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SESSION : 2024 - 25
 INTRODUCTION
The cell cycle is the series of events in which cellular components are doubled, and then
accurately segregated into daughter cells. In eukaryotes, DNA replication is confined to a
discrete Synthesis or S-phase, and chromosome segregation occurs at Mitosis or M-phase.
Two Gap phases separate S phase and mitosis, known as G1 and G2. These are not periods
of inactivity, but rather periods where cells obtain mass, integrate growth signals,
organize a replicated genome, and prepare for chromosome segregation.

The central machines that drive cell cycle progression are the cyclin-dependent kinases
(CDKs). These are serine/threonine protein kinases that phosphorylate key substrates to
promote DNA synthesis and mitotic progression. The catalytic subunits are in molar
excess, but lack activity until bound by their cognate cyclin subunits, which are tightly
regulated at both the levels of synthesis and ubiquitin-dependent proteolysis. Cyclin-
binding allows inactive CDKs to adopt an active configuration akin to monomeric and
active kinases. Layered on top of this regulation, CDK activity can also be negatively
regulated by the binding of small inhibitory proteins, the CKIs, or by inhibitory tyrosine
phosphorylation which blocks phosphate transfer to substrates.

Checkpoints emerged as a series of cell cycle dependencies. In seminal studies in the

fission yeast Schizosaccharomyces pombe, Mitchison and colleagues determined that cell
size was a determinant of cell division. Further, Rao and Johnson used human cell fusion
experiments , and determined a dependency between S phase and mitosis. That is, nuclei
undergoing S phase could delay mitotic entry of a G2 nucleus, whereas mitotic cells
stimulated nuclei to prematurely enter mitosis. In addition, studies in oocytes had
determined a similar relationship between S phase and mitosis .
 INDEX

INTRO Introduction
2
Acknowledgement
4

The Checkpoints: the basics

The Different Types Of Checkpoints
The G1 checkpoint
The G2 checkpoint
The spindle checkpoint
How do the checkpoints actually
work?
Project
cell cycle regulators
Analysis Cyclins
Cyclin-dependent kinasesCyclin-
dependent kinases
Maturation-promoting factor (MPF)
The anaphase-promoting
complex/cyclosome (APC/C)
Checkpoints and regulators
Cell Size Control
DNA Damage Responses

FINAL WORDS
Conclusion
Bibliography
 ACKNOWLEDGEMENTS
I would like to extend my gratitude to the board for selecting this topic and then my school which
indirectly enabled me to attempt this particular topic. Then I would like to thank and
acknowledge my subject teachers for assigning this topic to me.

I would also like to thank my friends who helped me get through some portions of the project
thus enabling the completion of it. I would also like to gratify my parents who were there with
me through my highs and my lows during the preparing time.

THE CHECKPOINTS: THE BASICS

There has been enormous progress in the molecular dissection of various
cell cycle checkpoint pathways. In many cases, this is very detailed with
close dissection of posttranslational modifications, structural biology,
enzyme kinetics, and so on. It would take a textbook to adequately detail all
these events, which we do not attempt to do here. Rather, we will focus on
the key concepts and regulatory events, and refer the reader to excellent
articles that describe the molecular details of these pathways
Cell cycle checkpoints are control mechanisms in the eukaryotic cell
cycle which ensure its proper progression. Each checkpoint serves as a
potential termination point along the cell cycle, during which the conditions
of the cell are assessed, with progression through the various phases of the
cell cycle occurring only when favorable conditions are met. There are many
checkpoints in the cell cycle, but the three major ones are: the G1
checkpoint, also known as the Start or restriction checkpoint or Major
Checkpoint; the G2/M checkpoint; and the metaphase-to-anaphase
transition, also known as the spindle checkpoint. Progression through these
checkpoints is largely determined by the activation of cyclin-dependent
kinases by regulatory protein subunits called cyclins, different forms of
which are produced at each stage of the cell cycle to control the specific
events that occur therein.

THE DIFFERENT TYPES OF CHECKPOINTS

 There are a number of checkpoints, but the three most important
ones are:

 The G1 checkpoint, at the G1/S transition.

 The G2checkpoint, at the G2/M transition.
 The spindle checkpoint, at the transition from metaphase to anaphase.

THE G 1 CHECKPOINT
 The G1 checkpoint is the main decision point for a cell – that is, the primary
point at which it must choose whether or not to divide. Once the cell passes the
G1 checkpoint and enters S phase, it becomes irreversibly committed to division.
That is, barring unexpected problems, such as DNA damage or replication errors,
a cell that passes the G1 checkpoint will continue the rest of the way through the
cell cycle and produce two daughter cells.

At the G1 checkpoint, a cell checks whether internal and external conditions are
right for division. Here are some of the factors a cell might assess:

 Size. Is the cell large enough to divide?

 Nutrients. Does the cell have enough energy reserves or available nutrients
to divide?
 Molecular signals. Is the cell receiving positive cues (such as growth
factors) from neighbors?
 DNA integrity. Is any of the DNA damaged?
These are not the only factors that can affect progression through the
G1 checkpoint, and which factors are most important depend on the type of cell.
For instance, some cells also need mechanical cues (such as being attached to a
supportive network called the extracellular matrix) in order to divide.

If a cell doesn’t get the go-ahead cues it needs at the G1 checkpoint, it may
leave the cell cycle and enter a resting state called G0 phase. Some cells stay
permanently in G0, while others resume dividing if conditions improve.

THE G 2 CHECKPOINT
 To make sure that cell division goes smoothly (produces healthy daughter cells
with complete, undamaged DNA), the cell has an additional checkpoint before M
phase, called the G2 checkpoint. At this stage, the cell will check:

 DNA integrity. Is any of the DNA damaged?

 DNA replication. Was the DNA completely copied during S phase?
If errors or damage are detected, the cell will pause at the G2 checkpoint to allow
for repairs. 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 SPINDLE CHECKPOINT

The M checkpoint is also known as the spindle checkpoint: here, the cell
examines 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 all the chromosomes are
firmly attached to at least two spindle fibers from opposite poles of the cell.

How does this checkpoint work? It seems that cells don't actually scan the
metaphase plate to confirm that all of the chromosomes are there. Instead, they
look for "straggler" chromosomes that are in the wrong place (e.g., floating
around in the cytoplasm). If a chromosome is misplaced, the cell will pause
mitosis, allowing time for the spindle to capture the stray chromosome.

HOW DO THE CHECKPOINTS ACTUALLY

WORK?
This article gives a high-level overview of cell cycle control, outlining the factors
that influence a cell’s decision to pause or progress at each checkpoint.
However, you may be wondering what these factors actually do to the cell, or
change inside of it, to cause (or block) progression from one phase of the cell
cycle to the next.

The general answer is that internal and external cues trigger signaling pathways
inside the cell that activate, or inactivate, a set of core proteins that move the cell
cycle forward.

.
 CELL CYCLE REGULATORS
WHY cell cycle transitions the factors that a cell considers when deciding
whether or not to move forward through the cell cycle? These include both
external cues (like molecular signals) and internal cues (like DNA damage).

Cues like these act by changing the activity of core cell cycle regulators inside
the cell. These core cell cycle regulators can cause key events, such as DNA
replication or chromosome separation, to take place. They also make sure that
cell cycle events take place in the right order and that one phase (such as G1)
triggers the onset of the next phase (such as S).

In this article, we'll look at a few of the most important core cell cycle regulators:
proteins called cyclins, enzymes called Cdks, and an enzyme complex called the
APC/C.
 CYCLINS
Cyclins are among the most important core cell cycle regulators. Cyclins are a
group of related proteins, and there are four basic types found in humans and
most other eukaryotes: G1 cyclins, G1/S cyclins, S cyclins, and M cyclins.

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\[^{1,2}\].

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.
 CYCLIN-DEPENDENT KINASES
In order to drive the cell cycle forward, a cyclin must activate or inactivate many
target proteins inside of the cell. Cyclins drive the events of the cell cycle by
partnering with a family of enzymes called the cyclin-dependent
kinases (Cdks). A lone Cdk is inactive, but the binding of a cyclin activates it,
making it a functional enzyme and allowing it to modify target proteins.

How does this work? Cdks are kinases, enzymes that phosphorylate (attach
phosphate groups to) specific target proteins. The attached phosphate group
acts like a switch, making the target protein more or less active. When a cyclin
attaches to a Cdk, it has two important effects: it activates the Cdk as a kinase,
but it also directs the Cdk to a specific set of target proteins, ones appropriate to
the cell cycle period controlled by the cyclin. For instance, G1/S cyclins send
Cdks to S phase targets (e.g., promoting DNA replication), while M cyclins send
Cdks to M phase targets (e.g., making the nuclear membrane break down).

In general, Cdk levels remain relatively constant across the cell cycle, but Cdk
activity and target proteins change as levels of the various cyclins rise and fall. In
addition to needing a cyclin partner, Cdks must also be phosphorylated on a
particular site in order to be active (not shown in the diagrams in this article), and
may also be negatively regulated by phosphorylation of other sites.

Cyclins and Cdks are very evolutionarily conserved, meaning that they are found
in many different types of species, from yeast to frogs to humans. The details of
the system vary a little: for instance, yeast has just one Cdk, while humans and
other mammals have multiple Cdks that are used at different stages of the cell
cycle. (Yes, this kind of an exception to the "Cdks don't change in levels" rule!)
But the basic principles are quite similar, so that Cdks and the different types of
cyclins can be found in each species.
 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). The name
dates back to the 1970s, when researchers found that cells in M phase contained
an unknown factor that could force frog egg cells (stuck in G2 phase) to enter M
phase. This mystery molecule, called MPF, was discovered in the 1980s to be a
Cdk bound to its M cyclin partner.

MPF provides a good example of how cyclins and Cdks can work together to
drive a cell cycle transition. Like a typical cyclin, M cyclin stays at low levels for
much of the cell cycle, but builds up as the cell approaches the G2/M transition.
As M cyclin accumulates, it binds to Cdks already present in the cell, forming
complexes that are poised to trigger M phase. Once these complexes receive an
additional signal (essentially, an all-clear confirming that the cell’s DNA is intact),
they become active and set the events of M phase in motion.

The MPF complexes add phosphate tags to several different proteins in the
nuclear envelope, resulting in its breakdown (a key event of early M phase), and
also activate targets that promote chromosome condensation and other M phase
events. The role of MPF in nuclear envelope breakdown is shown in simplified
form in the diagram below.
 THE ANAPHASE-PROMOTING
COMPLEX/CYCLOSOME (APC/C)
In addition to driving the events of M phase, MPF also triggers its own
destruction by activating the anaphase-promoting
complex/cyclosome (APC/C), a protein complex that causes M cyclins to
be destroyed starting in anaphase. The destruction of M cyclins pushes the cell
out of mitosis, allowing the new daughter cells to enter G1. The APC/C also
causes destruction of the proteins that hold the sister chromatids together,
allowing them to separate in anaphase and move to opposite poles of the cell.

How does the APC/C do its job? Like a Cdk, the APC/C is an enzyme, but it has
different type of function than a Cdk. Rather than attaching a phosphate group to
its targets, it adds a small protein tag called ubiquitin (Ub). When a target is
tagged with ubiquitin, it is sent to the proteasome, which can be thought of as
the recycle bin of the cell, and destroyed. For example, the APC/C attaches a
ubiquitin tag to M cyclins, causing them to be chopped up by the proteasome and
allowing the newly forming daughter cells to enter G.phase.

The APC/C also uses ubiquitin tagging to trigger the separation of sister
chromatids during mitosis. If the APC/C gets the right signals at metaphase, it
sets off a chain of events that destroys cohesin, the protein glue that holds
sister chromatids together.

 The APC/C first adds a ubiquitin tag to a protein called securin, sending it for
recycling. Securin normally binds to, and inactivates, a protein called separase.
.


Checkpoints and regulators

 CELL SIZE CONTROL

In order to maintain cell size and ensure that each daughter cell is endowed with the
appropriate amount of genetic and biosynthetic material, cells must, on average, exactly
double their contents before division. Control of cell size is critical for regulating nutrient
distribution for the cell and for regulating organ size and function in multicellular
organisms. The existence of cell size checkpoints has been proposed for allowing cells to
coordinate cell size with cell cycle progression. Cell size checkpoints have been observed
in G1 and G2. Early evidence for these checkpoints came from observations that the size of
new daughter cells after mitosis affects cell cycle progression: large daughter cells speed
up progression through G1 and/or G2, and small daughter cells delay exit from these
growth phases .However, different species and cell types vary widely in the location of
these checkpoints within the cell cycle, and thus in how the cell cycle is affected in
response to change in cell size.

Not surprisingly, much of what is known about size checkpoints at the molecular level is
based on regulation of the proteins involved in G1 and G2/M progression. Control of the
G1 cell size checkpoint has been studied most extensively in budding yeast, where the
cyclin Cln3, which activates Start, regulates cell size. Control of the G2/M cell size
checkpoint has been studied most extensively in fission yeast, where Cdc25 and Wee1
respond to cell size and nutritional status in their control of the Cdc2-cyclin B complex .

One proposed mechanism for control of cell size is via the monitoring of protein
translation. Ribosomal mass, and thus translational activity, should correlate with the size
of the cell, so it is thought that there is some product of translation called a “translational
sizer” that increases in abundance with cell size and that exerts control over the cell cycle
after a certain amount has accumulated . Cln3 and Cdc25 are both proposed translational
sizers. This hypothesis also offers an explanation for how cell size and the cell cycle
respond to nutritional status.

DNA DAMAGE RESPONSES

Throughout interphase, DNA damage elicits a cell cycle arrest that allows time for repair
pathways to operate prior to commitment to subsequent phases of the cell cycle. The
source of DNA damage may be intrinsic, such as intermediates of metabolism, attrition of
telomeres, oncogene overexpression, and DNA replication errors. Alternatively, there are
many extrinsic sources of DNA damage ranging from sunlight, to carcinogens, ionizing
radiation or other anticancer therapeutics. While there are many lesion-specific responses
for DNA repair, different lesions in genomic DNA activate common checkpoint pathways
whose goal is to maintain CDKs in an inactive state until the lesion is removed. Broadly
speaking, DNA damage checkpoints can be separated into those controlled by the tumor
suppressor and transcription factor p53, and those ultimately under the control of the
checkpoint kinase Chk1, and we will consider the latter first.

The Chk1 pathway is highly conserved from yeast to man. The components of the pathway
have come largely from genetic screens in the yeasts among damage-sensitive mutants,
with some additional components identified in mammalian cells . Chk1 is activated by all
known forms of DNA damage, though this is more efficient in S- and G2-phase than in G1,
and restricted to post-replicative lesions. The diversity of activating lesions suggested a
common intermediate, which is single- stranded DNA coated by Replication Protein A
(RPA), and containing a primer template junction.

In higher organisms, the transcription factor p53 is a critical component of DNA damage
checkpoints , particularly in G1 phase.
By ensuring that cells don't divide when their DNA is damaged, p53 prevents mutations
(changes in DNA) from being passed on to daughter cells. When p53 is defective or
missing, mutations can accumulate quickly, potentially leading to cancer. Indeed, out of
all the entire human genome, p53 is the single gene most often mutated in cancers.p53
and cell cycle regulation are key topics of study for researchers working on new
treatments for cancer.
 CONCLUSIONS
We have described here the basic principles behind the common cell cycle checkpoints.
They share the feature of detecting a defect in the division program, and then sending
signals forward to alter the oscillations of CDK activity and therefore cell cycle events.
Some aspects of checkpoint signaling remain to be clarified or determined (known
unknowns), either as a simple principle, or in the context of human development and
disease.

With this I come to the end of the project.

A project is a way to step aside of traditional learning and take a peek into a more
lively and interactive aspect of it. It helps us to not only know about a topic but to
actually engage in active learning, making our monotonous academic life a bit
more amusing and enjoyable.
The main objective of the project was to make me aware of the diverse machinery
on check points of cell cycle and in my opinion that particular goal has been
achieved. I am quite pleased with the outcome. The intensive research has allowed
me to not only sharpen my academic skills but it also has helped me hon. my other
skills like preparing a formal research paper (sort of) and other aspects which I
believe will be beneficial for me in the future.
~ Utsa Sarkar
 BIBLIOGRAPHY
Books used:
Ncert Biology Textbook (Std Xi)

WEBSITES USED:
 https://en.wikipedia.org/wiki/Cell_cycle_checkpoint
 https://www.khanacademy.org/science/biology/cellular-molecular-biology/stem-cells-and-
cancer/a/cell-cycle-regulators