Cell Cycle

Cell cycle can be defined as the entire sequence of events that leads to the duplication and division of a cell
and consist of three major phases namely mitosis (M phase) or the nuclear division, cytokinesis or the
division of the cell and interphase where replication of genetic material occurs. Recognition of these phases
began in 1882, when Walther Flemming named the process of nuclear division mitosis (from the Greek mito,
or “thread”) after he first observed the condensed chromosomes. Mitosis was a clear cell cycle landmark,
and the rest of the cell cycle between mitosis was called interphase.

The interphase can be further divided into G1 (gap phase 1), S (synthesis) and G2 (gap phase 2) phases.
This division of interphase into three separate phases based on the timing of DNA synthesis was first
proposed in 1953 by Alma Howard and Stephen Pelc of Hammersmith Hospital, London, based on their
experiments on plant meristem cells. Cell cycles can range in length from as short as 30 minutes in a
cleaving frog embryo, whose cell cycles lack both G1 and G2 phases, to several months in slowly growing
tissues, such as the mammalian liver.

Each cell is born at the completion of the M phase, which includes mitosis, the partitioning of the
chromosomes and other cellular components, and cytokinesis, the division of the cytoplasm. The
chromosomal DNA is replicated during S phase (synthetic phase). The remaining two phases are gaps
between mitosis and the S phase. The G1 phase (first gap phase) is the interval between mitosis and the
start of DNA replication. The G2 phase (second gap phase) is the interval between the
completion of DNA replication and mitosis. All cycling cells have an M phase and an S phase. The G1
and G2 phases vary in length and are very short in some early embryos. The M phase lasts only for an
hour in a period of 24 hour required for a eukaryotic cell to divide. Cell cycles can range in length from
as short as 30 minutes in a cleaving frog embryo, whose cell cycles lack both G1 and G2 phases, to several
months in slowly growing tissues, such as the mammalian liver.

Cells that are no longer capable of division, whether temporarily or permanently, remain in G0 phase. A cell
must receive a growth-promoting signal to proceed from the quiescent stage or G0 into G1 phase and thus
reenter the cell cycle.
Events of Interphase: The interphase is characterized by the following features: The nuclear envelope
remains intact. The chromosomes occur in the form of diffused, long, coiled and indistinctly visible
chromatin fibres. The DNA amount becomes double. Due to accumulation of ribosomal RNA (rRNA) and
ribosomal proteins in the nucleolus, the size of the latter is greatly increased. In animal cells, a daughter pair
of centrioles originates near the already existing centriole and, thus, an interphase cell has two pairs of
centrioles. In animal cells, net membrane biosynthesis increases just before cell division (mitosis). This extra
membrane is stored as blebs on the surface of the cells about to divide. Events in interphase take place in
three distinct phases.

G1 Phase: After the M phase of previous cell cycle, the daughter cells begin G1 of interphase of new cell
cycle. G1 is called first gap phase, as no DNA synthesis takes place during this stage. It is also known as
the first growth phase, since it involves synthesis of RNA, proteins and membranes which leads to the
growth of nucleus and cytoplasm of each daughter cell towards their enhancing size. During G1 phase,
chromatin is fully extended and not distinguishable as discrete chromosomes with the light microscope.
Thus, it involves transcription of three types of RNAs, namely rRNA, tRNA and mRNA; rRNA synthesis is
indicated by the appearance of nucleolus in the interphase (G1 phase) nucleus. Proteins synthesized
during G1 phase (a) regulatory proteins which control various events of mitosis (b) enzymes (DNA
polymerase) necessary for DNA synthesis of the next stage and (c) tubulin and other mitotic apparatus
proteins. G1 phase is most variable as to duration it either occupies 30 to 50 per cent of the total time
of the cell cycle. Terminally differentiated somatic cells (end cells such as neurons and striated muscle
cells) that no longer divide, are arrested usually in the G1 stage, such a type of G1 phase is called G0 phase.

S phase: During the S phase or synthetic phase of interphase, replication of DNA and synthesis of histone
proteins occur. New histones are required in massive amounts immediately at the beginning of the S period
of DNA synthesis to provide the new DNA with nucleosomes. At the end of S phase, each chromosome has
two DNA molecules and a duplicate set of genes. During replication, the duplicated DNA molecules, called
sister chromatids, become linked to each other by a protein complex called cohesin. Cohesin is a member of
the SMC, or structural maintenance of chromosomes, family of proteins. SMC proteins are DNA-binding
proteins that affect chromosome architectures; indeed, cells that lack SMC proteins show a variety of defects
in chromosome stability or chromosome behavior. This pairing of sister chromatids is important for their
orderly segregation later in mitosis. . S phase occupies roughly 35 to 45 per cent time of the cell cycle.

G2 phase: This is a second gap or growth phase of interphase. During G2 phase, synthesis of RNA and
proteins continues which is required for cell growth. Of particular significance to the cell cycle, most
microtubules – proteins that are required during mitosis – are produced during G2. It may occupy 10 to 20
per cent time of cell cycle. As the G2 phase draws to a close, the cell enters the M phase.

Dividing phase: There are two types of cell division possible. Mitosis and meiosis. The mitosis (Gr., mitos
=thread) occurs in the somatic cells and it is meant for the multiplication of cell number during
embryogenesis and blastogenesis of plants and animals. Fundamentally, it remains related with the growth
of an individual from zygote to adult stage. Mitosis starts at the culmination point of interphase (G2 phase).
It is a short period of chromosome condensation, segregation and cytoplasmic division. Mitosis is important
for growth of organism, replacement of cells lost to natural friction or attrition , wear and tear and for wound
healing. Hence, mitosis is remarkably similar in all animals and plants. It is a smooth continuous process and
is divided into different stages or phases.

Different phases of mitotic cell cycle

Duration in hours
Phases
Vicia faba (broad bean) Mouse liver cells Human Hela cells
G1 12 12 12
S 6 6-8 10
G2 12 3-6 3
M 1 1 1
G0 and Growth Control: Most cells of multicellular organisms differentiate to carry out specialized
functions and no longer divide. Such cells are considered to be in the G0 phase. Cells often enter G0 directly
as they exit their last mitosis. These nondividing cells (such as neurons and striated muscle cells) that no
longer divide are arrested usually in the G1 stage, such a type of G1 phase is called G0 phase, a branch of
the G1 phase. G0 cells are not dormant; indeed, they are often actively engaged in protein synthesis and
secretion, and they may be highly motile. Many G0 cells have a nonmotile primary cilium, which is an
important sensory organelle. The G0 phase is not necessarily permanent. In some specialized cases, G0 cells
may be recruited to reenter the cell cycle in response to specific stimuli. Cells that are no longer capable of
division, whether temporarily or permanently, remain in G0 phase, where they remain metabolically active
but no longer proliferate unless called on to do so by appropriate extracellular signals. A cell must receive a
growth-promoting signal to proceed from the quiescent stage or G0 into G1 phase and thus reenter the cell
cycle. This process must be highly regulated, as the uncontrolled proliferation of cells in a multicellular
organism can lead to cancer.

Cell cycle checkpoints

A checkpoint is a stage in the eukaryotic cell cycle at which the cell examines internal and external cues and
"decides" whether or not to move forward with division. These checkpoints occur near the end of G1 (The
G1/Restriction point), G2 Checkpoint at the G2/M transition, and M (Metaphase) Checkpoint/
Spindle Assembly checkpoint during metaphase to anaphase transition.

The G1 Checkpoint: The G1 checkpoint determines whether all conditions are favorable for cell division to
proceed.The G1 checkpoint, also called the restriction point (in yeast), is a point at which the cell
irreversibly commits to the cell division process. External influences, such as growth factors, play a large
role in carrying the cell past the G1 checkpoint. The cell will only pass the checkpoint if it is an appropriate
size and has adequate energy reserves. At this point, the cell also checks for DNA damage. A cell that
does not meet all the requirements will not progress to the S phase. The cell can halt the cycle and attempt
to remedy the problematic condition, or the cell can advance into G0 (resting) phase. Some cells stay
permanently in G0 phase, while others resume dividing if conditions improve.

The G2 Checkpoint: The G2 checkpoint bars entry into the mitotic phase if certain conditions are not met.
As with the G1 checkpoint, cell size and protein reserves are assessed. However, the most important role of
the G2 checkpoint is to ensure that all of the chromosomes have been accurately replicated without
mistakes or damage. If the checkpoint mechanisms detect problems with the DNA, the cell cycle is halted
and the cell attempts to either complete DNA replication or repair the damaged DNA. If the damage is
irreparable, the cell may undergo apoptosis, or programmed cell death. This self-destruction mechanism
ensures that damaged DNA is not passed on to daughter cells and is important in preventing cancer.

The M/ Spindle Checkpoint: The M checkpoint occurs near the end of the metaphase stage of mitosis.
The M checkpoint is also known as the spindle checkpoint because it determines whether all the sister
chromatids are correctly attached to the spindle microtubules. Because the separation of the sister
chromatids during anaphase is an irreversible step, the cycle will not proceed until the kinetochores of each
pair of sister chromatids are firmly anchored to at least two spindle fibers arising from opposite poles of the
cell.
Regulators Of The Cell Cycle

The Nobel Prize in Physiology or Medicine for 2001 jointly to Leland H. Hartwell, R. Timothy (Tim)
Hunt and Paul M. Nurse for their discoveries of "key regulators of the cell cycle". Among the above
three Nobel Laureates, Hartwell was responsible for the discovery of cell-division-cycle genes
(designated CDC genes) in budding yeast (Saccharomyces cerevisiae), Nurse was responsible for
the discovery of cell-division-cycle genes (designated cdc genes) in fission yeast
(Schizosacharomyces pombe) and Hunt was responsible for the discovery of cyclins in sea urchin,
frog and mammals

Cdk and Cyclin

Cyclin-dependent kinases (Cdks) belong to a group of protein kinases and are the central components that
coordinate activities throughout the cell cycle whose activities in turn are regulated by cyclin (a Cdk
regulatory protein) binding. The cyclin-Cdk complex causes the cyclical changes in the phosphorylation of
intracellular proteins initiate regulate the major events in the cell cycle such as DNA replication, mitosis or
cytokinesis etc. CDKs phosphorylate proteins on Serine and Threonine amino acid residues: they are
Serine/Threonine kinases. Cyclins were originally named because they undergo a cycle of synthesis and
degradation in each cell cycle. The levels of the cdk proteins, by contrast, are constant.

Cyclins can be divided into four classes.

1. G1/S cyclins: They bind to Cdk at the end of G1 and commit the cell to DNA replication. Their levels
fall in the S phase.
2. S cyclins: They bind to Cdk during S-phase and require for initiation of DNA replication and their
level remain high until mitosis.
3. M cyclins: Activate Cdks that stimulate entry into mitosis at the G2/M checkpoint.
4. G1 cyclins: governs the activities of G1/S cyclins i.e. promote cell cycle entry

As the names suggest, each cyclin is associated with a particular phase, transition, or set of phases in the cell
cycle and helps drive the events of that phase or period. For instance, M cyclin promotes the events of M
phase, such as nuclear envelope breakdown and chromosome condensation. The levels of the different
cyclins vary considerably across the cell cycle, as shown in the diagram at right. A typical cyclin is present
at low levels for most of the cycle, but increases strongly at the stage where it's needed. M cyclin, for
example, peaks dramatically at the transition from G2 to M phase. G1 cyclins are unusual in that they are
needed for much of the cell cycle.

Table: Major Cyclins and Cdks of Verterbrates and Budding yeast

Cyclin-Cdk Vertebrates Budding yeast
complex Cyclin Cdk partner Cyclin Cdk partner
G1-Cdk Cyclin D Cdk 4, Cdk 6 Cln 3 Cdk 1
G1/S-Cdk Cyclin E Cdk 2 Cln 1,2 Cdk 1
S-Cdk Cyclin A Cdk 2 Clb 5,6 Cdk 1
M-Cdk Cyclin B Cdk 1 Ckb 1,2,3,4 Cdk 1

In yeast cells, a single Cdk protein binds all classes of cyclins and triggers different cell-cycle events
by changing cyclin partners at different stages of the cycle. In vertebrate cells, by contrast, there are
four Cdks. Two interact with G1cyclins, one with G1/S- and S-cyclins, and one with M-cyclins.
 Fig.Cell cycle control by cyclin-CDK

CYCLIN-CDK COMPLEXES TRIGGER DIFFERENT CELL-CYCLE EVENTS

Each cyclin-Cdk complex phosphorylates a different set of substrate proteins. The same cyclin-Cdk
complex can also induce different effects at different times in the cycle, probably because the
accessibility of some Cdk substrates changes during the cell cycle. Certain proteins that function in
mitosis, for example, may become available for phosphorylation only in G2.

Studies of the three-dimensional structures of Cdk and cyclin proteins have revealed that, in the absence
of cyclin, the active site in the Cdk protein is partly obscured by a slab of protein, like a stone
blocking the entrance to a cave (Figure A). Cyclin binding causes the slab to move away from the
active site, resulting in partial activation of the Cdk enzyme (FigureB). Full activation of the cyclin-
Cdk complex then occurs when a separate kinase, the Cdk-activating kinase (CAK), phosphorylates
an amino acid near the entrance of the Cdk active site. This causes a small conformational change that
further increases the activity of the Cdk, allowing the kinase to phosphorylate its target proteins
effectively and thereby induce specific cell-cycle events (Figure C).

Fig. The structural basis of Cdk activation. The enzyme

is shown in three states. (A) In the inactive state,
without cyclin bound, the active site is blocked by a
region of the protein called the T-loop (red). (B) The
binding of cyclin causes the T-loop to move out of
the active site, resulting in partial activation of the
Cdk2. (C) Phosphorylation of Cdk2 (by Cdk-
activating kinase (CAK) at a threonine residue in the
T-loop further activates the enzyme by changing the
shape of the T-loop, improving the ability of the
enzyme to bind its protein substrates.
INHIBITORY PHOSPHORYLATION AND CDK INHIBITORY PROTEINS (CKLS) CAN
SUPPRESS CDK ACTIVITY

The rise and fall of cyclin levels is the primary determinant of Cdk activity during the cell cycle. Several
additional mechanisms, however, fine-tune Cdk activity at specific stages of the cycle.

Phosphorylation at a pair of amino acids in the roof of the kinase active site inhibits the activity of a cyclin-
Cdk complex. Phosphorylation of these sites by a protein kinase known as Weel inhibits Cdk activity,
while dephosphorylation of these sites by a phosphatase known as Cdc25 increases Cdk activity. This
regulatory mechanism is particularly important in the control of M-Cdk activity at the onset of mitosis.

During G1 and S phase, the CDK1 subunit of MPF is inactive due to an inhibitory enzyme, Wee1. Wee1
phosphorylates the Thr-14 residues in yeast and Tyr-15 residues in humans of CDK1, rendering MPF
inactive. During the transition of G2 to M phase, cdk1 is de-phosphorylated by CDC25. The CDK1
subunit is now free and can bind to cyclin B, activate MPF, and make the cell enter mitosis.

Binding of Cdk inhibitor proteins (CKIs) also regulates cyclin-Cdk complexes. The three-dimensional
structure of a cyclin-Cdk-CKl complex reveals that CKI binding stimulates a large rearrangement in the
structure of the Cdk active site, rendering it inactive. Cells use CKIs primarily to help govern the activities
of G1/S- and S-Cdks early in the cell cycle.
The activity of Cdk's during cell cycle progression is regulated by four molecular mechanisms.

 As already discussed for Cdkl, the first level of regulation involves the association of Cdk's with
their cyclin partners. Thus the formation of specific Cdk/cyclin complexes is controlled by cyclin
synthesis and degradation.

 Second, activation of Cdk/cyclin complexes requires phosphorylation of a conserved Cdk threonine

residue around position 160. This activating phosphorylation of the Cdk's is catalyzed by an enzyme
called CAK (for Cdk-Activating Kinase).

 In contrast to the activating phosphorylation by CAK, the third mechanism of Cdk regulation
involves inhibitory phosphorylation of tyrosine residues near the Cdk amino terminus, catalyzed by
the Weel protein kinase. In particular, both Cdkl and Cdk2 are inhibited by phosphorylation of
tyrosine-15, and the adjacent threonine-14 in vertebrates. These Cdk's are then activated by
dephosphorylation of these residues by members of the Cdc25 family of protein phosphatases.

 In addition to regulation of the Cdk's by phosphorylation, their activities are also controlled by the
binding of inhibitory proteins (called Cdk Inhibitors or CKIs)

The Cell-Cycle Control System Depends on Cyclical Proteolysis

Whereas activation of specific cyclin-Cdk complexes drives progression through the start and G2/M
checkpoints; progression through metaphase to anaphase transition is triggered not by protein
phosophorylation but by protein destruction, leading to the final stages of cell division.

The third major checkpoint is the metaphase-to-anaphase transition, which leads to sister- chromatid
segregation, completion of mitosis and cytokinesis. Progression through this checkpoint occurs when M-
phase cyclin-Cdk complexes stimulate an enzyme called the anaphase promoting complex, or cyclosome
(APC/C), which causes the proteolytic destruction of cyclins and of proteins that hold the sister chromatids
together.

The key regulator of the metaphase-to-anaphase transition is the anaphase promoting complex, or
cyclosome (APC/C), a member of the ubiquitin ligase family of enzymes. Many of these enzyrnes are used
in numerous cell processes to stimulate the proteolytic destruction of specific regulatory proteins. They
transfer multiple copies of the small protein ubiquitin in a process known as ubiquitination, to specific
target proteins (such as cyclins, Cdk inhibitor proteins and other cell-cycle regulators) , resulting in
the ubiquitinated proteins to recognized and destroyed by giant protease complexes called
proteasomes.

Ubiquitin is a small (8.5 kDa) regulatory protein found in most tissues of eukaryotic Ubiquitination affects
proteins in many ways: it can mark them for degradation via the proteasome, alter their cellular location,
affect their activity, and promote or prevent protein interactions.organisms, i.e. it occurs ubiquitously.

Proteasomes are protein complexes which degrade unneeded or damaged proteins by proteolysis, a
chemical reaction that breaks peptide bonds. Enzymes that help such reactions are called protease

The APC/C catalyzes the ubiquitylation and destruction of two major proteins. The first is securin, which
normally protects the protein linkages that hold sister chromatid pairs together in early mitosis. Destruction
of securin at the metaphase-to-anaphase transition activates a protease separase that cleaves the cohesins
complexes that hold the sisters chromatid together. The S- and M-cyclins are the second major targets of
the APC. Destroying these cyclins inactivates most Cdks in the cell. As a result, the many proteins
phosphorylated by Cdks from S phase to early mitosis are dephosphorylated by various phosphatases that
are present in the anaphase cell.

This dephosphorylation of Cdk targets is required for Dephosphorylation of these substrates is required for
spindle disassembly and the completion of mitosis, and for cytokinesis.

Following its activation in mid-mitosis, the APC/C remains active in G1, thereby providing a stable period
of Cdk inactivity. When G1/S-Cdks are activated in late G1, the APC is turned off, thereby allowing cyclin
accumulation to start the next cell cycle.

The cell-cycle control system also uses another ubiquitin ligase called SCF (after the names of its three
subunits SCF, whose name is derived from three of its central components—Skpl, cullin and the F-box.). It
ubiquitylates certain CKI proteins in late G1and thereby helps control the activation of S-cdks and DNA
replication.

The APC/C and SCF are both large, multisubunit complexes with some related components, but they are
regulated differently. APC/C activity changes during the cell cycle, primarily as a result of changes in its
association with an activating subunit-either Cdc20 during anaphase or Cdhl from late mitosis through early
G1.These subunits help the APC/C recognize its target proteins.

SCF activity also depends on subunits called F-box proteins, which help the complex recognize its target
proteins. Unlike APC activity, however, SCF activity is constant during the cell cycle. Ubiquitylation by
SCF is controlled instead by changes in the phosphorylation state of its target proteins, as F-box subunits
recognize only specifically phosphorylated proteins.

Maturation-promoting factor (MPF)

A famous example of how cyclins and Cdks work together to control cell cycle transitions is that
of maturation-promoting factor (MPF). In 1971, two independent teams of researchers (Yoshio
Masui and Clement Markert, as well as Dennis Smith and Robert Ecker) found that frog oocytes arrested in
G2 could be induced to enter M phase by microinjection of cytoplasm from oocytes that had been
hormonally stimulated with progesterone.[Because the entry of oocytes into meiosis is frequently referred to
as oocyte maturation, this cytoplasmic factor was called maturation promoting factor (MPF). This
mystery molecule, called MPF, was discovered in the 1980s to be a Cdk bound to its M cyclin partner.
Overview of functions
 Triggers the formation of mitotic spindle through microtubule instability.
 Promotes mitosis i.e. chromatin condensation through phosphorylation of condensins.
 The three lamins present in the nuclear lamina, lamin A, B & C, are phosphorylated by MPF at
serine amino residues. This leads to depolymerisation of the nuclear lamina & breakdown of nuclear
envelope into small vesicles.
 Causes phosphorylation of GM130, which leads to the fragmentation of the Golgi and the ER
 The following are affected by MPF.
 condensins, which enable chromatin condensation (see prophase)
 various microtubule-associated proteins involved in mitotic spindle formation
 lamins, interaction contributing to degradation of the nuclear envelope
 Histones, H1 and H3
 Golgi matrix, to cause fragmentation
MPF is composed of two subunits:
Cyclin-dependent kinase 1 (CDK1), the cyclin-dependent kinase subunit. It uses ATP to phosphorylate
specific serine and threonine residues of target proteins.
Cyclin, a regulatory subunit. The cyclins are necessary for the kinase subunit to function with the
appropriate substrate. The mitotic cyclins can be grouped as cyclins A & B.
These cyclins have a nine residue sequence in the N-terminal region called the “destruction box”, which can
be recognized by the ubiquitin ligase enzyme which destroys the cyclins when appropriate.
Activation of MPF
 MPF must be activated in order for the cell to transition from G2 to M phase. There are three amino
acid residues responsible for this G2 to M phase transition. The Threonine-161 (Thr-161) on CDK1
must be phosphorylated by a Cyclin Activating Kinase (CAK). CAK only phosphorylates Thr-161
when cyclin B is attached to CDK1.
 In addition, two other residues on the CDK1 subunit must be activated by dephosphorylation.
CDC25 removes a phosphate from residues Threonine-14 (Thr-14) and Tyrosine-15 (Tyr-15) and
adds a hydroxyl group. Cyclin B/CDK1 activates CDC25 resulting in a positive feedback loop.