Genetic regulation of cell

cycle (s. cerevisiae)

Biotechnology
Msc III sem

Submitted by
Unnati Banarjee
Guided by
Dr Sanjana Bhagat.
Introduction

The cell cycle is the succession of

events whereby a cell grows and divides
into two daughter cells that each contain
the information and machinery
necessary to repeat the process. •
Between one cell division and the next, Division cycle of most cells consist of
Cell growth
all essential components of cell must be four coordinated processes-
duplicated, the most important
component is genetic material which
must be accurately replicated and the
two copies carefully segregated to two
daughter cells.

Distribution of the duplicated

DNA replication Cell division.
chromosomes to daughter cells
 In eukaryotes cell cycle is more complex. •
Although cell growth is usually a
continuous process, DNA is synthesized
during only one phase of the cell cycle and
the replicated chromosomes are then
distributed to daughter nuclei by a complex
series of events preceding cell division. •
Progression between these stages of the
cell cycle is controlled by a conserved
regulatory apparatus.
Phases of cell cycle   Cell cycle is divided into two basic parts-
 Interphase
 Mitosis
 INTERPHASE - Chromosomes are decondensed and distributed
throughout the nucleus, so nucleus appears morphologically uniform.
At molecular level interphase is the time during which both cell
growth and DNA replication occur in an orderly manner in
preparation for cell division. It consist of 3 phases- 1. G1 phase(gap
1) - Corresponds to interval (gap) b/w mitosis and initiation of DNA
replication. - Cell is metabolically active and continuously grows but
does not replicate its DNA. 2. S phase (synthesis) - DNA replication
takes place. 3. G2 phase (gap 2) - cell growth continues and proteins
are synthesized in preparation for mitosis. - The duration of these
cycle phases varies considerably in different kinds of cells. - In
budding yeast, all 4 stages of cell cycle gets completed in approx. 90
mins.
Mitosis  In mitosis the nuclear envelope breaks down, the contents of the
nucleus condense into visible chromosomes, and the cell's
microtubules reorganize to form the mitotic spindle that will
eventually separate the chromosomes. • As mitosis proceeds, the cell
seems to pause briefly in a state called metaphase, in which the
chromosomes, already duplicated, are aligned on the mitotic spindle,
poised for segregation. • The separation of the duplicated
chromosomes marks the beginning of anaphase, during which the
chromosomes move to the poles of the spindle, where they
decondense and re-form intact nuclei. • The cell is then pinched in
two by a process called cytokinesis, which is traditionally viewed as
the end of the mitotic phase, or M phase, of the cell cycle.
 Regulation of cell cycle by cell growth and extracellular signals

Progression of the cells through the division cycle is regulated by

extracellular signals from the environment as well as by internal signals
that monitor and coordinate the various processes that take place during
different cell cycle phases.

Cell cycle progression is accomplished by a series of control points that

regulate progression through various phases of the cell cycle. A major cell
cycle regulatory point in many types of cells occurs late in G1 and controls
progression from G1 to S.
 This regulatory point was first defined by studies of budding yeast (Saccharomyces
cerevisiae) where it is known as START. Once cells have passed START, they are
committed to enter S phase and undergo one cell division cycle. Passage through
START is highly regulated event in yeast cell cycle where it is controlled by
external signals, such as availability of nutrients, mating factors, as well as by cell
size.
 Eg. If yeasts are faced with a shortage of nutrients, they arrest their cell cycle at
START and enter a resting state rather than proceeding to S phase. Polypeptide
factors that signal yeast mating also arrest the cell cycle at START, allowing
haploid yeast cells to fuse with one another instead of progressing to S phase.
 Also, START is a point at which cell growth is coordinate with DNA replication
and cell division. • It is evident in budding yeast in which cell division produce
progeny cells of very different sizes- a large mother cell and a small daughter cell. •
In order for yeast cell to maintain a constant size, the small daughter cell must grow
more than the large mother cell does before they divide again. • The cell size must
be monitored in order to coordinate cell growth with other cell cycle events.
Cell cycle
checkpoints
The events that take place during different stages of cell cycle must be
coordinated with one another, so that they occur in appropriate order. •
Cell cycle checkpoints prevent entry into the next phase of the cell cycle
until the events of the preceding phase have been completed. • Several
cell cycle checkpoints function to ensure that incomplete or damaged
chromosomes are not replicated and passed onto daughter cells . • These
checkpoints sense unreplicated or damaged DNA and coordinate further
cell cycle progression with the completion of DNA replication or repair.

Various checkpoints are- 1. G2 phase checkpoint – Checkpoint in G2

prevents initiation of mitosis until DNA replication is completed. – It
senses unreplicated DNA, which generate a signal that leads to cell cycle
arrest. – Thus operation of G2 checkpoint prevents initiation of M phase.
It also senses DNA damage, such as that resulting from irradiation. 2.G1
phase checkpoint – Arrest at the G1 checkpoint, allows repair of damage
to take place before cell enters S phase, where the damaged DNA would
be replicated.
. S-phase checkpoint • Provides continual monitoring of the integrity
of DNA to ensure that damaged DNA is repaired before it is
replicated. • It also provides a quality control monitor to promote
repair of any errors that occur during DNA replication. 4.Spindle
assembly checkpoint • Maintains integrity of genome, occurs
towards the end of mitosis. • Monitors the alignment of
chromosomes onto the mitotic spindle, ensuring that a complete set
of chromosomes is distributed accurately to daughter cells.

Cell cycle arrest at G1, S and G2 checkpoints is mediated by two

related protein kinases, ATM and ATR that recognize damaged or
unreplicated DNA and are activated in response to DNA damage. •
ATM and ATR activate a signalling pathway that leads not only to
cell cycle arrest, but also to activation of DNA repair and in some
programmed cell death.
 Restriction of DNA replication to once per cycle . It is important to ensure
that the genome is replicated only once per cell cycle. Thus once a
segment of DNA has been replicated in S phase, control mechanisms must
exist to prevent re-initiation of DNA replication until cell cycle has been
completed. Thus this mechanism involves action of MCM helicase proteins
that bind to replication origins together with the origin recognition
complex(ORC) proteins. MCM proteins act as ‘licensing factors’ that
allow replication to initiate.

MCM proteins bind to replication origins during G1, allowing DNA

replication to initiate when cell enters S phase. Once initiation has
occurred, MCM proteins are displaced from the origin, so replication
cannot initiate again.
Regulation of cell  Regulation of cell cycle progression • Cell cycle of all eukaryotes is
controlled by a conserved set of protein kinases, which are
cycle progression
responsible for triggering major cell cycle transitions. Protein kinases
and cell cycle regulation
 I. Maturation promoting factor(MPF)
 2. Second approach was genetic analysis of yeasts. • Pioneered by
Hartwell and colleagues in early 1970s. • Investigators identified
temperature sensitive mutants of Saccharomyces cerevisiae that were
defective in cell cycle progression. • These mutants called cdc
mutants underwent growth arrest at specific ponts in the cell cycle. •
Eg. Cdc28 mutants in S.cerevisiae caused the cell cycle to arrest at
START, indicating cdc28 protein is required for passage through this
critical regulatory point in G1.
The protein kinase encoded by the yeast cdc2 and cdc28 genes has since been
shown to be a conserved cell cycle regulator in all eukaryotes, which is known as
cdk1.

. 3. Third line of investigation • It converged with identification of MPF and

yeast genetics.

Studies with protein synthesis inhibitors had revealed that entry into M phase of
these embryonic cell cycle requires new protein synthesis.

In 1983, Tim Hunt identified 2 proteins. These proteins accumulate throughout

interphase and rapidly degraded toward end of each mitosis. • Hunt called these
proteins cyclins ( cyclin A and B) and suggested that they might function to
induce mitosis, with their periodic accumulation and destruction controlling entry
and exit from M phase.
 When MPF was purified from frog eggs in 1988, its molecular
characterization showd that this conserved regulator of cell cycle is
composed of 2 key subunits: cdk1 and cyclinB.
 Cyclin B is a regulatory subunit required for catalytic activity of
cdk1 protein kinase.
 MPF activity is controlled by periodic accumulation and degradation
of cyclin B during cell cycle progression.
 Once activated, the cdk1 protein kinase phosphorylates a variety of
target proteinsthat initiate the events of M phase.
 cdk1 activity triggers the degradation of cyclin B (ubiquitin
mediated proteolysis). This proteolytic destruction of cyclin B then
inactivates cdk1, leading the cell to exit mitosis, undergo cytokinesis
and return to interphase.

Families of cyclins and cyclin-
dependent kinases
cdk1 controls passage through
G2 to M transition is driven by cdk1 Passage through START is
START as well as entry into mitosis
+mitotic B type cyclins (clb1, clb2, controlled by cdk1 +G1 cyclinsor
in yeast. It does so, however in
clb3 and clb4). cln’s.
association with distinct cyclins.

These associations of cdk1 with

distinct B typeand G1 cyclins direct
Activity of cdk’s during cell cycle
cdk1+clb5 and clb6- required for cdk1 to phosphorylate different 1. Association of cdk’s with their
progression is reglated by four
progression through S phase. substrate proteins. • Required for cyclin partners.
molecular mechanisms
progression through specific phases
of cell cycle.

2. Activation of cdk/cyclin
complexes require phosphorylation
of a conserved cdk threonine
residue. Phosphorylation of cdk’s is
catalyzed by an enzyme called CAK
(cdk activating kinase).
3. Inhibitory phosphorylation of 4. cdk’s activities are also controlled
tyrosine residues near cdk amino by binding of inhibitory
terminus, catalyzed by wee1 protwin proteins( called cdk inhibitors or
kinase. CKIs).
DNA damage  DNA damage checkpoints • DNA
checkpoints
damage checkpoints play a critical role
in maintaining the integrity of genome
by arresting cell cycle progression in
response to damaged or incompletely
replicated DNA. • They allowtime for
the damage to be repaired before DNA
replication or cell division proceeds
Mitosis phase

During mitosis, the chromosomes codense, the nuclear envelope of most cells breaks down, the cytoskeleton
reorganizes to form the mitotic spindle and the chromosomes move to opposite poles, chromosomes segregation is
then usually followed by cell division(cytokinesis). Stages of mitosis

1. Prophase - marked by appearance of condensed chromosomes, each of which consists of two sister chromatids. -
daughter DNA molecules produced in S phase. - these newly replicated DNA molecules remain intertwined
throughout S and G2, becoming untangled during process of chromatin condensation. - end of prophase corresponds
to breakdown of nuclear envelope. - but in yeast, closed mitosis occurs in whih the nuclear envelope remains intact.

In closed mitosis, daughter chromosomes migrate to opposite poles of nucleus, which then divides in two. • In these
cells, the spindle pole bodies are embedded within the nuclear envelope and nucleus dividesin two following
migration of daughter chromosome to opposite poles of the spindle.

2. Prometaphase • Transition between prophase and metaphase. • Microtubules of mitotic spindle attach to
kinetochores of condensed chromosomes.

3. Metaphase • Chromosomes shuffle back and forth until they eventually align on metaphase plate in the center of
the spindle. 4. Anaphase • Breakage of link between sister chromatids which then separate and move to opposite
poles of the spindle. 5. Telophase • Nuclei reform and chromosomes decondense.
Mitosis
Cytokinesis • Begins during late anaphase and completed by the end of telophase. • Resulting in
formation of two interphase daughter cells.

Cdk/cyclinB and progression to metaphase • Events of M phase are initiated by activation of

cdk1/cyclinB protein kinase(MPF). Cdk1/cyclinB not only acts as a master regulator of M phase
transition, phosphorylating and activating other downstream protein kinase but also acts directly by
posphorylating some of the structural proteins involved in cellular organization.

Chromatin condensation is driven by protein omplexex called condensins, which are members of a
class of structural maintenance of chromatin(SMC) proteins that play key roles in organization of
eukaryotic chromosomes.

Another family of SMC proteins, called cohesins, contribute to chromosome segregation during
mitosis. • Cohesins bind toDNA in S phase and maintain linkage between sister chromatids
following DNA replication. • As cells enter M phase, condensins are activated by cdk1/cyclinB
phosphorylation.

Condensins replace cohesins along the length of chromosomes except at centromere.

 Cytokinesis • Cytokinesis of yeast and
animal cells is mediated by a
contractile ring of actin and myosinII
filaments that forms beneath the
plasma membrane. • Cleavage
proceeds as contraction of the actin-
myosin filaments pulls the plasma
membrane inward, eventually pinching
the cell in half.
References https://www.nature.com/articles/npjsba201516
The cell A molecular approach M.cooper
https://www.researchgate.net/publication/
327617218_Layers_of_regulation_on_cell-
cycle_gene_expression_in_the_budding_yeast
_Saccharomyces_cerevisiae
 Thankyou