commitment to the Cell Cycle and DNA Replication 891
Phosphorylation on multiple sites by G 1 CDKs prevents Rb
association with E2Fs and promotes its export out of the
nucleus. This allows E2Fs to activate transcription of genes
required for entry into S phase. Once the expression of genes
coding for G 1/S cyclins and CDK are induced by phosphoryla-
tion of some of the Rb molecules, the resulting G 1/S phase
CDK complexes further phosphorylate Rb in late G 1• This is
one of the principal biochemical events responsible for passage
through START. Since E2F also stimulates its own expression
and that of the G 1/S cyclin-CDKs, positive cross-regulation of
E2F and G 1/S cyclin-CDKs produces a rapid rise of both
activities in late G 1•
As they accumulate, S phase CDKs and mitotic CDKs
maintain Rb protein in the phosphorylated state throughout
the S, G 2, and early M phases. After cells complete anaphase
and enter early G 1 or G 0, a fall in all cyclin-CDK activities
leads to dephosphorylation of Rb. As a consequence, hypo-
phosphorylated Rb is available to inhibit E2F activity during
early G 1 of the next cycle and in G 0 -arrested cells. Thus G/S
phase CDK activity remains low until cells decide to enter a
new cell cycle and G 1 CDKs break the inhibitory grasp of Rb
over E2F.
Extracellular Signals Govern Cell Cycle Entry
Whether or not cells enter the cell cycle is influenced by extra-
cellular as well as intracellular signals. Unicellular organisms
such as yeast, for example, enter the cell cycle only when they
have reached an appropriate size, known as the critical cell
size. This critical size, in turn, is controlled by nutrients avail-
able in the environment. This coordination between cell size
and cell cycle entry will be discussed in Section 19. 7. Here we
restrict our discussion to the fact that G 1 cyclin synthesis is
responsive ro protein synthesis rate, which is in turn con-
trolled by pathways that are regulated by nutrients in the en-
vironment. This link between the macromolecule biosynthesis
and cell cycle machinery is best understood in budding yeast.
In this organism, the G 1 cyclin transcript CLNJ contains a
short upstream open-reading frame that inhibits translation
initiation at the Cln3 open reading frame when nutrients are
limited. This inhibition is diminished when nutrients are in
abundance. In the presence of sufficient nutrients, the TOR
pathway, which senses nutrients and growth factor signals, is
active and the pathway stimulates translational activity (see
Figure 8-30). Since Cln3 is a highly unstable protein, its con-
centration fluctuates with the translation rate of its mRNA.
Consequently, the amount and activity of Cln3-CDK com-
plexes, which depend on the concentration of Cln3 protein, is
largely regulated by nutrient levels.
In multicellular organisms, cells are surrounded by nutri-
ents, and as such, usually nutrients are not rate limiting for
proliferation. Rather, cell proliferation is controlled by the
presence of growth-promoting factors (mitogens) and growth
inhibitory factors (anti-mitogens) in the cell surroundings. Ad-
dition of mitogens to G 0-arrested mammalian cells induces-
as we discussed in Chapter 16-receptor tyrosine-kinase-linked
signal transduction pathways that initiate signal transduction
892 CHAPTER 19 • The Eukaryotic Cell Cycle
cascades that ultimately influence transcription and cell cycle
control. They do so in multiple ways.
Mitogens activate the transcription of multiple genes. Most
of these genes fall into one of two classes-early-response or
delayed-response genes-depending on how soon their en-
coded mRNAs appear. Transcription of early response genes is
induced within a few minutes after addition of growth factors
by signal transduction cascades that activate preexisting tran-
scription factors in the cytosol or nucleus (see Chapter 16).
Many of the early-response genes encode transcription factors,
such as c-Fos and c-Jun, that stimulate transcription of the de-
layed response genes. The early response transcription factors
AP-1 and Myc induce the transcription of genes encoding G 1
cyclins and CDKs. In addition to being controlled by transcrip-
tion of the genes encoding the G 1 cyclins, G 1 CDKs are regu-
lated by CKis. The CKI p15INK4b is a potent CDK inhibitor.
In some tissues mitogens inhibit the production of this CKI by
inhibiting its transcription.
Cell proliferation in many tissues is not only regulated by
proliferation promoting mitogens but also by anti-mitogens,
which prevent entry into the cell cycle. Similarly, during dif-
ferentiation, cells cease to divide and enter G 0 . Some differenti-
ated cells (e.g., fibroblasts and lymphocytes) cttn be stimulated
to re-enter the cycle and replicate. Many postmitotic differenti-
ated cells, however, never re-enter the cell cycle to replicate
again. Anti-mitogens and differentiation pathways prevent the
accumulation of G 1 CDKs. They antagonize the production of
G 1 cyclins and induce the production of CKis. Transforming
growth factor 13 (TGF-13) is an important anti-mitogen. This
hormone induces a signaling cascade that brings about G 1 ar-
rest by inducing the expression of p15INK4b. As we will see in
Chapter 24, the signaling pathways that regulate G 1 CDKs are
found mutated in most human cancers.
Degradation of an S Phase CDK Inhibitor
Triggers DNA Replication
Entry into S phase is defined by the unwinding of origins of
DNA replication. The molecular events leading to this event
are best understood in S. cerevisiae. G/S phase CDKs play
an essential role in this process. They turn off the degrada-
tion machinery that degrades S phase cyclins during exit
from mitosis and G 1 and induce the degradation of a CKI
that inhibits S phase CDKs.
One of the important substrates of the G 1/S phase cyclin-
CDK complexes is Cdh l. During late anaphase, this substrate
targeting factor directs the APC/C to ubiquitinylate substrate
proteins including S phase and mitotic cyclins, marking them
for proteolysis by proteasomes. The APC/C-Cdh 1 remains ac-
tive throughout GI> preventing the premature accumulation of
S phase and mitotiL cydin~. Phosphorylation of Cdhl by G 1/S
cyclin-CDKs causes it to dissociate from the APC/C complex,
inhibiting further ubiquitinylation of S phase and mitotic cy-
clins during late G 1 (Figure 19-16). This, combined with the
induced transcription of S phase cyclins during late Gl> allows
S phase cyclin proteins to accumulate as G 1/S cyclin-CDKs
levels rise. Later in the cell cycle, S phase and mitotic CDKs
Exit from mitosis S phase and mitosis
and G 1
G 1/S phase CDKs
Cdc14 phosphatase
S phase, mitotic S phase, mitotic
cyclins unstable cyclins stable
FIGURE 19-16 Regulation of S phase and mitotic cyclin levels in
budding yeast. In late anaphase, t he anaphase-promoting complex
(APC/C) ubiquitinylates S phase and mitotic cyclins. APC/C activity is
directed toward mitotic cyclins by a specificity factor, called Cdh 1. Cdh 1
activity is regulated by phosphorylation. During exit from mitosis and
G1, the specificity factor is dephosphorylated and active; during S
phase and mitosis, Cdhl is phosphorylated and dissociates from the
APC/C. and the ubiquitin ligase is inactive. The G1/S-phase CDKs, which
themselves are not APC/C-Cdhl substrates, phosphorylate Cdhl at the
G1-S phase transition. A specific phosphatase called Cdc74 removes the
regulatory phosphate from the specificity factor late in anaphase.
take over to maintain Cdhl in the phosphorylated and hence
inactive state . Only as mitotic CDKs decline and a protein
phosphatase known as Cdc14 is activated are these inhibitory
phosphates removed from Cdh 1, leading to its reactivation. In
mammalian cells similar mechanisms are responsible for sta-
bilizing S phase and mitotic cyclins, but the phosphatase in-
volved in dephosphorylation of Cdhl has not been identified.
In S. cerevisiae, as the S phase cyclin-CDK heterodimers
accumulate in late G 1 following the inactivation of APC/C-
Cdhl, they are immediately inactivated by binding of a CKI
called Sicl that is expressed late in mitosis and in early G 1
(Figure 19-17). Because Sicl specifically inhibits S phase and
M phase CDK complexes but has no effect on the G 1 CDK
START
Polyubiquitinylation of
phosphorylated Sic1;
proteasomal
degradation
fJ
FIGURE 19·17 Control of S phase onset inS. cerevisiae by
regulated proteolysis of the S phase inhibitor, Sicl. The S phase
cyclin-CDK complexes begin to accumulate in G 1 but are inhibited by
Sicl. This inhibition prevents initiation of DNA replication until the cells
have completed all G, events. G1/S phase CDKs assembled in late G1
phosphorylate Sicl at multiple sites (step 0 ), marking it for
and G 1/S phase CDK complexes, it functions as an S phase
inhibitor. Initiation of DNA replication occurs when the
Sicl inhibitor is precipitously degraded following its ubiqui-
tinylation by the SCF ubiquitin-protein ligase.
Degradation of Sicl is induced by its phosphorylation by
G/S phase CDKs (see Figure 19-17). It must be phosphory-
lated at at least six sites, which are relatively poor substrates
for the G/S phase CDKs, before it is bound sufficiently well
by SCF to be ubiquitinylated. Multiple, puur G 1/S phase CDK
phosphorylation sites lead to an ultrasensitive, switch-like re-
sponse in Sicl degradation and hence precipitous activation
of S phase CDKs (Figure 19-18). If Sicl were inactivated fol-
lowing the phosphorylation of a single site, Sicl molecules
would start to get phosphorylated as soon as the levels of
G/5 phase CDK activity begin to rise, leading to a gradual
decrease in Sicl levels. In contrast, when several sites need to
be phosphorylated, at low levels of G/S phase CDK activity
only a few sites are phosphorylated and Sicl is not destroyed.
Only when G 1/S phase CDK levels are high is Sicl sufficiently
phosphorylated at multiple sites to target it for degradation.
Thus Sicl degradation occurs only when G 1/S phase CDK
activity has reached its peak and virtually all other G tiS phase
CDK substrates have been phosphorylated.
Once Sicl is degraded, the S phase cyclin-CDK complexes
induce DNA replication by, as we will see shortly, phosphor-
ylating several proteins involved in activating the replicative
helicases. This mechanism for activating the S phase cyclin-
CDK complexes-that is, inhibiting them as the cyclins are
synthesized and then precipitously degrading the inhibitor-
permits the sudden initiation of replication at large numbers
of replication origins. An obvious advantage of proteolysis
for controlling passage through this critical point in the cell
cycle is that protein degradation is an irreversible process,
ensuring that cells proceed in o ne direction through the cycle.
The dependence of this event on phosphorylation of multiple
poor phosphorylation sites makes the degradation of Sicl,
and hence the activation of DNA replication, abrupt.
!
DNA
-----~
0
replication
~
S phase
ubiquitinylation by the SCF ubiquitin ligase and subsequent protea-
soma l degradation (step fJ). The activeS phase CDKs then trigger
initiation of DNA synthesis (step lll by phosphorylating and recruiting
MCM helicase activators to DNA replication origins. [Adapted from R. W.
King et at., 1996, Science 274:1652.]
19.4 Commitment to the Cell Cycle and DNA Replication 893
(a) One optimal G1/S phase CDK site in Sic1
Sic1 G 1/S phase S phase
protein CDK activity CDK activity
~
0 and Only Once During the Cell Cycle
"'
Qi
> As discussed in Chapter 4, eukaryotic chromosomes are rep-
~ licated from multiple replication origins. Initiation of repli-
c
0"'
a':
G,
(b) Six sub-optimal G 1/S phase CDK site in Sic1
Sic1 G 1/S phase S phase
protein CDK activity CDK activity
~
0
"'
Qi
~
> tiation of DNA replication is controlled and the role of S
c phase CDKs in the process before turning to the mechanisms
'iii
0 whereby these kinases prevent re-initiation.
a':
G 1 ~ S phase
FIGURE 19-18 Six suboptimal G1 /S phase CDK phosphorylation
sites in Sic1 create a switch-like cell cycle entry. (a) A single optimal
G/S phase CDK site in Sicl would result in a sluggish G1-S phase
transition. As G1/S phase CDKs accumulate during G1, Sicl would get
progressively degraded. As a result, S phase CDKs would slowly rise.
Instead of an abrupt rise in S phase CDK activity, initiation of S phase
would be a drawn-out event. (b) Six suboptimal phosphorylation sites
ensure that Sicl is only fully phosphorylated and hence recognized by
the SCF when G/S phase CDKs have reached high levels. This also
ensures that Sicl degradation occurs rapidly and when G/S phase
CDKs have accomplished all their other G1 tasks. [Adapted from Nash
et al., 2001 , Nature 414:514-521, and D. 0. Morgan, 2006.]
Entry into S phase in metazoan cells is regulated in a
similar manner as in budding yeast. Like Sicl, the CKI p2 7
prevents the premature activation of S phase CDKs during
G 1. Unlike Sicl, however, this CKI inhibits both S phase
CDKs and G 1/S phase CDKs and has additi onal cell cycle
functions. For example, while inhibiting G /S phase CDKs
and S phase CDKs, p27 helps assemble and hence activate G 1
CDKs. Similar to the situation in yeast, however, p27 is re-
moved from cyclin-CDK complexes by ubiquitin-dependent
protein degradation. Two pathways contribute to p27 degra-
dation. On stimulation with mitogens, mitogen-activated
protein kinases phosphorylate p27, promoting its export
from the nucleus into the cytoplasm, where one of the cellu-
lar ubiquitin ligases, KPC, is found. A second pathway, anal-
ogous to the one operating on Sicl, targets p2 7 for
degradation at the G 1-S phase transition. As G 1/S phase
CDKs and S phase CDKs reach high levels during late G 1 and
early S phase, they begin to phosphorylate p27, targeting the
CKI for ubiquitinylation by SCF. Degradation of p27 causes
activation of G /S phase and S phase CDKs. These kinases
894 CHAPTER 19 • The Eukaryotic Cell Cycle
then initiate S phase by phosphorylating proteins important
for the initiation of DNA replication.
Replication at Each Origin Is Initiated Once
cation from Lhe~e origins occurs th rougho ut S phase.
However, no eu karyotic o rigin initiates more than once per
S phase. Moreover, S phase continues until replication from
multiple origins along the length of each chromosome results
in complete replication of the entire chromosome. These two
factors ensure that the correct gene copy number is main-
tained as cells proliferate.
S phase CDKs play an essential role in the regula tion of
DNA replication. The kinases initiate DNA replication only
at the G 1-S phase transition and prevent re-initiation from
origins that have already fired. We will first discuss how ini-
The mechanisms underlying the initiation of DNA replica-
tion are best understood in budding yeast; so we will focus
our discussion on this organism. H owever, it is important to
note that the proteins and mechanisms controlling the initia-
tion of DNA synthesis are essentially the same in all eukary-
otic species. A protein complex known as the origin-recognition
complex (ORC) is associated with all DNA replication
origins. In budding yeast, replication origins contain an
11-base-pair conserved core sequence to w hich ORC binds. In
multicellular o rganisms, origins of DNA replication lack a rec-
ognizable consensus sequence. Instead, chromatin-associated
factors target ORCs to the DNA. ORC and two add iti onal
replication initiation factors, Cdc6 and Cdtl , associate with
the ORC at origins during G 1 to load the replicative helicases
known as the MCM helicase complex onto DNA (Figure
19-19, step 0 ). The MCM helicases fu nction to unwind the
DNA during the initiation of DNA replication.
To ensure that origins fire only once at the beginning of S
phase, MCM helicase complex loading and its activation occur
at two opposing phosphorylation states. The MCM helicases
can only load onto the DNA in a state of low CDK activity
that occurs when CDKs are inactivated during exit from mito-
.. '
sis and during early G 1• In other words, MCM helicases load
onto DNA when they are unphosphorylated . In contrast, acti-
vation of MCM helicases and the recruitment of DNA poly-
merases to the unwound origin DNA are triggered by S phase
CDKs. Recall that S phase CDKs only become active when
G 1/S phase CDK levels reach their peak and CKis of S phase
CDKs are destroyed. It is then that phosphorylation by S phase
CDKs and a second heterodimeric protein kinase, DDK, acti-
vates the MCM helicase and recruits the polymcrases to the
sites of replication initiation (Figure 19-19, steps fJ and 10).
So how do S phase CDKs and DDK collaborate to initi-
ate DNA replication? ORC and the two other initiation fac-
tors, Cdc6 and Cdt1, recruit the MCM helicases to sites of
 - ~~ -- ----~==----===
~~; £"'ff~..£<'.£<'.£'<'.ff.£.'f'.£<'..£.'<:
[ •.
state
MCM helicase
MCM helicase S phase CDK
activat ion
DD~ oJ~
~~~
·.
Polymerase loading ~
and replication
<§!)
FIGURE 19-1 9 The molecular mechanisms governing the
initiation of DNA replication. Step 0 : During exit from mitosis and
early G~< when CDK activity is low, the MCM loading factors ORC, Cdc6,
and Cdtl load the replicative helicase, the MCM complex, onto origin
DNA. Step f) : Activation of S phase CDKs and DDK mark the onset of
S phase. They pho5phorylate the MCM helicase, Sld2 and Sld3
(depicted as green phosphorylation events), to facilitate the loading of
MCM helicase activators-the Cdc45-Sid3 and the GINS complexes-
onto sites of replication initiation. This leads MCM helicases to unwind
replication initiation during Gh when CDK activity is low (see
Figure 19-19, step 0 ). When DDK and S phase CDKs are ac-
tivated in late G" DDK phosphorylates two subunits of the
MCM helicase. The S phase CDKs phosphorylate two proteins
called Sld2 and Sld3. T hese phosphorylation events have an
activating effect, promoting the recruitment of MCM helicase
activators to sites of replication initiation (the phosphorylation
events shown in green in Figure 19-19, steps 111 and II). The
helicase activators are called the Cdc45-Sld3 complex and the
GINS com plex. Exactly how they promote activation of the
MCM helicases is not yet clear. In addition to activating the
MCM helicase to unwind DNA, the Cdc45-Sld3 complex and
the GINS complex recruit polymerases to the DNA, poly-
merase£ to synthesize the leading strand and polymerase 8 to
synthesize the lagging strand (see Figure 19-19, step II). The
replication machinery then initiates DNA synthesis.
DNA. S phase CDKs also prevent reloading of MCM helicases by
phosphorylating the pre-RC components Cdc6 and Cdtl (shown as
yellow phosphorylation events), promoting their release from origins
and degradation by the SCF. S phase CDKs also phosphorylate MCM
helicases, which leads to their export from the nucleus when the
helicases disengage from the DNA when replication is complete.
Step il: DNA polymerases are recruited to origins, which leads to the
initiation of DNA synthesis (see Figure 4-31 ).
S phase CDKs are not only essential to initiate DNA repli-
cation, they are also responsible fo r ensuring that each origin
fires only once during S phase. Preventing re-firing of origins
during S phase is brought about by phosphorylation of several
components of the MCM helicase loading machinery and the
MCM helicase itself. To distinguish these phosphorylation
events from the ones required for the initiation of DNA replica-
tion, they are depicted in yellow in Figure 19-19. Concomitant
with activation of the MCM helicase, Cdc6 and Cdtl dissoci-
ate from the sites of DNA replication initiation. Once they dis-
sociate, their phosphorylation leads to their degradation by the
SCF ubiquitin ligase. Phosphorylation of the MCM helicase
leads to the export of these proteins from the nucleus after they
dissociate from the DNA on completion of DNA replication.
Thus only after CDK activity is lowered by APC/C-Cdhl dur-
ing exit from mitosis can the MCM helicases be reloaded onto
19.4 Commitment to the Cell Cycle and DNA Replication 895
DNA. As a result, hclicasc loading is restricted to late stages of
mitosis and early G 1 (sec figure 19-19, step 0).
The general mechanisms governing the initiation of DNA
replication in metazoan cells parallel those inS. cereL'isiae,
although small differences are found in vertebrates. Phosphor-
ylation of MCM helicase activators by G 1/S phase CDKs and
S phase CDKs likely promotes their recruitment to sites of
DNA replication initiation. As in yeast, phosphorylation of
MCM loadmg factors likely prevent~ reloading of MCM heli-
cases until the cell passes through mitosis, thereby ensuring
that replication from each origin occurs only once during each
cell cycle. In metazoans, a second small protein, geminin, con-
tributes to the inhibition of re-initiation at origins until cells
complete a full cell cycle. Geminin is expressed in late G 1; it
binds to and inhibits MCM helicase loading factors as they
are released from origins once DNA replication is initiated
during S phase (see Figure 19-19, step f)), contributing to the
inhibition of re-initiation at an origin. Geminin contains a de-
struction box at its N-terminus that is recognized by the APC/
C-Cdh 1, causing it to be ubiquitinylated in late anaphase and
degraded by proteasomes. This frees the MCM helicase load-
ing factors, which are also dephosphorylated as CDK activity
declines, to bind to ORC on replication origins and load
MCM helicases during the following G 1 phase.
Duplicated DNA Strands Become
Linked During Replication
During S phase, as chromosomes are duplicated to form sis-
ter chromatids, they become tethered to each other by pro-
tein links. The linkages between sister chromatids established
during S phase will be essential for their accurate segregation
during mitosis.
The protein ci>mplexes that establish cohesion between sis-
ter chromatids are called cohesins. The cohesin complex is
composed of four subunits: Smcl (sometimes also called
Rad21), Smc3, Scc1, and Sed. Smcl and Smd are members
of the SMC protein family, which is characterized by long
coiled-coil domains that are flanked by a globular domain con-
taining ATPase activity. The ATPase domains interact with
Sec I and Sed and, together, form a ring structure. The struc-
tural mechanism by which cohesins link sister chromatids to-
gether is not understood, but it is likely that rings of cohesin
embrace one or both coptes of the replicated DNA. However,
it is clear that cohesins are essential to hold the replicated DNA
molecules together. When Xenotms egg extracts were depleted
of cohesin by treatment with antibodies specific for the cohesin
SMC proteins, the depleted extracts were able to replicate the
DNA in added sperm nuclei, but the resulting sister chromatids
did not associate properly with each other.
Some details of how cohcsins arc loaded onto DNA to
mediate sister chromatid cohesion are being unraveled. We
know that establishment of cohesion between sister chroma-
tids is tightly tied to DNA replication. Cohesins associate
with chromosomes during G 1 (Figure 19-20, step 0). During
DNA replication, they arc loaded onto chromosomes in such
a way that they can hold sister chromatids together. Most
896 CHAPTER 19 • The Eukaryotic Cell Cycle
likely this occurs as replication forks replicate the DNA (fig-
ure 19-20, step f)). Converting DNA-bound G 1 cohesins into
cohesive complexes requires several cohesin loading fac-
tors-including a protein complex related to proteins that
load the sliding clamp at the replication fork-and the acety-
lation of the Smc3 subunit. As we will see in Section 19.6,
cohesins are essential to accurately attach the replicated sister
chromatids onto the mitotic spindle and for their segregation
during mitosis. Cells lacking cohesins or the factors that load
them onto chromosomes segregate chromosomes randomly.
KEY CONCEPTS of Section 19.4
Commitment to the Cell Cycle and DNA Replication
• START defines a stage in G 1 after which cells are irrevers-
ibly committed to the cell cycle. Molecularly, it is defined as
the point when GtfS phase CDK activity reaches levels suf-
ficient to initiate S phase.
• The molecular events promoting entry into the cell cycle
are conserved across species. G 1 CDKs inhibit a transcrip-
tional inhibitor. This allows the transcription of G 1/S phase
cyclins and other genes important for S phase.
• Extracellular signals such as nutritional state (in yeast)
and the presence of mitogens and anti-mitogens (in verte-
brates) regulate entry into the cell cycle.
• Various polypeptide growth factors called mitogens stim-
ulate cultured mammalian cells to proliferate by inducing
expression of early-response genes. Many of these encode
transcription factors that stimulate expression of delayed re-
sponse genes encoding the GtfS phase cyclins and E2f tran-
scription factors.
• The G 1/S phase CDKs phosphorylate and inhibit Cdh 1,
the specificity factor that directs the anaphase-promoting
complex (APC/C) to ubiquitinylate S phase and M phase cy-
clins. This allows S phase cyclins to accumulate in late G 1 •
• In yeast, S phase CDKs initially are inhibited by Sicl.
Phosphorylation marks Sic I for ubiquitinylation by the SCF
ubiquitin-protein ligase and proteosomal degradation, re-
leasing activated S phase CDKs that trigger onset of the S
phase (see Figure 19-17).
• DNA replication is initiated from helicase loading sites
known as origins of replication.
Loading and activation of the MCM helicase occur in mu-
tually exclusive cell cycle states: MCM helicasc loading can
only occur when CDK activity is low (during early Gd;
MCM helicases are activated when CDK activity is high.
S phase CDKs and DDK trigger the initiation of DNA rep-
lication by the recruitment of MCM hclicase activators to
origins (see Figure 1 9-19).
• Initiation of DNA replication occurs at each origin only
once during the cell cycle. This is achieved because S phase
CDKs activate the helicases and at the same time prevent
additional helicases from loading onto DNA.
 Cohesins Sister chromatids
~
~ ~
~
~
~
m pliooHoo
D fJ fork
B
Centromere
/
r G,
BJ
S phase
FIGURE 19-20 Model for establishing cohesin linkage of sister
chromatids. There is strong evidence that the cohesin complex is
circular, but it is not known whether a single cohesin ring links sister
chromatids or whether two rings, one each around the separate sister
chromatids, are linked to each other like links in a chain. For simplicity,
only one ring is shown here. Step 0 : Cohesins are loaded onto
chromosomes during G,, but they do not possess cohesive properties
(indicated as cohesins laterally associated with chromosomes). Step f):
Concomitant with DNA replication, and most likely closely behind the
replication fork, cohesins are converted into cohesive molecules, able
to hold sister chromatids together (indicated as cohesin rings
• Cohesins establish linkages between the replicated DNA
molecules. This linking mechanism is coupled to DNA
replication.
19.5 Entry into Mitosis
Once S phase has been completed and the entire genome has
been duplicated, the pairs of duplicated DNA chromosomes-
the sister chromatids-are segregated to the future daughter
cells. This process requires not only the formation of the
apparatus that faci litates this segregation-the mitotic
spindle-but essentially a complete remodeling of the cell.
Chromosomes condense and attach to the mitotic spindle,
the nuclear envelope is disassembled, and almost all organ-
elles are rebuilt or modified. All these events are triggered by
mitotic CDKs. This section will first discuss how the mitotic
CDKs are precipitously activated after the completion of
DNA replication, during G2 • We will then see how these pro-
~ II
" Polo kinase
Aurora B kinase
Prophase
encircling the replicated sister chromatids). This conversion into
cohesive cohesins requires cohesin loading factors. During G2, sister
chromatids are replicated and linked along their entire length by
cohesins. During this time, the Mei-5332/Sgo proteins recruit the
protein phosphatase 2A (PP2A) to centromeric regions. Step iJ: In
vertebrate cells, cohesins are released from chromosome arms during
prophase and early metaphase by the action of Polo kinase and Aurora
B kinase. By the end of metaphase, cohesins are retained only in the
region of the centromere, where PP2A prevents cohesin phosphoryla-
tion and hence dissociation through recruiting PP2A.
rein kinases bring about the dramatic changes in the cell nec-
essary to facilitate sister chromatid segregation during
anaphase, focusing on the events as they occur in metazoans.
Precipitous Activation of Mitotic
CDKs Initiates Mitosis
Mitotic CDKs initiate mitosis. Whereas levels of the catalytic
CDK subunit are constant throughout the cell cycle, mitotic
cyclins gradually accumulate during S phase. Most eukaryotes
contain multiple mitotic cyclins, which for historical reasons
are called cyclin A and cyclin B. As they assemble, mitotic
CDK complexes are maintained in an inactive state through
inhibitory phosphorylation on the CDK subunit. Recall from
Section 19-3 that two highly conserved tyrosine and threonine
residues in mammalian CDKs are subject to regulated phos-
phorylation. In CDKl, the mitotic CDK, phosphorylation of
tyrosine 15 and threonine 14 maintains mitotic cyclin-CDK
complexes in an inactivate state. The phosphorylation state of
T L4 and Y15 is controlled by a dual-specificity protein kinase
19.5 Entry into Mitosis 897
 Mitotic cyclin
?:
:~
-
t/)
Q) ...
<.)
> <tl
~~
cO
:.=u
<.) '
>C
u:.= <.)
>
<.)
Mitosis
Y15 T"
®®
Inactive Active
FIGURE 19-21 Phosphorylation on the CDK subunit restrains
mitotic CDK activity during S phase and G2• Mitotic cyclins are
synthesized during 5 phase and G2 and bind to CDK1. However, the
cyclin-CDK complex is not active because threonine 14 and tyrosine
15 of the CDK1 subunit are phosphorylated by the protein kinase Wee1.
Once DNA replication has been completed, the protein phosphatase
Cdc25 is activated, and the phosphatase dephosphorylates CDK1.
Active mitotic CDKs further stimulate Cdc25. At the same time, mitotic
CDKs inhibit Wee 1, the protein kinase that places the inhibitory
phosphorylation on the CDK subunit. Ongoing DNA replication inhibits
Cdc25 activity. How Cdc25 is initially activated upon completion of DNA
replication to put in motion these feedback loops is not yet known.
known as Weel and a dual-specificity phosphatase Cdc25
(Figure 19-21). !he regulation of mitotic CDKs by these ac-
tivities underlies the abrupt activation of their kinase activiry at
the GrM phase transition and explains the observation that
although mitotic cyclins gradually accumulate during S phase
and G 2, mitotic CDKs are not active until cells enter mitosis.
Studies in the fission yeast Schizosaccharornyces pornbe
unraveled the mechanisms that lead to the precipitous acti-
vation of mitotic CDKs during G 2 . The dual-specificity pro-
tein kinase Weel phosphorylates CDKs on the inhibitory
tyrosine 15. (Threonine 14 is not phosphorylated inS. pornbe
CDK 1.) Yeast cells with a defective weel gene activate mitotic
CDKs prematurely and hence experience premature entry
into mitosis. Weel mutants not only enter mitosis prema-
turely but are also smaller. This is because unlike most other
cukaryotes, which coordinate cell size and cell division dur-
ing Gh this coordination occurs during G 2 in fission yeast.
Fission yeast cells carrying a mutant CDK 1 gene in which
the tyrosine 15 residue is replaced hy phenylalanine (phenyl-
alanine is structurally similar to tyrosine but cannot be phos-
phorylated) show the same premature mitotic CDK
activation and entry into mitosis. The phosphatase that op-
poses Wee 1 is Cdc25. Fission yeast cells carrying mutations
in the cdc2S gene arrest in G 2 , indicating that the phospha-
tase is essential for entry into mitosis.
898 CHAPTER 19 • The Eukaryotic Cell Cycle
Vertebrates contain multiple Weel protein kinases and
multiple Cdc25 phosphatases that collaborate to control not
only mitotic CDK activity but also the activity of G 1/S phase
CDKs. One member of the Cdc25 family of phosphatases,
Cdc25A, is activated in late G 1• It rem oves the inhibitory
phosphorylation on tyrosine 15 of the G/S phase CDKs and S
phase CDK catalytic subunit to activate the kinases. Another
family member, Cdc25C, is active during G 2 and removes
the inhibitory phosphorylation on mitotic CDKs.
Activation of mitotic CDKs is the consequence of rapid
inactivation of Wee] and activation of Cdc25. Central to this
rapid transition are feedback loops, where mitotic CDKs acti-
vate Cdc25 and inactivate Wee 1 (see Figure 19-21 ). Phosphor-
ylation of Cdc25 by mitotic CDKs stimulates its phosphatase
activiry; phosphorylation ofWeel by mitotic CDKs inhibits its
kinase activity. Ongoing DNA replication inhibits Cdc25 ac-
tivity. A critical question that we know little about is how this
positive feedback loop is started once DNA replication has
been completed. CDKs that function ea rlier in the cell cycle
have been suggested to start the positive feedback loop.
Although it is not yet known how the precipitous activa-
tion of mitotic CDKs is initiated, it is clear that once active,
these protein kinases set in motion all the events necessary to
ready the cell for chromosome segregation. The activation of
mitotic CDKs is associated with changes in the subcellular
localization of these kinases. Mitotic CDKs initially associate
with centrosomes, where they are thought ro facilitate centro-
some maturation. They then enter the nucleus, where they
bring about chromosome condensation and nuclear envelope
breakdown. In what follows we will discuss how the mitotic
CDKs accomplish the coordinated execution of mitosis.
During DNA replication initiation, S phase CDKs work to-
gether with DDK to promote MCM helicase activation. As in
the initiation of DNA replication, CDKs collaborate with other
protein kinases to bring about the mitotic events. The Polo ki-
nase family is critical for formation of the mitotic spindle as
well as chromosome segregation. The family of Aurora kinases
plays key roles in mitotic spindle formation and ensuring that
chromosomes attach to the mitotic spindle in the correct way so
that they are segregated accurately during mitosis. Their contri-
bution to the various mitotic events will also be discussed.
Mitotic CDKs Promote Nuclear
Envelope Breakdown
During interphase, chromosomes are surrounded by the nu-
clear envelope. The centrosomes that form the mitotic spin-
dle are located in the cytoplasm. For chromosomes to
interact with the microtubules nucleated by the centrosomes,
the nuclear envelope needs to be dismantled.
The nuclear envelope is a double-membrane extension of
the endoplasmic reticulum containing many nuclear pore
complexes (see Figures 9-32 and 13-33). The lipid bilayer of
the inner nuclear membrane is associated with the nuclear
lamina, a meshwork of lamin filaments adjacent to the inside
face of the nuclear envelope (Figure 19-22a). The three
nuclear lamins (A, B, and C) present in vertebrate cells be-

Lamin tetramer
1 MPF
... c~ cc
Phosphorylated
lamin dimers
FIGURE 19-22 The nuclear lamina and its regulation by
phosphorylation. (a) Electro11 micrograph of the nuclear lamina
from a Xenopus oocyte. Note the regular mesh-like network of Ia min
intermediate filaments. This structure lies adjacent to the inner
nuclear membrane (see Figure 18-46). (b) Schematic diagrams of
the structure of the nuclear lamina. Two perpendicular sets of
10-nm-diameter filaments built of lamins A, B. and C form the nuclear
lamina (top). lndividuallamin filaments are formed by end-to-end
polymerization of Ia min tetramers, which consist of two Iamin
coiled-coi l dimers (middle). The red and blue circles represent the
·' globular N-tPrminal and (-terminal domains, respectively. Phosphor-
ylation of specific serine residues near the ends of the rod-like central
section of Ia min dimers causes the tetramers to depolymerize
(botrom). As a result, the nuclear lamina disintegrates. [Part (a) from U.
Aebi et al., 1986, Nature 323:560; courtesy of U. Aebi; part (b) adapted from A.
Murray and T. Hunt, 1993, The Cell Cycle: An Introduction, W. H. Freeman and
Company.]
long to a class of cytoskeletal proteins, the intermediate fila-
ments, that are critical in supporting cellular membranes.
Lamins A and C, which are encoded by the same transcnp-
tion unit and produced by alternative splicing of a single pre-
mRNA, are identical except for a 133-residue regwn at the
C-terminus of laminA, which is absent in lamin C. Lamin B,
encoded by a different transcription unit, is modified post-
transcriptionally by the addition of a hydrophobic isoprenyl
group near its carbo.>.yl terminus. This fatty acid becomes
embedded in the inner nuclear membrane, thereby ancho ring
the nuclear lamina to the membrane (see Figure 10-19). All
three nuclear lamins form dimers containing a rod-like a-
helical coiled-coil central section and globular head and tail
domains; polymerization of these dimers through head-to-
head and tail-to-tail associations generates the intermediate
filaments that compose the nuclear lamina (Figure 19-22b).
Once mitotic CDKs are activated at the end of G 2 , they
phosphorylate specific serine residues in all three nuclear lam ins.
This causes depolymerization of the lamin intermediate fila-
ments (see Figure 19-22b). The phosphorylated laminA and C
dimers are released into solution, whereas the phosphorylated
lamin B dimers remain associated with the nuclear membrane
via their isoprenyl anchor. Depolymerization of the nuclear lam-
ins leads to disintegration of the nuclear lamina meshwork and
contributes to disassembly of the nuclear env~lope.
Mitotic CDKs also affect other nuclear envelope compo-
nents (Figure 19-23). The CDKs phosphorylate specific
nu cleoporins, which causes nuclear pore complexes to dis-
sociate into subcomplexes during prophase. Phosphoryla-
tion of integral membrane proteins of the inner nuclear
membrane is thought to decrease their affinity for chromatin
and further contributes to the disassembl y of the nuclear
envelope. The weakening of the associations between the
inner nuclear membrane proteins and the nuclear lamina
and chromatin allows sheets of inner nuclear membrane to
retract into the endoplasmic reticulum, which is continuous
with the outer nuclear membrane.
Mitotic CDKs Promote Mitotic Spindle Formation
A key function of mitotic CDKs is to induce the formation of
the mitotic spindle, also known as the mitotic apparatus. As
we saw in Chapter 18, the mitotic spindle is made of micro-
tubules that attach to chromosomes via specialized protein
structures associated with chromosomes known as kincto-
chores. In most organisms, the mitotic spindle is organized
by centrosomes, sometimes called spindle pole bodies. They
contain a specialized tubulin, -y tubulin, which together with
associated proteins nucleates microtubules. Notable excep-
tions to centrosome-based spindle assembly mechamsms are
higher plants and metazoan oocytes. In these cells, (- ) end~
of microtubules are cross-linked and the microtubules self-
assemble into a spindle.
The function of the mitotic spindle is to segregate chromo
somes so that the sister chromatids separate from each other
and are moved to opposite poles of the mitotic spi ndle (see Fig-
ure 18-37). To achieve this, chromosomes have to attach to the
19.5 Entry into Mitosis 899

Cytoplasm
/ fJINM .
protems
FIGURE 19-23 Nuclear envelope proteins phosphorylated by
mitotic CDKs. Step 0: Components of the nuclear pore complex (NPC)
are phosphorylated by mitotic CDKs in prophase, causing NPCs to
dissociate into soluble and membrane-associated NPC subcomplexes.
Step fJ: Mitotic CDK phosphorylation of inner nuclear membrane (INM)
proteins inhibits their interactions with the nuclear lamina and
chromatin. Step 0 :Mitotic CDK phosphorylation of nuclear lam ins
causes their depolymerization and dissolution of the nuclear lamina.
Step B : Mitotic CDK phosphorylation of chromatin proteins induces
chromatin condensation and inhibits interactions between chromatin
and the nuclear envelope. [Adapted from B. Burke and J. Ellenberg, 2002,
Nat. Rev. Mol. Cell Bioi. 3:487.]
mitotic spindle so that one kinetochore of each sister chromatid
pair attaches to ll)icrotubules emanating from opposite poles.
Sister chromatids are then said to be hi-oriented. In what fol-
lows, we will discuss how the mitotic spindle forms, how chro-
mosomes attach to it, and how cells correct faulty attachments.
During Gh cells contain a single centrosome that func-
tions as the major microtubule nucleating center of the cell.
0 VIDEO: Normal Cell Division in C. elegans Embryogenesis
FIGURE 19-24 Chromosome attachment to the mitotic spindle.
Chromosomes attach to the mitotic spindle and congress to the
spindle center. They then attach via their kinetochores to the ends of
microtubules (called end-on attachments), and these attachments are
stabilized by additional microtubules. The final chromosome attach-
ment where the chromosome has stably bi-oriented on the mitotic
spindle is shown.
900 CHAPTER 19 • The Eukaryotic Cell Cycle
Mitotic spindle formation begins at the G 1-S phase transi-
tion with the duplication of ccn trosomes. The mechanism
whereby this duplication occurs is poorly understood, but at
the heart of this process is the duplication of the pair of cen-
trioles, sho rt microtubules arranged orthogonally to each
other. As discussed in Chapter 18, G 1 cells contain a single
pair of centrioles. Concomitant wi th entry into S phase and
triggered by the G 1/S phase CDKs, the two centrioles split
apart, and each centnole begins to grow a daughter centriole
(sec Figure 18-35). The new centrioles grow and mature dur-
ing S phase, each centriole pair begins to assemble centro-
somal material, and by G 2 the two centrosomes have formed.
Several additional protein kinases have been identified that
control centrosome dup lication . Chief among them is a
member of the conserved Polo kinase family, Plk4 . How
G 1/S phase CDKs and Plk4 promote centrosome duplication
is not yet understood but is thought to involve the phos-
phorylation of multiple centrosome components, which fa-
cilitates their duplication and growth. As we will see, the
Polo kinases not only play a key role in centrosome duplica-
tion but also participate in essentially all aspects of mitosis.
The key initiating step of mitotic spindle formation is the
severing of the ties that link the duplicated c~ntrosomcs. This
centrosome disjunction occurs in G 2 and is triggered by
mitotic CDKs (see Figure 18-35). As soon as this separation
occurs, microtubules arc nucleated from both centrosomes
and they move away from each other, pulled by the motor
protein dynein . The specifics of microtubule array formation
and mitotic spindle assembly were discussed in Chapter 18.
Here we will briefly consider how chromosomes attach to the
mitotic spindle and how mistakes in the process are corrected.
For chromosomes to be accurately segregated during mi-
tosis, the sister chromatid pair has to be stably hi-oriented on
the mitotic spindle (Figure 19-24). How is this accomplished?
Once centrosomes have moved apart from each other, micro-
tubules, in a search-and-capture mechanism, begin to interact
wi th kinetochores of sister chromatid pairs. Initially, chro-
mosomes glide along the length of microtuhules propelled by
motor proteins. When the chromosome reaches the end of a
.·
~ VIDEO: Abnormal Chromosome Segregation in C. elegans Embryo in the Absence of Kinetochore Protein KNL-3
FIGURE 19-25 Stable and unstable chromosome attachments.
When sister kinetochores attach to microtubules emanating from
opposite spindle poles, they are stably attached. This configuration
is called amphitelic attachment (a). Microtubules (green) pull
kinetochores (tan); cohesins resist this pulling force. This leads to
kinetochores being pulled away from the protein kinase Aurora B (red
zone), which localizes to the inter sister kinetochore space. As a result,
Aurora ~can no longer phosphorylate the microtubule binding factors
at the kinetochore and kinetochore-microtubule attachments are
stable. When one kinetochore attaches to microtubules emanating
from two opposite spind le poles (merotelic attachment, b), or both
sister kinetochores attach to microtubules emanating from the same
spindle pole (syntelic attachment, c), or only one of the two sister
kinetochores attaches to microtubules (monotelic attachment, d),
kinetochores are not pulled out of the Aurora B zone. As a result,
Aurora B phosphorylates the microtubule binding subunits of the
kinetochore and microtubule attachments are destabilized.
microtubule, the kinetochores attach to microtubules in an
end-on attachment, the final configuration in which chromo-
somes are linked to the mitotic spindle. Kinetochores of sister
chromatids then bind microrubules emanating from the op-
posite spindle pole. In the end, the sister chromatid pair is
said to be stably bi-oriented on the mitotic spindle.
The ultimate goal of chromosome attachment to the mi-
totic spindle is that each and every chromosome is attached to
the mitotic spindle in a bi-orientcd manner (also known as
amphitelic attachment; Figure 19-25a). How does the cell
"know" that this has occurred? Microscopic analysis of
chromosome attachment has shown that initially many chro-
mosomes attach to microtubules in faulty ways. A kinetochore
can attach to microtubules emanating from both poles, a situ-
ation called merotelic attachment (Figure 19-25b). Kineto-
chores of a sister chromatid pair can attach to microtubules
from the same pole (syntelic attachment; Figure 19-25c) or
only one kinetochore can attach to microtubules (monotelic
attachment; Figure 19-25d). Clearly, none of these attach-
ments are productive in the sense that they would not result in
accurate chromosome segregation. Thus mechanisms must be
in place that detect and correct such faulty attachments.
The sensing mechanism used by cells to detect incorrect
attachments is based on tension. When sister chromatids arc
correctly attached to microrubulcs, their kinetochores are
under tension (see Figure 19-25a). Microtubules attached to
the kinetochores pull at them and the cohesin molecules that
hold the sister chromatids together withstand these forces,
creating tension at kinctochores. Merotelic, syntelic, or
monotelic attachment leads to insufficient tension at kineto-
chores, allowing the cell to distinguish these faulty forms of
attachment from the correct amphitelic one.
How does the cell sense w hether or not kinctochores are
under tension? The protein kinase Aurora B and its associated
regulatory factors, together known as the chromosomal pas-
senger complex (CPC), sense kinetochores that are not under
tension and sever these microtubule attachments, giving cells a
(a) Amphitelic attachment (b) Merotelic attachment
~Cohesins
J Aurora B
~ti · ~~\~
Microtubules
§v
Sister chromatids
(d) Monotelic attachment
(c) Syntelic attachment
second chance to get the attachment right. The molecular basis
for this sensing mechanism is partly understood. Aurora B
phosphorylates a number of kinetochore components involved
in microtubule binding. When phosphorylated, these proteins
lose their microtubule-binding activity. Aurora B localizes to
the region of sister chromatids between the two kinetochores.
When kinetochores are not under tension, they are in close
proximity to Aurora Band the protein kinase can phosphory-
late the microtubule-binding subunits of the kinetochores, de-
stabilizing any kinetochore-microtubule attachments (see
Figure 19-25h-d). When microrubules are attached correctly
to kinetochores, microtubule forces pull kinctochores away
from Aurora Band the kinase can no longer reach its targets at
kinetochores (see Figure 19-25a).
Microtubules continuously pull on chromosomes. Once all
chromosomes have attached to microtubules in an amphitelic
manner, the only things that prevent chromosomes from segre-
gating to the poles and that hold them back in the middle of
the spindle are the cohesin complexes (see Figure 19-25a). As
we will see in Section 19.6, it is the severing of these cohesin
molecules that initiates anaphase chromosome segregation.
Chromosome Condensation Facilitates
Chromosome Segregation
Chromosome segregation not only requires the building of
the apparatus that segregates chromosomes, it also requires
that the DNA is compacted into travel-friendly structures.
An attempt to segregate the long and intertwined DNA-
protein complexes present in interphase cells would lead to
breakage of the DNA and hence loss of genetic material. To
19.5 Entry mto Mitosis 901

FIGURE 19-26 Scanning electron micrograph of a metaphase
chromosome. During metaphase, the chromosomes are fully
condensed and the two individual sister chromatids are visible.
[Biophoto Associates/Photo Researchers.)
avoid this fate, cells compact their chromosomes during pro-
phase into the dense structures we have become acquainted
with through light or electron microscopy (Figure 19-26).
Chromosome condensation results in a dramatic reduc-
tion in chromos9me length, up to 10,000-fold in vertebrates.
The second key aspect of the compaction process is the un-
tangling of the intertwined sister chromatids. This process is
called sister chromatid resolution and is mediated in part by
the decatenation activity of topoisomerase II and goes hand
in hand with the condensation process.
Condensation of chromosomes is not very well under-
stood, but central to the process is a protem complex known
as the condensin complex. This protein complex is related to
the cohesin complex that links sister chromatids together after
DNA replication and was first identified based on its ability to
promote chromosome condensation in frog extracts. Like the
cohcsin complex, condensins are composed of two large
coiled-coil SMC protein subunits that associate through thetr
ATPase domains with non-SMC subunits. When condensin
function is lost in cells, chromosomes do not condense and
sister chromatid tangles are not resolved. How condensins
compact chromosomes is not understood, but in analogy ro
cohesin rings, condensins may create intrachromosomallink-
ages that package chromosomes into the characteristic loops
seen in electron micrographs of compacted chromosomes.
Like all early mitotic events, chromosome condensation is
ultimately triggered by mitotic CDKs. The exact mechanisms
whereby these kinases induce chromosome condensation is not
902 CHAPTER 19 • The Eukaryotic Cell Cycle
known, but it appears that mitotic CDKs induce the process at
least in part through activating condensins. Two of the non-SMC
condensin su bunits are targets of mitotic CDKs. Their phosphor-
ylation stimulates condensins to supercoil DNA in vitro.
Finally, processes acting in parallel to condensins also
contribute to chromosome compaction. The dissociation of
cohesins from chromosomes is such a parallel process. A
large fraction of cohesins is removed from chromosomes
during prophase (see Figure 19-20, step lJ). This process is
mediated by phosphorylation of cohesins by Polo kinase and
Aurora B kinase. In most organisms, cohesins are only main-
tained around centromeres. Cohesins around centromeres
are protected from phosphorylation-dependent removal by
protein phosphatase 2A (PP2A). This phosphatase is re-
cruited to centromeric regions by a member of a family of
PP2A targeting factors known as the Mei-S332/Shugoshin
family of proteins. The protected 'pool of cohesins provides
the resistance to the pulling force exerted by microtubulcs
necessary to establish tension at bi-oriented kinetochores. As
we will see in Section 19.8, this protection mechanism also
plays an essential role in establishing the meiotic chromo-
some segregation pattern.
KfY CONCFOTS of Section 19.5
Entry into Mitosis
Mitotic CDKs induce entry into mitosis in all eukaryotes.
Mitotic CDKs are held inactive until the completion of
DNA replication by inhibitory phosphorylation on the CDK
subunit.
Mitotic CDKs promote their own activation through pos-
itive feedback loops leading to the rapid inactivation of
Wee! kinase and activation of Cdc25 phosphatase.
Mitotic CDKs induce nuclear envelope breakdown 111
most eukaryotes by phosphorylating lamins.
Centrosome duplication occurs during S phase. Mitotic
CDKs induce the separation of the duplicated centrosomes,
which initiates mitotic spindle formation.
Sister chromatids attach to the mitotic spindle via their ki-
netochores in a hi-oriented manner, with one sister kineto-
chore attaching to microtubules emanating from one spindle
pole and the other one to microtubules nucleated by the other
spindle pole.
Cells sense hi-orientation of sister chromatids through a
tension-based mechanism. When kinetochores are not under
tension, the protein kinase Aurora B phosphorylates the mi-
crotubule binding subunits of the kinetochore, which de-
creases their microtubule binding affinity.
Chromosomes must be compacted for segregation.
Condensins, protein complexes that are related to cohes-
ins, facilitate chromosome condensation and are activated
by mitotic CDKs.
 19.6 Completion of M itosis: Chromosome
Segregation and Exit from Mitosis
Once all chromosomes have condensed and correctly at-
tached to the mitotic spindle, chromosome segregation com-
mences. In this section, we will discuss how cleavage of
cohesins by a protease known as separase triggers anaphase
chromosome movement and how this cleavage is regulated.
We will then explore how thf' machinery that initiates cohc-
sin cleavage at the metaphase-anaphase transition, the
APC/C, also initiates mitotic CDK inactivation. Next, we
will see how phosphatases activated at the end of mitosis
participate in mitotic CDK inactivation, bringing about the
disassembly of mitotic structures and the resetting of the cell
tO the G 1 state. We will end this section with a discussion of
cytokinesis, the process that produces two daughter cells.
Separase-Mediated Cleavage of Cohesins
Initiates Chromosome Segregation
As mentioned in the previous section, each sister chromatid
of a metaphase chromosome is attached to microtubules via
its kinetochore (see Figure 19-24). At metaphase, the spindle
is in a state of tension, with forces pulling the two kineto-
chores toward the opposite spindle poles. Sister chromatids
do not separate because the} arc held together at thetr cen-
tromeres by cohesin complexes. In all organisms analy1.ed to
date, loss of cohesins from chromosomes triggers anaphase
chromosome movement. The mechanism that brings about
this loss of cohesins from chromosomes is conserved too. A
protease known as separase cleaves a subunit of cohesin
called Sccl or Rad21, breaking the protein circles linking
sister chromatids (Figure 19-27). Once this link is broken,
anaphase begins as poleward force exerted on kinetochores
moves the split sister chromatids toward spindle poles.
Smc3
Scc3
Separase
·. FIGURE 19·27 Regulation of cohesin cleavage. Separase, a
protease that can cleave the Sccl subunit of cohesin complexes, is
inhibited before anaphase by the binding of securin. Mitotic CDKs also
inhibit separase by phosphorylating it. When all the kinetochores have
attached to spindle microtubules and the spindle apparatus is properly
assembled and oriented, the Cdc20 specificity factor associated with
19.6 Completion of Mitosis: Chromosome Segregation and Exit from Mitosis
Cohesin cleavage was discovered in budding yeast. Anal-
ysis of the Sec I subunit by Western blot analysis showed
that the protein migrated in SDS PAGE gels according to its
predicted molecular weight from G 1 until metaphase, but
during anaphase the protein ran considerably faster 111 SDS
PAGE, indicating the protein became somehow smaller. Sub-
sequent studies showed that the faster-migrating form of
Sccl was indeed a cleavage product. Insight into the identity
of the protein that was rf'~ponsihle for the cleavage of cohc-
sin came from the analysis of previously identified yeast mu-
tants that failed to segregate chromosomes during anaphase.
A mutant in the gene encoding £SP7-what we now know
to be scparase-failed to produce the cleavage fragment.
Subsequent analyses revealed not only that separase is a pro-
tease but that cleavage of cohesin is essential for chromo-
some segregation. Cells expressing a form of Sccl with its
cleavage sites mutated fail to segregate their chromosomes.
Given the irreversible nature of Sccl cleavage, it is absolutely
essential that scparase activity is tightly controlled. In what
follows we will discuss this regulation.
The APC/C Activates Separase Through
Securin Ubiquitinylation
Prior to anaphase, a protein known as securin binds to and
inhibits separase (see Figure 19-2 7). Once. all kinetochores
have attached to spindle microtubules in the correct hi-
oriented manner, the APC/C ubiquitin ligase directed by
specificity factor Cdc20 ubiquitinylates securin (note that
this specificity factor is distinct from Cdh 1, which targets
APC/C substrates for degradation later during mitosis).
Polyubiquitinylated securin is rapidly degraded by protea-
somes, thereby releasing separase.
APC/CcdclO is activated in prophase by mitotic CDK
phosphorylation of several APC/C subunits. However, this
Ub
/
Ub
/
Ub
d
the APC/C directs it to ubiquitinylate securin and mitotic cyclins.
Following securin degradation and a decrease in mitotic CDK activity,
the released and dephosphorylated separase cleaves the Sccl subunit,
breaking the cohesin circles and allowing sister chromatids to be
pulled apart by the spindle apparatus that is pulling them toward
opposite spindle poles.
903
phosphorylated APC/CcdclO is not active until all chromo-
somes have hi-oriented on the mitotic spindle. As we will sec
in Section 19.7, APC/CCJc20 is inhibited by a checkpoint path-
way that ensures that mitosis does not continue until all chro-
mosomes have achieved proper attachment to the mitotic
apparatus. Cdc20 is inhibited until every kinetochore has at-
tached to microrubules and tension is applied to the kineto-
chores of all sister chromatids, pulling them toward opposite
spindle poles. In vertebrate cells, separase is also regulated by
phosphorylation. Mitotic CDK activity inhibits scparasc dur-
ing prophase and metaphase. Only when mitotic CDK activity
begins to decline ar the metaphase-anaphase transition
through APC/CCdclo_mcdiated protein degradation can sepa-
rase become active and trigger chromosome segregation.
Once cohesins are cleaved, anaphase chromosome move-
ment ensues. As discussed in Chapter J 8, chromosome segrega-
tion is mediated by microtubule depolymerization and motor
proteins as spindle poles move away from each other. Decline
in mitotic CDK activity is important for these anaphase chro-
mosome movements. When mitotic CDK inactivation is inhib-
ited, anaphase does occur, but it is abnormal. Dephosphorylation
of a number of microtubule-associated proteins that affect mi-
crotubule dynamics appears important for this process. In bud-
ding yeast, this dephosphorylation is brought about by the
protein phosphatase Cdcl4, which we will see, plays an essen-
tial role in the final cell cycle stage, exit from mitosis.
Mitotic CDK Inactivation Triggers
Exit from Mitosis
Anaphase spindle elongation and the events associated with
exit from mitosis-mitotic spindle disassembly, chromo-
some decondensation, and nuclear envelope re-formation-
are brought about by the dephosphorylation of CDK
substrates. In other words, exit from mitosis can be viewed
as a reversal of entry into mitosis. The phosphorylation
events that triggered the different mitotic events need to be
undone for the cell to reverse to the G 1 state.
Dephosphorylation of mitotic CDK substrates is caused
by the inactivation of mitotic CDKs. In most organisms, mi-
tottc CDK inactivation is triggered by APC/CCd.:lO_mcdiated
degradation of mitotic cyclins. As mitotic CDKs activate
APCtc<ddO, they initiate their own demise. In budding yeast
only about 50 percent of mitotic cyclins are degraded by
APC/CCJdo. As we will sec in Section 19.7, a pool of mitotic
cyclins is protected from APC/Ccd,lO ro allow for enough
time to position the mitotic spindle accurately within the
cell. How a fraction of mitotic cyclins is protected from
APC!CcJ,lO is not known, but it is clear that a second mitotic
CDK-inactivating step i~ needed for exit from mitosis to
occur. The conserved protein phosphatase Cdc14 brings
about this second step in mitotic CDK inhibition.
In budding yeast, complete inactivation of mitotic CDKs
requires the destruction of mitotic cyclins by APC/CCJhl and
the accumulation of the CDK inhibitor Sicl, which-re-
call-holds S phase CDKs in check until cells enter the cell
cycle. Both APCtc<Jhl and Sicl are inhibited by mitotic
904 CHAPTER 19 • The Eukaryotic Cell Cycle
Mitosis G,
----11 Sic1
Mitotic CDKs f - - - - APC/C-Cdhl
t
Cdc14
t
Mitotic exit network
FIGURE 19 · 28 The protein phosphatase Cdc:14 triggers exit
from mitosis i n budding yeast. During mitosis, mitotic CDK activity
inhibits its inhibitors, APC/CCdht and Sicl. During G,, APC/Ccdht and Sicl
inhibit mitotic CDKs. During exit from mitosis, the protein phosphatase
Cdc14 throws the switch between these two antagonistic states. The
mitotic exit network activates the phosphatase during anaphase,
allowing it to dephosphorylate APC/Ccdht, thereby activating it. The
phosphatase also promotes the accumulation of Sicl. In addition,
Cdc14 dephosphorylates the many mito~ic CDK substrates, which
leads to rapid exit from mitosis.
CDKs. Conversely, APC/Ccdhi and Sicl inhibit mitotic CDKs
(Figure 19-28). The protein phosphatase Cdc14 throws the
switch between these two mutually antagonistic states dur-
ing anaphase. Cdcl4 is kept inactive during most of the cell
cycle, but the phosphatase is activated during anaphase by a
GTPase signaling pathway known as the mitotic exit net-
work (MEN). This signaling cascade is, as we will sec in
Section 19.7, responsive to spindle position and only be-
comes active in anaphase, when the anaphase spindle is
properly positioned within the cell. Once activa ted during
anaphase, Cdcl4 dephosphorylates APC/Ccdhl and Sicl to
promote mitotic cyclin degradation and mitotic CDK inacti-
vation, respectively. This leads to exit from mitosis.
Phosphatase activity is also essential for exit from mitosis
in vertebrates. Simple inactivation of mitotic CDKs is nor suf-
ficient to trigger the timely exit from mitosis. It is not yet clear
which phosphatase dephosphorylates CDK substrates to reset
the cell to the G 1 stage. Both protein phosphatase 1 and pro-
tein phosphatase 2A have been implicated in the process.
Ultimately, reversal of mitotic CDK phosphorylation
changes the activities of many proteins back to their inter-
phase state. Dephosphorylation of condensins, histone Hl,
and other chromatin-associated proteins leads to the decon-
densation of mitotic chromosomes in telophase. The targets of
CDKs whose dephosphorylation is important for mitotic spin-
dle disassembly arc not known, but likely multiple proteins are
targets. More is known about how the nuclear envelope re-
forms. Dephosphorylated inner nuclear membrane proteins
are thought to bind to chromatin once again. As a result, mul-
tiple projections of regions of the ER membrane containing
these protems are thought to associate with the surface of the
dPcondensing chromosomes and then fuse with one another
directed by an unknown mechanism to form a continuous
double membrane around each chromosome (Figure 19-29).
Dephosphorylation of nuclear pore subcomplexes allows them
to reassemble into complete NPCs traversing the inner and
outer membranes soon after fusion of the ER projections.
Ran-GTP, required for driving most nuclear import and
·.
 ~ VIDEO: Nuclear Envelope Dynamics During Mitosis
... FIGURE 19-29 Model for reassembly of the nuclear envelope
during telophase. Extensions of the endoplasmic reticulum (ER)
associate with each decondensing chromosome and then fuse with
one another, forming a double membrane around the chromosome.
Dephosphorylated nuclear pore subcomplexes reassemble into nuclear
pores, forming individual mini-nuclei called karyomeres. The enclosed
chromosome further decondenses, and subsequent fusion of the
nuclear envelopes of all the karyomeres at each spindle pole forms
a single nucleus containing a full set of chromosomes. NPC, nuclear
pore complex. [Adapted from B. Burke and J. Ellenberg, 2002, Nature Rev. Mol.
Cell Bioi. 3:487.]
export (sec Chapter 13), stimulates both fusion of the ER pro-
jections to form daughter nuclear envelopes and assembly of
.· NPCs (see Figure 19-29). The Ran-GTP concentration is high-
est in the microvicinity of the decondensing chromosomes be-
cause the Ran-guanine nucleotide-exchange factor (Ran-GEF)
is bound to chromatin. Consequently, membrane fusion is
stimulated at the surfaces of decondensing chromosomes,
forming sheets of nuclear membrane with inserted NPCs.
Cytokinesis Creates Two Daughter Cells
When chromosome segregation is completed, the cytoplasm
and organelles arc distributed between the two future daugh-
ter cells. This process is called cytokinesis. With the excep-
tion of higher plants, the division of the cell is brought about
by a contractile ring made of actin and the actin motor myo-
sin (see Figure 17-36). During cytokinesis, the ring contracts
in a manner similar to muscle contraction, pulling the mem-
brane inward and eventually dosing the connection between
the two daughter cells.
Cytokinesis must be coordinated with other cell cycle
events in space·and time. For cell division to produce two
daughter cells each containing the components necessary for
survival, the division plane must be placed so that each cell
receives approximately half of the mother cell's cytoplasmic
content and exactly half of the genetic content. Cytokinesis
must also be coordinated with the temporal sequence of cell
cycle events, the completjon of mitosis. In what follows, we
will explore both these aspects of cytokinesis regu lation.
In animal cells, the contractile ring forms during ana-
phase and is placed in the middle of the anaphase spindle.
This ensures that each daughter cell receives half of the ge-
netic mate rial. Despite the importance of this coordination,
surprisingly little is known about it. Some experiments
support the idea that signals sent from the spindle midzone
to the cell cortex are important to coordinate the site of
cywkinesis with spindle position. Other research suggests
that microtubules of t he spindle interact with the cell cor-
tex, positioning the cleavage furrow with respect to spindle
pole position. Most likely, a combination of these path-
ways governs the formation of the cleavage furrow during
cyrok inesis.
19.6 Completion of Mitosis: Chromosome Segregation and Exit from Mitosis
Cytosol
Chromatin
Membrane!
recruitment
Membrane fusion,
NPC assembly
l
:"--- ....-_:) -~
l( Outernuclear~ ~
.......-'-n.-"' .-J ~ - m~r-a~~ l
Inner nuclear
membrane '
In budding yeast and fission yeast, the site of cytokmesis is
determined prior to mitosis. In budding yeast, this occurs during
G~. when the bud site is determined. In fission yeast, proteins
important for contractile ring formation accumulate in the mid-
dle of the cell during G2• Irrespective of the sequence of events-
site of mitosis first, then site of cytokinesis or vice versa-it is
clear that these events must be tightly coordinated. As we will
see in Section 19. 7, cells have developed surveillance mecha-
nisms that ensure that the site of cytokinesis is coordinated with
spindle position. This is especially important during asymmetric
cell divisions, which give rise to cells of different size and/or fate.
Such cell divisions are essential during development and in stem
cell divisions (see Chapter 21). Cytokinesis must also be coordi-
nated with other cell cycle events. The major signal for cytokine-
sis is the inactivation of mitotic CDKs. Cells expressing a
stabilized version of mitotic cyclins progress through anaphase
but do not undergo cytokinesis. The CDK targets in the cytoki-
nesis machinery have not yet been discovered.
This concludes our discussion of the molecular events of
cell division. As we saw, cyclin-dependent kinases and ubiq-
uitin-mediated protein degradation are at the center of this
control (Figure 19-30). In what follows, we will discuss the
mechanisms that ensure a subsequent cell cycle stage is not
initiated until the previous has been completed and that each
cell cycle step occurs accurately.
905
 Phosphatases activate

8
Cdh1 and APC/C-Cdh1/
proteasome degrades ~
mitotic cyclins

~8
APC/C-Ccic?O/ Telophase and cytokinesis

Anaphase DNA prereplication

securin
complexes assemble
Early at origins
G, G 1/S phase-CDKs inactivate Cdh1
M id-late G1/S phase-C DKs activate
G, expression of S phase cyclin
CDKs components
'
START G 1/S phase-CDKs phosphorylate
S phase inhibitor
Cdc25 phosphatase s
Metaphase activates mitotic CDKs,
which activate early
mitotic events

S phase CDKs activate

prereplication complexes

DNA replication
FIGURE 19-30 Fundamental processes in the eukaryotic cell cycle. See text for discussion.

KEY CONCEPTS of Section 1 9.6 19.7 Surveillance Mechanisms

Completion of Mitosis: Chromosome Segregation
and Exit from Mitosis
Cleavage of cohesin by separase induces chromosome seg-
regation during anaphase.
At the onset of anaphase, the APC/C is directed by Cdc20
to ubiquitinylate securin, which is subsequently degraded by
proteasomes. T h is activates separase (see figure 19-27).
Exit from mitosis is triggered by mitotic CDK inactivation
mainly brought about by mitotic cyclin degrad ation.
Ex1t from mitosis requires the activity of protein phospha-
tases such as Cdcl4 to remove mitotic phosphorylations
from many different proreins, permitting mitotic spindle dis-
assembly, the decondensation of c h romosome~, and the re-
assembly of the nuclear envelope.
• Cytokinesis finalizes cell fission and must be coordinated
with the site of nuclear division. This coordination is espe-
cially important in cells undergoing an asymmetric division.
in Cell Cycle Regulation
Su rveillance mechanisms known as checkpo int path ways op-
erate to ensure that t he next cell cycle event is not initiated
until the previous one has been completed. Checkpoint path-
ways consist of sensors that monitor a pa rticular cell ular
event, a signaling cascade that ini tiates the response, and an
effector that ha lts cell cycle p rogression and acti,·ates repair
pathways when necessary. Cell cycle even ts moni tored by
checkpoint pathways include grow th , DNA replication,
DNA damage, kinetochore attachment to t he mitotic spin-
d le, and position of the spind le within t he cell. They are re-
sponsible for the extraordinarily high fidelity of cell division,
ensuring that each daughter cell receives t he correct number
of accurately replicated chromo~omes. They function by
controlling t he protei n kinase activities of the cyclin-CDKs
through a variety of mechanisms: regulation of the synthesis
and degradation of cyclins, phosphorylation of CDKs at
inhibitory ~ites, regulation of the synthesis and stability of

906 CH APTER 19 • The Eukaryotic Cell Cycle

 CDK inhibitors (CKis) that inactivate cyclin-CDK com- the permissive temperature, the temperature-sensitive pro-
plexes, and regulation of the APC/C ubiquitin-protein ligase. rein is functional again, and cells continue to proliferate.
Thus, although the cdc13 mutant cells failed to di\ide at the
restrictive temperature, they retained their viability and so
Checkpoint Pathways Establish Dependencies
were able to resume proliferation once they were returned to
and Prevent Errors in the Cell Cycle the permissive temperature where the temperature-sensitive
The experiments that led to the idea of surveillance mecha- cdc13 protein was functional again.
nisms or checkpoint pathways establishing dependencies in To characterize the cdc13 mutant in more detail, Hartwell
the cell cycle were simple and beautiful in their interpretation. and colleagues examined the effecr~ of introducing a second
Recall that Lee Hartwell and colleagues isolated temperature- mutation in another gene, a deletion in the RAD9 gene. The
sensitive cdc mutants in S. cerevisiae. Their characterization RAD9 gene is not essential for viability, but when it is deleted,
and the study of the genes affected in these mutants pro- cells are extremely sensitive to DNA damaging agents such as
foundly shaped our understand ing of the eukaryotic cell x-rays. This mutation on its own does not affect growth of
cycle. It was the characterization of one of these cdc mu- cells at any temperature but had a dramatic effect on cdc13
tants, cdc 13, that led Hartwell and colleagues to formulate mutants. When the researcher~ examined the cdc13 rad9 dou-
the checkpoint pathway concept. ble mutant at the restrictive temperature, they observed that
For the purpose of our discussion here, the only thing the mutant no longer arrested tn G 2 bur instead the cells con-
that is important to know about CDC7J function is that the tinued to divide for a few divisions (Figure 19-3lb). When
gene is required for telomere replication and, in its absence, they returned these cells to the permissive temperature, the
large stretches of incompletely replicated telomeric DNA double mutant failed to resume proliferation. This indicates
persist in cells. Cells carrying a temperature-sensitive allele that while the cells continued to divide a few times at the re-
of the cdc13 gene as the sole source of CDCJJ arrest with a strictive temperature, they lost viability.
G 2 DNA content when shifted to the restrictive temperature Hartwell and colleagues proposed the following explana-
(Figure 19-3 1a). This arrest is indicative of a defect in late S tion for this observation: cdcl 3 mutants arrest at the restnc-
phase or entry into mitosis. When the cells are returned to tive temperature because they harbor incompletely replicated
DNA. This damaged DNA signals the cell tO arrest cell cycle
progression and induce repair of the damage because mitosis
(a) cdc13 mutant Cell cycle arrest
of cells with damaged DNA would almost certainly lead to
Resume cell death. The RAD9 gene is part of the machinery that con-
proliferation; veys this cell cycle arrest. In cells lacking RAD9, the "halt cell
f ---+ colony cycle progression" signal does not work and cells undergo
will form
mitosis despite incompletely replicated DNA. This kills the
cells. Hartwell and his colleagues called this surveillance
Permissive Restrictive Permissive mechanism a checkpoint pathway.
temperature temperature temperature
(25 •cJ (37 •cJ (25 •cJ Today we know that cells harbor multiple checkpoint
pathways to ensure that a cell cycle phase does not commence
before the pre\ ious one has been completed. In addition to
(b) cdc13 rad9.l No cell cycle arrest;
mutant few divisions establishing dependencies, the checkpoint pathways ensure
that each aspect of chromosome replication and division oc-
Q
---+
G
W
V 7

---+
Cells are dead
because of cell
division with
curs with accuracy. For example, a single kinetochore that fails
to attach to the mitotic spindle can halt cell cycle progression
~
\_u
0 (i'r(\
~
incompletely
replicated DNA
in metaphase by activating the spindle assembly checkpoint.
Each checkpoint is built in the same manner. A sensor
Permissive Restrictive Permissive detects a defect in a particu lar cellular process and in re-
temperature temperature temperature sponse to this defect activates a signal transduction pathway.
(25 •cJ (37 •cJ (25 •cJ
Effectors activated by the signaling pathway initiate repair
EXPERIMENTAL FIGURE 19-31 An experiment that led to of the defect and halt cell cycle progression until the defect is
the checkpoint pathway concept. (a) When shihed to the restrictive
corrected. In what follows we will discuss the major check-
temperature, cdc 13 mutants arrest cell cycle progression because
point pathways that govern cell cycle progression.
of incomplete DNA replication. When the cells are returned to the
permissive temperature, they resume proliferation because they
maintained viability during the cell cycle arrest. (b) cdc13 rad9.l double The Growth Checkpoint Pathway Ensures
mutants do not arrest when shifted to the restrictive temperature That Cells Only Enter the Cell Cycle After
because these cells cannot sense that their DNA is incompletely
Sufficient Macromolecule Biosynthesis
replicated. Cells undergo mitosis, and this leads to cell death because
genetic information is lost. Therefore, cells quickly lose viability at the Cell proliferation requires that cells multiply through the pro-
restrictive temperature and can no longer resume proliferation when cess of cell division and that the indi,·idual cells grow through
they are returned to the permissive temperature. macromolecule biosynthesis. Cell growth and division are

19.7 Surveillance Mechan isms in Cell Cycle Regulation 907

separate processes, but for cells to maintain a constant size as
they multiply, cell growth and division must be tightly coor-
dinated. For example, when nutrients are limited, cells reduce
their growth rate, and cell division must be down-regulated
accordingly. This type of coordination between cell growth
and division is especially important in unicellular organisms
that experience changes in nutrient availability as part of
their natural life cycle. It is therefore not surprising that sur-
veillance mechanisms exist th:~t adjmt cell division rate ac-
cording to growth rate.
ln budding yeast, cell growth and division are coordi-
nated in G 1• In this stage of the cell cycle, the growth and
division cycles are linked by making the activity of G 1 CDKs
dependent on cell growth. Which aspect of growth is linked
to the cell cycle? Classical experiments using protein synthe-
sis inhibitors indicate that growth rate and hence cell cycle
control by growth is determined by protein synthesis. How
protein synthesis controls G 1 CDK activity is an area of ac-
tive investigation. The G 1 cyclin Cln3 is subject to transla-
tional control, making levels of this cyclin especially sensitive
to protein synthesis rate. However, it is clear that this is not
the only mechanism. Although the molecular pathways that
coordinate cell division with cell growth are not yet under-
stood, it appears that this control is highly plastic. The length
of G 1 and the crittcal cell size, that is, the size at which cells
enter the cell cycle, change with nutrient availability.
S. pombe grows as a rod -shaped cell that increases in
length as it grows and then divides in the middle during mi-
tosis to produce two daughter cells of equal size (see Figure
19-4 ). Unlike budding yeast and most metazoan cells that
grow primarily during G" this yeast does most of its grow-
ing during the G1 phase of the cell cycle and it is entry into
mitosis that is carefully regulated in response to cell size.
Recall entry into mitosis is regulated by the protein kinase
Wee I, which inliibits CDK 1 by phosphorylating tyrosine 15.
When nutrients are limited, Weel phosphorylates CDKl;
hence cells remain in G 2 until they reach the critical size for
mitotic entry. This size control is brought about by regulated
protein localization. The protein kinase Porn 1 forms a gradi-
ent from each pole toward the middle of the cell. The CDK
inhibitor Weel and its inhibitor Cdr2 localize in patches to
the cell cortex in the middle of the cell (Figure 19-32 ). Poml
prevents Cdr2 from inhibiting Weel. When cells are small,
Porn] inhibits Cdr2. Weel is active and prevents entry into
mitosis. As cells grow, the local concentration of Pom 1 in
the middle of the cell declines and Cdr2 becomes active and
inhibits Weel. Cells can now enter mitosis. Hence in this
organism, cell length is measured by a protein gradient.
Nutrients are usually not limiting in multicellular organ-
isms. Instead, cell growth is controlled by growth factor sig-
naling pathways such as the Ras, AMPK, and TOR pathways
(see Chapters 8 and 16). These pathways also appear to be
important for coordinating cell growth and division. Muta-
tions in key components of growth factor signaling path-
ways, such as Myc, cause dramatic changes in cell size in
Drosophzla. Myc regulates the transcription of many genes
important for macromolecule biosynthesis and also, more
908 CHAPTER 19 • The Eukaryotic Cell Cycle
(a) Small cells (b) Large cells
Cdr2
Entry into mitosis Entry into
inhibited by Pom1 mitosis promoted
FIGURE 19-32 A protein gradient measures cell length in
S. pombe. The protein kinase Pom1 forms a gradient from each pole
toward the middle of the cell. Wee1 and its inhibitor Cdr2 are localized
to patches of cell cortex in the middle of the cell. Pom 1 inhibits Cdr2,
and hence the pathway that inhibits Wee1. (a) In small cells, the
concentration of Pom 1 in the middle of the cell is higher. Cdr2 is
inhibited, allowing Wee1 to remain active and prohibit entry into
mitosis. (b) As cells grow in length, the c~ncentration of Pom1 in the
middle of the cell declines. Pom1 inhibition of Cdr2 decreases, and the
Cdr2 signaling pathway is now able to inhibit Wee1, promoting entry
into mitosis. [Adapted from Moseley et al.. 2009; Nature 459:857-860.)
indirectly, G 1 CDKs. Thus this transcription factor appears
to integrate cell growth and division. However, the details of
this coordination remain to be elucidated.
The DNA Damage Response Halts Cell Cycle
Progression When DNA Is Compromised
The complete and accurate duplication of the genetic ma-
terial is essential for cell division. If cells enter mitosis when
DNA is not completely replicated or otherwise damaged, ge-
netic changes occur. In many instances, those changes will
lead to cell death, but as we will see in Chapter 24, they can
also lead to genetic alterations that result in loss of growth
and division control and eventually cancer. This is under-
scored by the finding that many proteins involved in sensing
DNA damage and its repair are frequently found mutated in
human cancers.
The enzymes that replicate DNA are highly accurate, but
this exactness is not enough to ensure complete accuracy
during DNA synthesis. Furthermore, environmental insults
such as x-rays and UV light can cause DNA damage, and
this damage must be repaired before a cell's entry into mito-
sis. Cells have a DNA damage response system in place that
senses many different types of DNA damage and responds
by activating repair pathways and halting cell cycle progres-
sion until the damage has been repaired. Cell cycle arrest can
occur either in G" S phase, or G 2 , depending on whether
DNA damage occurred before cell cycle entry or during
DNA replication. [n multicellular organisms, the strategy to
deal with particularly severe DNA damage is different.
Rather than attempting to repair the damage, cells undergo
programmed cell death or apoptosis, a mechanism that we
will discuss in detail in Chapter 21.
DNA damage exists in many different forms and ranges in
severity. A break of the DNA helix, known as a double-strand
break, is perhaps the most severe form of damage since such a
lesion would almost certainly lead to DNA loss if mitosis en-
sued in its presence. More subtle defects include single-strand
breaks, structural changes in nucleotides, or DNA mis-
matches. For our discussion here, it is important to note that
cells have sensors for all these different types of damage. These
sensors scan the genome and, when they detect a lesion, as-
semble signaling and repair factors onto the site of the lesion.
Central ro the detection of the different lesions is a pair of
homologous protein kinases called ATM and ATR. These
protein kinases get recruited to sites of DNA damage. They
then initiate the sequential recruitment of adapter proteins
and another set of protein kinases called Chk I and Chk2.
These kinases then activate repair mechanisms and cause cell
cycle arrest or apoptosis in animals (Figure 19-33). ATR and
ATM recognize different types of DNA damage. ATM is very
specialized in that it only responds to double-strand breaks.
ATR is able to recognize more diverse types of DNA damage,
such as stalled replication forks, damaged nucleotides, and
double-strand breaks. ATR recognizes these diverse types of
damage because all of them contain some amount of single-
stranded DNA, either as part of the damage itself or because
repair enzymes create single-stranded DNA as part of there-
pair process. Stalled replication forks, for example, are recog-
nized by ATR. The association of ATR with stalled forks is
Stalled . (see Figure 19-34 ).
Double-strand re lication DNA Nucleotide
bc•:'.:...............':~~'?'9'
~
[£hk2, Chk})
Repair
±
Cdc25
l
CDKs Apoptosis p21
FIGURE 19-33 The DNA damage response system. The protein
kinases ATM and ATR are activated by damaged DNA. ATR responds to
a variety of DNA damage-most likely to the single-stranded DNA that
exists either as a result of the damage itself or as a result of repair. ATM
is specifically activated by double-strand breaks. Because double-
strand breaks are converted into single-stranded DNA as a result of
repair, they also, albeit indirectly and hence depicted as a dashed line,
activate ATR. ATM and ATR, once activated by DNA damage, activate
another pair of related protein kinases, Chkl and Chk2. These kinases
then induce the DNA repair machinery and cause cell cycle arrest by
inhibiting Cdc25. In metazoan cells, when the DNA damage is severe,
Chkl and Chk2 also activate the transcription factor p53. p53 induces
cell cycle arrest by inducing transcription of the CKI p21 and apoptosis.
thought to activate its protein kinase activity, leading to the
recruitment of adapter proteins whose function is to recruit
and help activate the Chk 1 kinase. Active Chk 1 then induces
repair pathways and inhibits cell cycle progression.
Chkl and Chk2 halt the cell cycle. The protem kinascs
phosphorylate Cdc25, thereby inactivating it (sec Figure 19-33).
When the DNA damage occurs during G" Cdc25A inhibi-
tion results in inhibition of G 1/S phase CDKs and S phase
l.DKs (Figure 19-34). As a result, these kinases cannot initi-
ate DNA replication. When the DNA damage occurs during
S phase or in G 2, Cdc25C inhibition by Chkl/2 results in the
mhibition of mitotic CDKs and hence arrest in G 2 • Acnve
DNA replication also inhibits entry into mitosis. ATR con-
tinues to inhibit Cdc25C via Chk 1 until all replication forks
complete DNA replication and disassemble. This mechanism
makes the initiation of mitosis dependent on the completion
of chromosome replication. Finally, cells also sense DNA
replication stress that results in stalled or slowing of replica-
tion forks. lt triggers activation of the ATR-Chk l check-
point pathway and results in down-regulation of S phase
CDK activity and prevents firing of late-replicating origins.
Chkl-mediated inhibition of the Cdc25 famil) of phos-
phatases is not the only mechanism whereby DNA damage
or incomplete replication inhibits cell cycle progression. As
we will see below, DNA damage leads to the activation of
the transcription factor p53, which transcribes the CDK in-
hibitor p21. p21 binds to and inhibits all metazoan cyclin-
CDK complexes. As a result, cells arc arrested in G 1 and G 2
ATM recognizes double-strand breaks (sec Figure 19-33 ).
The protein kinase gets directly recruited to the DNA ends b;
a complex known as the MRN complex, which binds to bro-
ken ends and holds them together. Activated ATM then phos-
phorylates and activates Chk2 and recruits repair protems.
These repair proteins initiate homologous recombination, as
discussed in Chapter 4. This process involves the creation of
single-stranded overhangs, which in turn recruit and activate
ATR and its effectors, further enhancing the DNA damage
response. ATM can also recruit an alternative repair pathwa}
where two double-strand breaks are directly fused with each
other in a repair process known as nonhomologous end join-
ing. Like ATR, ATM activation also halts cell cycle progres-
sion by a Chk2-mediatcd inhibition of Cdc25, thus preventing
activation of CDKs. This inhibition can occur in G 1 or G 2 •
A key effector of the DNA damage response in metazoan
cells is the transcription factor p53 (sec Figure 19-33 ). It is
known as a tumor suppressor because its normal function is
to limit cell proliferation in the face of DNA damage. The
protein is extremely unstable and generally does not accu-
mulate to high enough levels to stimulate transcription under
normal conditions. The instability of p53 results from its
ubiquitinylation by a ubiquitin-protein ligase called Mdm2
and subsequent proteasomal degradation. The rapid degra-
dation of p53 is inhibited by ATM and ATR, which phos-
phorylate p53 at a sire that interferes with Mdm2 binding.
This and other modifications of p53 in re~ponsc to DNA
damage greatly increase its ability to activate transcription of
19.7 Surveillance Mechanisms in Cell Cycle Regulation 909
 Ongoing DNA damage DNA damage
DNA
replication ! !
ATM/R ATM/R

!
ATR
+
Chkl/2
I
~
p53 p53
!
! 1 ! ! DNA damage

Chk1 ~ Cdc25C p21 CIP p21 CIP

!
! 1 1 ATM/R--+ Chk1/2
Mitotic CDKs

!
M phase
entry
+ 1
p21 CIP Cdc25A

1 !
S phase +-- G 1/S phase and S phase CDKs
Late entry
replication S phase

i
S phase CDKs

i
Cdc25A

.__--Chkl
T
i
ATR

i
Replication stress
FIGURE 19-34 Overview of DNA damage checkpoint controls in ATR protein kinases (ATM/R) inhibit Cdc25 via the Chkl / 2 protein
the cell cycle. During G1, the p53-p21 CIP pathway inhibits G1 CDKs. kinases. They also activate p53, which induces production of the CKI
During ongoing DNA replication and in response to replication stress p2 1. During G,, the DNA damage checkpoint pathway inhibits Cdc2SA
(slow DNA replication fork movement or DNA replication fork collapse), inhibiting G1/S phase CDKs and S phase CDKs, thereby blocking entry
the ATR-Chkl protein kinase cascade phosphorylates and inactivates into or passage through S phase. During G2, ATM/R-Chkl /2 inhibit
Cdc25C, thereby preventing the activation of mitotic CDKs and Cdc25C. The p53-p21 °P pathway is also activated. Red symbols indicate
inhibiting entry into mitosis. In response to DNA damage, the ATM or pathways that inhibit progression through the cell cycle.

specific genes that help the cell cope with DNA damage. One
of th ese genes encodes the CKI p21 (see Figure 19-34 ).
Under some circumstances, such as when DNA damage
ts extensive, p53 also activates expression of genes that lead
to apoptosis, the process of programmed cell death that nor-
mally occurs in specific cells during the development of mul-
ticellular animals. In metazoans, the p53 response evolved to
induce apoptosis in the face of extensive DNA damage, pre-
sumably to prevent the accumulation of multiple mutations
that might convert a normal cell into a cancer cell. The dual
role of p5 3 in both cell cycle arrest and the induction of
apoptosis may account for the observation that nearly all
cancer cells have mutations in both alleles of the p53 gene or
in the pathways that stabilize p53 in response to DNA damage
(see Chapter 24). The consequences of mutations in p53,
A TM, and Chk2 provide dramatic examples of the signifi-
cance of cell cycle checkpoint pathways to the health of a
multicellular organism.
The Spindle Assembly Checkpoint Pathway
Prevents Chromosome Segregation Until
Chromosomes Are Accurately Attached
to the Mitotic Spindle
The spindle assembly checkpoint pathway prevents entry into
anaphase until every kinetochore of every chromatid is prop-
erly attached to spindle microtubules. If even a single kineto-
chore is unattached or not under tension, anaphase is
inhibited. Clues about how this checkpoint operates initially
came from the isolation of yeast mutants with a defective re-
sponse to benomyl, a microtubule-depolymerizing drug. Low
concentrations of benomyl increase the time required for
yeast cells to assemble the mitotic spindle and attach kineto-
chores to microtubules. Wild-type cells exposed to benomyl
do not begin anaphase until these processes are completed
and then proceed on through mitosis, producing normal
daughter cells. In contrast, mutants defective in the spindle

910 CHAPTER 19 • The Eukaryotic Cell Cycle

assembly checkpoint pathway proceed through anaphase be-
fore assembly of the spindle and attachment of kinetochores
is complete; consequently, they mis-segregate their chromo-
somes, producing abnormal daughter cells that die.
We now know that cells harbor a surveillance mecha-
nism that prevents anaphase entry in the presence of unat-
tached kinetochores. Components of the spindle assembly
checkpoint recognize and bind to unoccupied microtubule
binding sites at kinetochores and create an anaphase inhibi-
tory signal (Figure 19-35a). A protein known as Mad2 (mitotic
arrest defective 2) is central to the creation of this inhibitory
signal. Mad2 regulates Cdc20, the specificity factor required
to target the APGC to securin. Recall that APGCCJdo_mediated
ubiquitinylation of sccurin and its subsequent degradation is
required for activation of separase and entry into anaphase (see
(a) Checkpoint activation
a
~~~~:,";~ I!J: a ~~~
~ ~riiSO-.:
~
11
Mad1 \ 1:.1
~cse
ottoohm~
L.ck of •
--~--------::t~ --~
li3 Mad2 in open conformation
@ Mad2 in closed conformation
FIGURE 19-35 The spindle assembly checkpoint pathway. The
spindle assembly checkpoint pathway is active until every single
kinetochore has attached properly to spindle microtubules. (a) The Mad2
protein exists in two conformations, one "open" (red squares) and the
other "closed" (orange circles). According to the current model, Mad1
and the closed Mad2 form a tetramer that binds to unattached kineto-
chores via the Mad1 subunit (step 0 ). Open Mad2 can bind transiently to
closed Mad2 bound to Mad 1 at the kinetochore (step 6 ). This interac-
tion with closed Mad2 stimulates open Mad2 to bind a Cdc20. Open
Mad2 can bind Cdc20 only while it is interacting with a closed Mad2. This
converts the open Mad2 protein to the closed conformation, causing it
to dissociate from the closed Mad2 at the kinetochore (step 0 ). The
stable interaction of closed Mad2 with Cdc20 prevents Cdc20 from
binding to the APC/C. Further, the closed Mad2 bound to (dc20 can
interact transiently with another Mad2 in the open conformation (step!)),
causing it to bind another Cdc20 molecule. This converts the open Mad2
to the closed conformation bound to Cdc20. This newly formed closed
Mad2-Cdc20 complex dissociates from the first Mad2-Cdc20 pair,
generating two Mad2-Cdc20 complexes (step 1,'1). Thus free Mad2 in the
open conformation is quickly converted to closed Mad2 bound to Cdc20
Figure 19-27). Mad2 is recruited to kinetochores that arc not
attached to microrubules. Madl appears critical for this pro-
cess. Kinerochore-bound Mad2 rapidly exchanges with a solu-
ble form of Mad2 that inhibits all the Cdc20 in the cell (Figure
19-35a). When microtubules attach to kinetochores, the kinet-
ochores release the bound Mad2 and cease the process by
which the inhibitory, soluble form of Mad2 is produced (figure
19-35b). However, when even a single kinetochore is unat-
tached to microtubules from the opposite spindle pole of its
sister, sufficient soluble inhibitory Mad2 is produced at the un-
attached kinetochore to inhibit all the Cdc20 in the cell. This
elegant model for the spindle assembly checkpoint pathway
can account for the ability of a single unattached kinetochore
to inhibit all the cellular Cdc20 until the kinetochore becomes
properly associated with spindle microtubules.
(b) Checkpoint inactivation
8&8_/'9 •
IJ
eso~ .,
Release of 9,1
Mad1-Mad2 lO
tetra mer from
kinetochore
Attachment
completed
'-----
Mad1
Microtubules
as this cycle repeats (step 1';1). The source of closed Mad2 that initiates this
chain reaction is the closed Mad2 bound to Mad 1 associated with a
kinetochore, explaining how a single unattached kinetochore can cause
inactivation of all the Cdc20 in the cell through the formation of closed
Mad2-Cdc20 complexes. How are unattached kinetochores that recruit
Mad 1-Mad2 complexes generated? Either microtubules fail to attach or
Aurora B severs kinetochore-microtubule attachments that are not under
tension (see Figure 19-25). (b) Silencing of the spindle assembly
checkpoint pathway: Attachment of microtubules (green) to kinetochores
causes the displacement of the Mad 1-Mad2 tetra mer. Mad2 in the
displaced tetra mer cannot interact with open Mad2 but rather binds to
p31 comet. p31 comet is always active and disassembles Mad2-Cdc20
complexes, releasing active Cdc20 (step fJ). However, a small number of
Mad1 Mad2 tetramers bound to kinetochores can generate enough
Mad2-Cdc20 complexes by the mechanism shown in (a) to overcome the
activity of p31. Once all kinetochores have attached to microtubules,
causing the release of all Mad 1-Mad2 tetramers, p31 activity predomi-
nates, releasing active Cdc20, which binds to the APC/C, resulting in
ubiquitinylation and proteasomal degradation of securin and the onset
of anaphase. [Modified from A. De Antoni et al., 2005, Curr. Bioi. 15:214.]
19.7 Surveillance Mechanisms in Cell Cycle Regulation 911
 Entry into anaphase is also inhibited when the attach-
ments of microtubules to kinetochores are faulty. As we saw
in Section 19.5, kinetochores of sister chromatids often at-
Kin 4
tach to microtubules emanating from the same pole (syntelic Tem1-GDP ---,.,..__,
attachment) or a single k inetochore attaches to microtubules (mactive)
that originate from two different poles (merotelic attach- M etaphase -'----\-- Nucleus
ment). These faulty attachments result in insufficient or no ten- Nucleolus Spindle
sion at sister kinetochores, and such microtubule-kinetochore Cdc14 lf - -'--f - microtubule
(inactive)
interactions are quick ly dest::~hilin·d hy Aurora B phosphory-
lation of the microtubule-binding proteins of the kineto-
chore. This leads to the generation of unattached kinetochores,
which are recognized by the spindle assembly checkpoint. In Proper nucl ear D ~ fJ Improper nuclear
this manner Aurora B and the spindle assembly checkpoint positio n / ~ position

collaborate during every cell cycle to accurately attach every

•
single pair of sister chromatids on the mitotic spindle in the
correct, hi-oriented manner.
The spindle assembly checkpoint pathway is essential for NI.Jcleolus
viability in mice, highlighting the importance of this quality- Cdc14
control pathway during every cell division. If anaphase is Anaphase
(inactive)
initiated before both kinetochorcs of a rep licated chromo-
some become attached to microtubules from opposite spin- Cdc14
(ACTIVE)
dle poles, daughter cells are produced that have missing or
extra chromosomes, an outcome called twndisjunction.

~
When nondisjunction occurs in mitotic cells, it can lead to
the misregulation of genes and contribute to the develop-
Exit from Late mitotic
ment of cancer. When nondisjunction occurs during the mei- mitosis arrest
otic division that generates a human egg or sperm, trisomy
of any chromosome can occur. Down syndrome is caused by Checkpoint IJ
failure
trisomy of chromosome 21, resulting in developmental ab-
normalities and mental retardation. Every other trisomy re-

An"'~
sults in embryonic lethality or death shortly after birth.
·.
The Spindle Position Checkpoint Pathway
Ensures That the Nucleus Is Accurately multinucleate cells

Partitioned Between Two Daughter Cells

The coordination of the site of nuclear division with that of
cytokinesis is essential for the production of two identical
daughter cells. If cytokinesis occurred in such a way that
each daughter cell failed to receive a complete genetic com-
plement, chromosome loss or gain would ensue. In many
systems, surveillance mechanisms have been described that
ensure cytokinesis does not occur when the mitotic spindle is
not correctly positioned in the cell. This surveillance mecha-
nism, known as the spindle position checkpoint pathway, is
best understood in budding yeast. l n budding yeast, the site
of bud formation and therefore the site of cytokinesis is de-
termined during G 1• Thus the axis of division is defined prior
to mitosis and the mitotic spindle must be aligned along this
mother-bud axis during every cell division (Figure 19-36,
step 0 ). When this process fails, the spindle position check-
point prevents mitotic CDK inactivation, giving the cell an
opportunity to reposition the spindle prior to spindle disas-
sembly and cytokinesis (Figure 19-36, step H ). If the spindle
position checkpoint fails, cells that misposition their spindles
give rise to mitotic products with too many or too few nuclei
(Figure 19-36, step !)).
FIGURE 19-36 The spindle position checkpoint in budding
yeast. Cdcl4 phosphatase activity is required for exit from mitosis.
Top: In 5. cerevisiae, during interphase and early mitosis, Cdcl4 (red dots)
is sequestered and inactivated in the nucleolus. Inactive Tem1 -GDP
(purple) associates with the spindle pole body (SPB) nearest to the bud
as soon as the mitotic spindle forms. If chromosome segregation
occurs properly (step O J, extension of the spindle microtubules inserts
the daughter SPB into the bud, causing Teml to be activated by an
unknown mechanism. Teml-GTP activates a protein kinase cascade,
which then promotes the release of active Cdcl4 from the nucleolus
and exit from mitosis. If the spindle apparatus fails to place the
dilughter SPB in the bud (step f) ), Kin4 (c.yan), an inhibitor ofTeml, IS
recruited from the mother cell cortex to t he mother-cell-located SPB
and maintains Teml in the GOP bound form and mitotic exit does not
occur. Ltel (orange) is an inhibitor of Kin4 and localized to the bud.
Ltel prevents Kin4 that leaks into the bud from inhibiting Teml.lfthe
checkpoint fails (step !D), cells with mispositioned spindles inappropri-
ately exit from mitosis and produce anucleate and multi-nucleate cells.

912 CHAPTER 19 • The Eukaryotic Cel l Cycle

 Recall that in budding yeast, a pool of mitotic cyclins are
spared from degradation by APCICCdclO to facilitate the
sometimes difficult process of aligning the mitotic spindle in
such a way that half the nucleus squeeze~ through the tiny
bud neck during anaphase spindle elongation. Recall further
that inactivation of this protected pool of mitotic cyclin-CDK
complexes is triggered by the protein phosphatase Cdc l 4,
which in turn is activated h} a signal transduction pathway
known as the mitotic exit 11etwork (see rigure 19-28). The
mitotic exit network is controlled by a small (monomeric)
GTPase called Tern 1. This member of the GTPase superfam-
ily of switch proteins controls the activity of a protein kinase
cascade similarly to the way Ras controls MAP kinase path-
ways (see Chapter 16). Tern 1 associates with spindle pole
bodies (SPBs) as soon as they form. An inhibitor of the GTPase
called Kin4 localizes to the mother cell but is absent from the
bud (sec Figure 19-36). An inhibitor ofKin4 called Ltel local-
izes to the bud but is absent from the mother cell and inhibits
any residual Kin4 that leaks into the bud. When spindle micro-
tubule elongation at the end of anaphase has correctly posi-
tioned segregating daughter chromosomes into the bud,
Teml inhibition by Kin4 is relieved. As a consequence, Teml
is converted into its active GTP-bound state, activating the
protein kinase signaling cascade. The terminal kinase in the
cascade then phosphorylates the nucleolar anchor that binds
and inhibits Cdcl4, releasing the Cdc14 phosphatase into the
cytoplasm and nucleoplasm in both the bud and mother cell
(see rigure 19-36, step 0 ). Once active Cdc14 is available,
mitotic CDKs are inactivated and cells exit from mitosis.
When the spindle fails to position correctly, the Teml-bear-
ing spindle pole body fai ls to enter the bud and the mitotic
exit network cannot be activated. Cells arrest in anaphase.
Thus spatial restriction of inhibitors and activators of a signal
transduction pathway allows the cell to sense a spatial cue,
spindle position, and translate it into regulation of a signal
transduction pathway.
'-'~V CO C"EPTc; of c;('>rtio--- 19 7
Surveillance Mechanisms in Cell Cycle Regulation
• Surveillance mechanisms known as checkpoint pathways
establish dependencies among cell cycle events and ensure
that cell cycle progression does not occur prior to the com-
pletion of a preceding event.
• Checkpoint pathways consist of sensors that monitor a
particular cellular event or defect therein, a signaling path-
way, and an effector that halts cell cycle progression and
activates repa ir pathways when necessary.
Growth and cell division are integrated during G 1 in most
systems. Reduced macromolecule biosynthesis delays cell cycle
entry.
Cells arc able to detect and respond to a wide variety of
DNA damage, and the response differs depending on the cell
cycle stage that cells are in.
In response to DNA damage two related protein kinases
AT~l and ATR are recruited to the site of damage, where
they activate signaling pathways that lead to cell cycle arrest,
repa ir, and, under some circumstances, apoptosis.
The spindle assembly checkpoint pathway, which prevents
premature initiation of anaphase, utilizes Mad2 and other
proteins to regulate the APC/C specificity factor Cdc20, which
t;Irget~ <;ecurin and mitotic cyclins for ubiquitinylation.
The spindle position checkpoint pathway prevents mitotic
CDK inactivation when the spindle is mispositioned. ln thts
pathway localized activators and inhibitors and a sensor that
shuttles between them allows the cells to sense spindle position.
19.8 Meiosis: A Special Type
of Cell Division
In nearly all diploid eukaryotes, meiosis generates haploid
germ cells (eggs and sperm), which can then fuse wtth a germ
cell from another individual to generate a diploid zygote that
develops into a new individual. Meiosis is a fundamental as-
pect of the biology and evolution of all eukaryotes because it
resu lts in the reassortment of the chromosome sets received
from an individual's two parents. Both chromosome reas-
sortmcnt and homologous recombination between parental
DNA molecules during meiosis guarantees that each haploid
germ cell generated will receive a unique combination of
gene alleles that is distinct from each parent as well as from
every other haploid germ cell formed.
The mechanisms of meiosis are analogous to those of mi-
tosis. However, several key differences in meiosis allow this
process to generate haploid cells with generic diversity (see
Figure 5-3). In mitosis, each S phase is followed by chromo-
some segregation and cell division. In contrast, during meio-
sis, one round of DNA replication is followed by two
consecutive chromosome segregation phases. This leads to
the formation of haploid rather than diploid daughter cells.
During the two divisions, maternal and paternal chromo-
somes are shuffled and divided so that the daughter cells are
different in genetic makeup from the parent cell. In this sec-
tion, we will discuss the similarities between mitosis and
meiosis as well as the meiosis-specific mechanisms that
transform the canonical mitotic cell cycle machinery so that
it brings about the unusual cell division that leads to the
formation of haploid daughter cells.
Extracellular and Intracellular Cues
Regulate Entry into Meiosis
The signals triggering entry into the meiotic divisions in
metazoans are a very active area of research and much is snll
unknown. However, the same basic principles govern the de-
cision to enter the meiotic program in all organisms where
this has been studied. Extracellular signals induce a tran-
scriptional program that produces meiosis-specific cell cycle
19.8 Me1osis: A Special Type of Cell Division 913
factors that bring about the unusual meiotic cell divisions.
This modification of the cell cycle goes hand in hand with a
developmental program that induces the features character-
istic of gametes, such as the development of a flagellum in
sperm or the production of a stress-resistant cell wall during
spore formation in fungi. At least one of the extracellular
signals inducing entry into the meiotic divisions in mammals
is retinoic acid, a steroid hormone that functions in many
differc>nr developmental processes. The cellular targets of
this hormone and how it functions to specify the meiotic fate
remain to be dtscovered.
The molecular mechanisms underlying the decision to
enter the meiotic divisions are well understood in S. cerez,r-
srae. The decision to enter the meiotic divtsions is made in
G 1• Depletion of nitrogen and carbon sources induces diploid
cells to undergo meiosis instead of mitosis, yielding haploid
spores (see Figure 1-17). During the meiotic divisions, bud-
ding is repressed and pre-meiotic S phase and the two mei-
otic divisions occur within the confines of the mother cell.
Spore walls are then produced around the four meiotic prod-
ucts. Recall that budding and the initiation of DNA repl ica-
tion are induced by G/S phase CDKs. Their expression
needs to he inhibited tu prevem budding. Nutritional starva-
tion represses expression of G 1/S phase cyclins, inhibiting
budding. However, DNA replication also relies on GtfS
phase CDKs. How can pre-meiotic DNA replication occur in
the absence of G 1/S phase CDKs? The sporulation-specific
protein kinase lme2 takes over the role of G tfS phase CDKs

Row Mitosis Meiosis

In somatic cells In cells in the sexual cycle

One cell division,

Parental Q ---+ 0 Daughter
Two cell divisions,
0 0 ___. 0 0 Products

0 00
resulting in
cell
resulting in four Meiocyte ---+
two daughter cells o cells products of meiosis of meiosis

Chromosome number @ Chromosome number

0 00
2 per nucleus maintained
{e.g., for a diploid cell}
(§---+
@)
halved in the products
of meiosis e ---.. 0 ---.. 00

i~ ~ i~ ~
One premitotic .,a..,"
~II)

One premeiotic .,
~

a..,"
(/)

<(<3 <(<3
3 S phase per cell Z=> S phase for both z"c:
division oc: cell divisions 0

G1 S G2 M Gt s M, Mu

4
Normally, no pairing
of homologous
Chrom7 / Full synapsis of
of homologous Bivalent
{:!
~ ...:-- Chromatid
chromosomes in prophase chromosomes in prophase
1-;>- ...} Chromosome
Chromosome/

At least one recombination

Normally, no recombination
5
in prophase
between nonsister Chiasma- 8::
::;;._._ ,.:_
chromatids

Bi-oriented sister Co-orientation of

++
6 sister kinetochores in meiosis I
kinetochores

Loss of cohesion Maintenance of cohesion

7 between sister chromatid between sister chromatid arms
arms during prophase during prophase of meiosis I

Centromeres Centromeres do not

......--.....
~
8 divide at
anaphase
divide at anaphase I
but do at anaphase II ......--.....
~
Anaphase I Anaphase II

Conservative process: daughter cells' genotypes

Promotes variation among the products of meiosis
identical with parental genotype

Cell undergoing mitosis can be diploid or haploid Cell undergoing meiosis is diploid or multiple thereof

FIGURE 19-37 Comparison of the main features of mitosis and meiosis. [Adapted from A. J. F. Griffiths et al., 1999, Modern Genetic Analysis,
W. H. Freeman and Company.]

914 CHAPTER 19 • The Eukaryotic Cell Cycle

 in promoting DNA replication. lme2 promotes (1) phos-
.· phorylation of the APC/C specificity factor Cdh 1, inactivat-
ing it so that S phase and M phase cyclins can accumulate;
(2) phosphorylation of transcription factors to induce genes
required for S phase, including DNA polymerases and
S phase cyclins; and (3) phosphorylation of the S phase CDK
inhibitor Sic I, leading to release of activeS phase CDKs and
the onset of pre-meiotic DNA replication.
Several Key Features Distinguish Meiosis
from Mitosis
The meiotic divisions differ in several fundamental aspects from
the mitotic divisions. These are summarized in Figure ] 9-37.
During meiosis, a single round of DNA replication is followed
by two cycles of cell division, termed meiosrs I and meiosis 11
(Figure 19-38). Meiosis II resembles mitosis in that sister
chromatids are segregated. However, meiosis I is very differ-
ent. During this division, homologous chromosomes-the
chromosome inherited from your mother and the same chro-
mosome inherited from your father-arc segregated. This
unusual chromosome segregation requires three meiosis-
specific modifications to the chromosome segregation ma-
chinery. In what follows we will discuss these and explain
why they are needed.
The tension-based sensing mechanism responsible for ac-
curately attaching chromosomes to the spindle during mitosis is
also responsible for segregating chromosomes during meiosis I.
Thus homologous chromosomes must be linked so that the
tension-based mechanism of accurate attachment to the spindle
can function. Homologous recombination between homolo-
gous chromosomes creates these linkages (see Figure 19-38).
The molecular mechanisms of homologous recombination
are discussed in detail in Chapter 4. Here, we will restrict our
discussion to the importance of homologous recombination
for the meiotic divisions to occur successfully.
In G 2 and pr'o phase of meiosis I, the two replicated chro-
matids of each chromosome are associated with each other
by cohesin complexes along the full length of the chromo-
some arms, just as they arc following DNA replication in a
mitotic cell cycle (see Figure 19-38). In prophase of meiosis
I, homologous chromosomes (i.e., the maternal and paternal
chromosome I, the maternal and paternal chromosome 2,
etc.) pair with each other and undergo homologous recom-
bination. Significantly, at least one recombination event oc-
curs between a maternal and a paternal chromosome. The
crossing over of chromatids produced by recombination can
be observed microscopically in the first meiotic prophase
and metaphase as structures called chiasmata (singular, chi-
asma). In contrast, no pairing between homologous chromo-
somes occurs during mitosis, and recombination between
nonsister chromatids is rare. Concomitant with homologous
recombination, homologous chromosomes associate with
·. each other in a process known as synapsis. In most organ-
isms this synapsis is mediated by a proteinaceous complex
known as the synaptonemal complex (SC). Homologous
chromosomes linked through chiasmata are called bivalents
(see Figure 19-38). The chiasmata now provide the resis-
tance to the pulling force exerted by microtubules on the
metaphase I spindle (Figu rc 19-39).
The recombination between nonsister chromatids that
occurs in prophase of meiosis I has at least two functional
consequences: First, it holds homologous chromosomes to-
gether during meiosis I metaphase. Second, it contributes to
genetic diversity among individuals of a species b} ensuring
new combinations of gene alleles in different individuals.
(Note: genetic diversity primarily arises from the indepen-
dent reassortment of maternal and paternal homologs dur-
ing the meiotic divisions). The homologs connected through
at least one chiasma, which formed during prophase of mei-
osis I, must now align on the meiosis I spindle so that mater-
nal and paternal chromosomes are segregated away from
each other during anaphase of meiosis I. This requires that
the kinetochorcs of sister chromatids attach to spindle fibers
emanating from the same spindle pole rather than from op-
posite spindle poles as in mitosis (see Figure 19-39). Sister
chromatids arc said to be co-oriented. However, the kineto-
chores of the maternal and paternal chromosomes of each
bivalent attach ro spindle microtubules from opposite spin-
dle poles; they are hi-oriented.
Finally, to facilitate two consecutive chromosome segre-
gation phases, cohesins have to be lost from chromosomes in
a stepwise manner. Recall that during mit(>sis, all cohesins
are lost by the onset of anaphase (Figure 19-40a). In con-
trast, during meiosis, cohesins are lost from chromosome
arms by the end of meiosis I, but a pool of cohesins around
kinetochores is protected from removal (Figure 19-40b).
This pool of cohesins persists throughout meiosis I but is
removed at the onset of anaphase II. As we will see next, loss
of cohesins from chromosome arms is required for homolo-
gous chromosomes to segregate away from each other dur-
ing meiosis I.
The mechanisms that remove cohcsins during meiosis arc
the same as during mitosis. Securin degradation releases sep-
arase, which then cleaves the cohesins holding the chromo-
some arms together. This allows the recombined maternal
and paternal chromosomes to separate, bur each pair of
chromatids remains associated at the centromere. During
metaphase II, sister chromatids align on the metaphase II
spindle and separase is activated yet again, cleaving the re-
sidual cohesin around centromeres, facilitating anaphase II
(see Figure 19-40b).
Recombination and a Meiosis-Specific Cohesin
Subunit Are Necessary for the Specialized
Chromosome Segregation in Meiosis I
As discussed earlier, in metaphase of meiosis I, both sister
chromatids in one (replicated) chromosome associate with mi-
crotubules emanating from the same spindle pole rather than
from opposite poles as they do in mitosis (see Figure 19-39).
Two physical links between homologous chromosomes resist
the pulling force of the spindle until anaphase: (a) crossing
over between chromatids, one from each pair of homologous
19.8 Meiosis: A Special Type of Cell DivisiOn 91 s
0 FOCUS ANIMATION: Meiosis

FIGURE 19-38 Meiosis. Pre-meiotic cells have Paternal

two copies of each chromosome (2n), one derived homolog
Premeiotic
from the paternal parent and one from the maternal
cell (2n)
parent. For simplicity, the paternal and maternal Maternal
homologs of only one chromosome are dia- homolog
grammed. Step 0 : All chromosomes are replicated
during S phase before the first meiotic division,
giving a 4n chromosomal complement. Cohesin
complexes (not shown) link the sister chromatids
composing each replicated chromosome along
Repl icated
their full lengths. Step f) : As chromosomes chromosomes (4n)
condense during the first meiotic prophase,
replicated homologs pair and undergo homologous
recombination, leading to at least one crossover
event. At metaphase, shown here, both chromatids
of one chromosome associate with microtubules
emanating from one spindle pole, but each
D
1
Recombination and synapsis
betw~en homologs in prophase

member of a homologous chromosome pair

associates with microtubules emanating from Metaphase I
opposite poles. Step iJ : During anaphase of meiosis II)

I, the homologous chromosomes, each consisting of 'iii

0
two chromatids, are pulled to opposite spindle 'iii
::iE
poles. Step [1 : Cytokinesis yields the two daughter
cells (now 2n). which enter meiosis II without
undergoing DNA replication. At metaphase of meio-
sis II, shown here, the sister chromatids associate
with spindle microtubules from opposite spindle
poles, as they do in mitosis. Steps lit and r!l: Anaphase I
Segregation of sister chromatids to opposite
spindle poles during the second meiotic anaphase
followed by cytokinesis generates haploid gametes
(1 n) containing one copy of each ch romosome.
Micrographs on the left show meiotic metaphase I
and metaphase II in developing gametes from
Li/ium (lily) ovules. Chromosomes are aligned at the
metaphase plate. [Photos courtesy of Ed Reschke/Peter
Arnold, Inc.] Daughter cells
in metaphase II
(2n)

=
II)
'iii
0 Anaphase II
'Gi
::iE

ln ln 1n 1n
 Centriole (a) Mitosis
___.-(spindle pole)
<:J{' Metaphase Anaphase

' ·- ·. Spindle microtubules

..!,
Separase
\

".!.
/.--·~.....
, .... -
-~~
.
()'- :-.~
'
.' ......
. '\
~

;- -
~ ;

(b) Meiosis
Metaphase I Anaphase I

,;;;~- _..... ~\; ..!,

·' FIGURE 19-39 Chiasmata and cohesins distal to them link
homologous chromosomes in meiosis I metaphase. Connections
between chromosomes during meiosis I are most easily visualized in
organisms with acrocentric centromeres, such as the grasshopper. The
kinetochores at the cent rome res of sister chromatids attach to spindle
microtubules emanating from the same spindle pole, with the
kinetochores ofthe maternal (red) and paternal (blue) chromosomes
attaching to spindle microtubules from opposite spindle poles. The
maternal and paternal chromosomes are attached at the chiasmata
which are formed by recombination between them and the cohesion
between sister chromatid arms that persists throughout meiosis I
metaphase. Note that elimination of cohesion between sister chroma-
tid arms is all that is required for the homologous chromosomes to
separate at anaphase. [Adapted from L. V. Paliulis and R. B. Nicklas, 2000,
J. Cell Bioi. 150:1223.]
chromosomes, and (b) cohesins distal to the crossover point
(see figure 19-40b, top) . Evidence for the function of rccombi
nation during meiosis comes from the observation that when
recombination is blocked by mutations in proteins essential
for the process, chromosomes segregate randomly during
meiosis I; that is, homologous chromosomes do not neces-
sari ly segregate to opposite spindle poles.
At the onset of meiotic anaphase 1, cohesins between chro-
mosome arms are cleaved by separase. This cleavage is re-
quired for homologous chromosomes to segregate. If cohesins
were not lost from chromosome arms, the recombined sister
chromatid would rip during anaphase I. The maintenance of
centromeric cohesion during meiosis I is necessary for the
proper segregation of sister chromatids during meiosis II.
Studies in many organisms have shown that a specialized
cohesin subunit, Rec8, is necessary for the stepwise loss of
cohesins from chromosomes during meiosis. Expressed only
during meiosis, Rec8 is homologous to Sccl, the cohesin
subunit that closes the cohesin ring in the cohesin complex
of mitotic cel ls. Immunolocalization experiments revealed
that during early anaphase of meiosis I, Rec8 is lost from
chromosome arms but is retained at centromeres. However,
during early anaphase of meiosis II, centromeric Rec8 is
:_
-.~. \
Separase
~~- ~·'
Sgoi-PP2A Rec8
Metaphase II Anaphase II
,~lid
_.!. :-:..
~~~!.
\ ..! ,
, \\::....... - - . (4,\ Separase
'""'-·
"·
Rec8
FIGURE 19-40 Cohesin function during mitosis and meiosis.
(a) During mitosis, sister chromatids generated by DNA replication inS
phase are initially linked by cohesin complexes along the length of the
chromatids. During chromosome condensation, cohesin complexes
(yellow) become restricted to the region of the centromere at meta-
phase. Mei-5332/Sgo 1 (purple) recruits PP2A to centromeres, where they
antagonize Polo kinase and Aurora B, preventing the dissociation of
cohesins from centromeric regions. Dissociation of Mei-5332/Sgo 1 from
centro meres and activation of separase leads to removal of cohesins at
the centromere. Sister chromatids now separate, marking the onset of
anaphase. (b) In prophase of meiosis I, maternal and paternal chroma-
tids establish linkages between each other by homologous recombina-
tion. By metaphase I, the chromatids of each replicated chromosome
are cross-linked by cohesin complexes along their full length. Rec8, a
meiosis-specific homolog of Sec 1, is cleaved along chromosome arms
but not around the centromere, allowing homologous chromosome
pairs to segregate to daughter cells. Centromeric Rec8 is protected from
cleavage by PP2A, recruited to centromeric regions by the PP2A
regulator Mei-5332/Sgol (shown in purple). By metaphase II, the
Mei-5332/ Sgo 1-PP2A complex dissociates from chromosomes. Cohesins
can now be cleaved during meiosis II, allowing sister chromatids to
segregate. [Modified from F. Uhlmann, 2001 , Curr. Opin. Cell Bioi. 1 3:754.]
cleaved by separase, so the sister chromatids can segregate,
as they do in mitosis (see figure 19-40b, bottom). Conse-
quently, understanding the regulation of Rec8-cohesin com-
plex cleavage is central to understanding chromosome
segregation in meiosis I.
The mechanism that protects Rec8 from clea\·age at cen-
tromeres during meiosis I is similar to the mechanism that
protects Scc1 at centromeres during mitosis. Recall that dur-
ing mitotic prophase, protein kinascs, chief among them Polo

19.8 Meiosis: A Special Type of Cell Division 917

kinase, phosphorylate cohesins in the chromatid arms, caus-
ing them to dissociate and eliminating cohesion on chromatid
arms by metaphase. However, cohesion at the centromeres is
maintained because a specific isoform of protein phosphatase
2A (PP2A) is localized to centromeric chromatin by members
of a family of proteins known as the Mei-S332/Shugoshin.
PP2A keeps cohesin in a hypophosphorylated state that does
not dissociate from chromatin (see Figure 19-40a). During
mt:taphase II, Mei-S332/Sgol dissociates from chromo-
somes. In addition, when the last kinetochore is properly as-
sociated with spindle microtubules, Cdc20 is derepressed
and associates with the APC/C, causing ubiquitinylation of
securin. This releases separase activity, which cleaves Sccl
whether it is phosphorylated or not, eliminating cohesion at
the centromere and allowing chromatid separation in ana-
phase (see Figure 19-40a).
Cohesin removal differs for meiosis I because when
Rec8 replaces Sccl in the cohesin complex, the complex
does not dissociate in prophase when it is phosphorylated.
The meiotic cohesin complex can only be removed from
chromatin via the action of separase. Rec8 also differs from
Sec I in that it must be phosphorylated by several protein
kinases to be cleaved by separase. During meiosis I, the
centromere-specific isoform of PP2A targeted to centromeric
chromatin by Mei-5332/Shugoshin prevents this phosphor-
ylation. The PP2A targeting factor and PP2A then dissociate
from chromosomes by metaphase II, allowing separase
cleavage of Rec8.
Co-orienting Sister Kinetochores Is Critical
for Meiosis I Chromosome Segregation
As discussed earlier, in mitosis and meiosis 11, sister kineto-
chores attach to. spindle microtu buies emanating from op-
posite spindle poles; the kinetochores are said to be
br-orzented. This is essential for segregation of sister chroma-
tids to different daughter cells. In contrast, at meiosis I meta-
phase, sister kinetochores attach to spindle microtubules
emanating from the same spindle pole; the sister kineto-
chores are said to be co-oriented (see Figure 19-39). Obvi-
ously, attachment of sister kinetochores to the proper
microtubules in meiosis I and II is critical for correct meiotic
segregation of chromosomes.
Proteins required for meiosis I sister kinetochore co-
orientation were first identified in S. cerevisiae. We now
know that a protein complex known as the monopolin com-
plex associates with kinetochores during meiosis I and links
sister kinetochores to favor attachment to microtubules em-
anating from the same spindle pole. In organisms where ki-
netochores attach to multiple microtubules, Rec8-containing
cohesins are essential for sister kinetochore co-orientation.
These meiosis-specific cohesins impose a rigid kinetochore
structure, restricting the movement of sister kinetochores
and thereby favoring attachment to microtubules from the
same spindle pole.
Like during mitosis and meiosis II, correct attachment of
meiosis I chromosomes is mediated by a tension-based mecha-
918 CHAPTER 19 • The Eukaryotic Cell Cycle
nism. During meiotic metaphase I, kinetochore-associated mi-
crotubules are also under tension (even though the co-oriented
kinetochores of sister chromatids attach to microtubules com-
ing from the same spindle pole) because chiasmata generated
by recombination between homologous chromosomes and the
cohesins distal to the chiasmata prevent them from being
pulled to the poles (see Figure 19-39). Since kinetochore-
microtubule attachments are unstable in the absence of tension
(due to Aurora B-mediated phosphorylatiOn), kinetochores
that attach to the wrong spindle fibers release the incorrect
microtubules, enabling them to bind microtubules again
until attachments are made that generate tension. As in mi-
tosis, once tension is generated, microtubule attachment to
the kinetochores is stabilized.
DNA Replication Is Inhibited, Between
the Two Meiotic Divisions
The mechanism by which DNA replication is suppressed be-
tween meiosis I and II is currently an active area of investiga-
tion, but it is thought that a change in the regulation of CDK
activity is at least in part responsible for this suppression. The
same S phase CDKs that promote DNA replication prior to
mitosis are needed for pre-meiotic DNA repli~ation. The same
mitotic CDKs that promote mitosis also promote the meiotic
divisions, except we now call mitotic CDKs meiotic CDKs
since they promote the meiotic divisions and not mitosis.
So how is DNA replication prevented between the two
meiotic divisions? Following anaphase of meiosis l, meiotic
CDK activity does not fall as low as it does following mitotic
anaphase. This partial drop in CDK activity is thought to be
sufficient to promote the disassembly of the meiosis I spindle
but insufficient to promote MCM helicase loading (recall a
state of very low or no CDK activity is needed to load MCM
helicases). During prophase of meiosis II, meiotic CDK activ-
ity rises again and the meiosis II spindle forms. After all
sister kinetochores have attached to microtubules from
opposite spindle poles, separase is activated and cells pro-
ceed through meiosis II anaphase, telophase, and cytokinesis
to generate haploid germ cells.
KEY CONCEPTS of Section 19.8
Meiosis: A Special Type of Cell Division
Meiosis is a specialized division in which meiosis-specific
gene products modulate the mitotic cell division program
(see Figure 19-38).
The meiotic division comprises one cycle of chromosome
replication followed by two cycles of cell division to produce
haploid germ cells from a diploid pre-meiotic cell. During
meiosis I, homologous chromosomes are segregated; during
meiosis li sister chromatids separate.
• Specialized environmental conditions induce a develop-
mental program that leads to the meiotic divisions.

• During prophase of meiosis I, homologous chromosomes
undergo recombination. At least one recombination event
occurs between chromatids of homologous chromosomes,
linking the homologous chromosomes.
• Cohesins distal to the chiasma are responsible for holding
the homologous chromosomes together during prophase
and metaphase of meiosis I.
• At the omt!t of anaphase of meiosis I, cohesms on chromo-
some arms are phosphorylated and as a result cleaved by
separase, but cohesins in the region of the centromere are
protected from phosphorylation and cleavage. This protec-
tion is brought about by a meiosis-specific cohesin subunit
and a protein phosphatase that associates with centromeres.
As a result, the chromatids of homologous chromosomes re-
main associated during segregation in meiosis I.
• Cleavage of centromeric cohesins during anaphase of
meiosis IT allows individual chromatids to segregate into
germ cells.
• A complex of meiosis-specific kinetochore proteins, known
as the monopolin complex, promote the co-orientation of sis-
ter chromatids during meiosis I. Both sister chromatids attach
to microtubules emanating from the same spindle pole.
• Incomplete CDK inactivation between the two meiotic di-
visions inhibits DNA replication.
the spindle assembly checkpoint. Many questions remain
about how the plane of cytokinesis and the localization of
daughter chromosomes are determined in cells that divide
symmetrically and asymmetrically as is often seen as part of
development of complex tissues and organ structures. How
the cell cycle machinery is modulated by developmental cues
to bring about specialized divisions is also an intense area of
investigation.
Understanding the::.e detailed aspects of cell cycle control
will have significant consequences, particularly for the treat-
ment of cancers. Cancer cell!. often have defects in cell cycle
checkpoints that led to the accumulation of multiple muta-
tions and DNA rearrangements that result in the cancer
phenotype. However, the absence of these checkpoints can
make specific types of cancers particularly vulnerable to ex-
tensive DNA damage induced with radiation therapy or
chemotherapy. Normal cells activate cell cycle checkpoints
that arrest the cell cycle until the DNA damage is repaired.
Bur these cancer cells do nor and as a consequence suffer
sufficient genetic damage to induce apoptosis. If more were
understood about cell cycle controls and checkpoint path-
ways, it might be possible to design ever more effective ther-
apeutic strategies, especially against types of cancer that are
largely resistant to today's conventional therapies. It seems
very likely that better understanding of tht: molecular pro-
cesses involved will allow the design of more effective treat-
ments in the future.

Perspectives for the Future

The remarkable pace of cell cycle research over the last 25 Key Terms
years has led to a detailed model of eukaryotic cell cycle con- anaphase-promoting maturation-promoting
trol. A beautiful logic underlies these molecular controls. complex/cyclosome factor (MPF) 880
Each regulatory event has two important functions: to acti- (APC/C) 888 meiosis 913
vate a step of the cell cycle and to prepare the cell for the
APC/C specificity factor 888 mitogen 892
next event of th~ cycle. This strategy ensures that the phases
of the cycle occur in the proper order. ATM/ATR 909 mitosis 875
Although the general logic of cell cycle regulation now Aurora B kinase 901 mitotic CDKs 883
seems well established, many critical details remain to be dis- Cdc14 phosphatase 913 monopolin complex 918
covered. For instance, how cell growth and division are co- Cdc25 phosphatase 898 p53 protein 909
ordinated and how the metabolic stare of a cell feeds into its CDK-activating kinases Polo kinases 898
cell cycle machinery remain to be discovered. A number of (CAK) 888
key nutrient and growth-factor-sensing pathways such as the Rh protein 891
CDK inhibitor (CKls) 888 SCf (Skp 1, Cullin, F-box
AMPK, Ras, and TOR pathways have been identified and
their inner workings recently revealed. Understanding how checkpoint pathway 874 proteins) 888
they impact the cell cycle machinery will be a critical ques- cohesin 896 S phase CDKs 883
tion to be addressed in the years to come. Substantial prog- condensin 902 START 875
ress has been made recently in identifying substrates critical cell size 892 securin 903
phosphorylated by different CDKs, but much work remains crossing over (or cross sensor 906
to be done ro understand how the modification of these pro- ovcr)915
teins leads to the multiple events that these CDKs trigger. separase 903
cyclin 874 sister chromatids 875
Much has been discovered recently about the operation
of cell cycle checkpoint pathways, but the mechanisms that cyclin-dependent kinase synaptonemal complex 915
activate ATM and ATR in the DNA damage checkpoint are (CDK) 874
Weel protein-tyrosine
poorly understood. Likewise, much remains to be learned E2F transcription factor 887 kinase 898
about the control and mechanism of regulation of Mad2 in G 1 CDKs 883

Key Terms 919


Review the Concepts
1. What cellular mechanism(s) ensure that passage through
rhe cell cycle is unidirectional and irreversible? What mo-
lecular machinery underlies these mechanism(~)?
2. What types of experimental strategies do researchers em-
ploy to study cell cycle progression? How can these strate-
gies differ based on generic versus biochemical approaches?
3. Tim Hunt shared the 2001 Nobel Prize in Physiology or
Medicine for his work in the discovery and characterization
of cyclin proteins in eggs and embryos. Describe the experi-
mental steps that led him to his discovery of cyclins.
4. What experimental evidence indicates that cyclin B is re-
quired for a cell to enter mitosis? What evidence indicates
that cyclin B must be destroyed for a cell to exit mitosis?
5. What physiological differences between S. pombe and S.
cerevisiae make them useful yet complementary tools for
studying the molecular mechanisms involved in cell cycle
regulation and control?
6. ln Xenofms, one of the substrates of mitotic CDKs is the
Cdc25 phosphatase. When phosphorylated by mitotic
CDKs, Cdc25 is activated. What is the substrate of Cdc25?
How does this information help to explain the rapid rise in
mitotic CDK activity as cells enter mitosis?
7. Explain how CDK activity is modulated by the following
proteins: (a ) cyclin, (b) CAK, (c) Weel, (d) p21.
8. Explain the role of CDK inhibitors. If cyclin-CDK com-
plexes are required to allow regulated progression through
the eukaryonc cell cycle, what would be the physiological
rationale for CDK inhibitors?
9. What is the functional definition of START? Cancer cells
typically lose START control. Explain how the following
mutations, which arc found in some cancer cells, lead to a
bypass of START controls: (a) overexpression of cyclin D,
(b) loss of Rb function, (c) loss of p 16 function, (d) hyperac-
tive E2F.
10. The Rb protein has been called the "master brake" of
the cell cycle. Describe how the Rb protein acts as a cell cycle
brake. How is the brake released in mid- to late G 1 to allow
the cell to proceed to the S phase?
11. A common feature of cell cycle regulation is that the
events of one phase ensure progression into a subsequent
phase. InS. cerevisiae, G 1 and G 1/S phase CDKs promote
S-phase entry. Name two ways in which they promote the
activation of S phase.
12. For S phase to be completed in a timely manner, DNA
replication initiates from multiple origins in eukaryotes. InS.
cereuisiae, what role do S-phase CDKs and DDK complexes
play to ensure th;:~t the entire genome is replicated once and
only once per cell cycle?
13. In 2001, the Nobel Prize in Physiology or Medicine was
awarded to three cell-cycle scientists. Paul Nurse was recog-
nii'ed for his studies with the fission yeastS. pombe, in par-
ticular for the discovery and characterization of the weer+-
920 CHAPTER 19 • The Eukaryotic Cell Cycle
gene. What did the characterization of the wee 1 gene tell us
about cell cycle control?
14. Describe how cells know whether sister kinetochores are
properly attached to the mitotic spindle.
15. Describe the series of events by which the APC/C pro-
motes the separation of sister chromatids at anaphase.
16. Meiosis and mitosis are overall analogous processes in-
volving many of the same proteim. However, some proteins
function uniquely in each of these cell-division events. Ex-
plain the meio'>is-specific function of the following: (a) lme2,
(b) Rcc8, (c) monopolin.
17. Leland Hartwell, the third recipient of the 2001 Nobel
Prize in Physiology or Medicine, was acknowledged for his
characterization of cell cycle checkpoint pathways in the
budding yeastS. cerevisiae. What is a cell cycle checkpoint
pathway? When during the cell cycle do checkpoint path-
ways function? How do cell cycle checkpoint pathways help
to preserve the genome?
18. What role do tumor suppressors, including p53, play in
mediating cell cycle arrest for cells with DNA damage?
19. Individuals with the hereditary disorder ataxia telangi-
ectasia suffer from neurodegeneration, immunodeficiency,
and increased incidence of cancer. The g"enetic basis for
ataxia telangiectasia is a loss-of-function mutation in the
ATM gene (ATM 5; ataxia telangiectasia mutated ). Besides
p53, what other substrate is phosphorylated by ATM? How
does the phosphorylation of this substrate lead to inactiva-
tion of CDKs to enforce cell cycle arrest?
Analyze the Data
1. Many of the proteins that regulate transit through the cell
cycle have been characterized. Xnf7, identified in extracts of
Xenopus eggs, binds to the anaphase-promoting complex/
cyclosome (A PC/C). To elucidate the function of this pro-
tein, studies have been undertaken in which Xnf7 either has
been depleted from extracts using an antibody raised against
it or has been augmented in the extracts through addition of
extra Xnf7. The consequences on transit through mitosis
were then assessed (see J. B. Casaletto eta!., 2005, f. Cell Bioi.
169:61-71 ).
a. Xenopus egg extracts, arrested in metaphase, were
either depleted of Xnf7 or were mock depleted (subjected to
the same treatment as the first sample but without Xnf7
antibody), then released from metaphase arrest by addition
of Ca 2 . Aliquots of the extract were then sampled at vari-
ous times after Ca 2 • addition and the amounts of mitotic
cyclin determined, as shown on the Western blot below.
What information do these data provide about a possible
function for Xnf7?
Time (mon) 0 5 10 15 20 30 40 60
Mock depleted
Xnf7 depleted -
 b. In additional studies, exogenous Xnf7 was added to
Xenopus egg extracts, arrested at metaphase, so that the
total amount of this protein in the extracts was higher than
normal. The extracts, released from arrest by Cal+ addition,
were then assessed at various times after release for mitotic
cyclin ubiquitinylation (cyclin-Ub conjugates). What is the
rationale for examining ubiquitinylation? Using the follow-
ing figure, determine what information these studies add be-
·. yond that obtained from p:nr (a).
No addition Xnf7 addition
T1me (mon) 0 4 6 8 10 12 16 20 30 0 4 6 8 10 12 16 20 30
,.. ..,
41i11110,.,. ....
c. The spindle checkpoint pathway prevents cells with
unattached kinetochores from proceeding into anaphase.
Thus cells in which this checkpoint has been activated do not
enter anaphase and do not degrade mitotic cyclin. No-
codazole, a drug that prevents microtubule assembly, can be
used to activate the spindle checkpoint. Cells in nocodazole
become arrested in early mitosis because they cannot form a
spindle, and so all kinetochores remain unattached. To deter-
mine if Xnf7 is required for a functional spindle checkpoint,
Xenopus egg extracts, arrested in metaphase, were subjected
to various protocols (see the following figure): untreated (no
nocodazole) or treated with nocodazole and either mock de-
pleted (preimmune) or immunodepleted of Xnf7 (a-Xnf7).
The extracts were then treated with Ca2+ to overcome arrest,
and aliquots of the extracts were assessed at various times for
mitotic cyclin, a~ shown on the Western blot below. What
can you conclude about Xnf7 from these data?
Time (min) 0 10 20 30 40
- Nocodazole
Q)
2
('0
'8
Preimmune
--
zg a·Xnf7
--~
+
2. In this chapter, we have discussed that cyclins are a re-
quired component of cyclin-CDK complexes for regulated
progression through the eukaryotic cell cycle. Most cyclins
are progressively synthesized and then degraded systemati-
cally in a temporal fashion at various points in the cell cycle.
As we discussed in Chapter 7, cellular protein expression can
be regulated at several different points starting with initia-
tion of gene transcription.
a. What type of assay(s) could be employed to determme
whether the expression of cyclin B is regulated at the tran-
scriptional or translational level, if either?
b. Based on what you've learned in Chapter 19, is it pos-
sible that the activity of cyclin B could be regulated at the
post-translational level? Describe a cellular mechanism
through which this might happen.
c. How might it be possible for cyclin B expression and/
or activity to be at least partially regulated by events in the
cell's external environment?
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922 CHAPTER 19 • The Eukaryotic Cell Cycle
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