What is the Cell Cycle?

The cell cycle is required for cell growth and cell division into two daughter cells. A eukaryotic cell cannot
divide unless it replicates its genome (DNA) and then separates the duplicated genome. To achieve these tasks
cells must perform DNA synthesis and mitosis. The cell cycle is an ordered set of events. The G1 phase stands
for “GAP-1” and is required for cell growth and preparation of DNA synthesis. The S-phase stands for
“Synthesis” and replicates the genome. The G2 phase is “GAP-2” and needed for cell growth and preparation
for mitosis. The last phase is M and it stands for “Mitosis” in which cells segregate duplicated chromosomes.

What is the Checkpoint?

The cell cycle proceeds by a defined sequence of events where late events depend upon completion of early
events 1. The aim of the dependency of events is to distribute complete and accurate replicas of the genome to
daughter cells 2. To monitor this dependency, cells are equipped with the checkpoints that are set at various
stages of the cell cycle. When cells have DNA damages that have to be repaired, cells activate DNA damage
checkpoint that arrests cell cycle. According to the cell cycle stages, DNA damage checkpoints are classified
into at least 3 checkpoints: G1/S (G1) checkpoint, intra-S phase checkpoint, and G2/M checkpoint. Upon
perturbation of DNA replication by drugs that interfere with DNA synthesis, DNA lesions, or obstacles on
DNA, cells activate DNA replication checkpoint that arrests cell cycle at G2/M transition until DNA
replication is complete. There are more checkpoints such as Spindle checkpoint and Morphogenesis
checkpoint. The spindle checkpoint arrests cell cycle at M phase until all chromosomes are aligned on spindle.
This checkpoint is very important for equal distribution of chromosomes. Morphogenesis checkpoint detects
abnormality in cytoskeleton and arrests cell cycle at G2/M transition.

What happens if you lose one of checkpoints?

DNA replication and chromosome distribution are indispensable events in the cell cycle control. Cells must
accurately copy their chromosomes, and through the process of mitosis, segregate them to daughter cells. The
checkpoints are surveillance mechanism and quality control of the genome to maintain genomic integrity.
Checkpoint failure often causes mutations and genomic arrangements resulting in genetic instability. Genetic
instability is a major factor of birth defects and in the development of many diseases, most notably cancer.
Therefore, checkpoint studies are very important for understanding mechanisms of genome maintenance as
they have direct impact on the ontogeny of birth defects and the cancer biology.

DNA maintenance checkpoint

Accurate duplication of eukaryotic genome is a challenging task, given that environment of cell growth and
division is rarely ideal. Cells are constantly under the stress of intrinsic and extrinsic agents that cause DNA
damage or interference with DNA replication. To cope with these assaults, cells are equipped with DNA
maintenance checkpoints 3 to arrest cell cycle and facilitate DNA repair pathways. DNA maintenance
checkpoints include (a) the DNA damage checkpoints that recognize and respond to DNA damage, and (b)
the DNA replication checkpoint that monitors the fidelity of copying DNA 3.
(a)  DNA damage checkpoint
DNA damage checkpoints ensure the fidelity of genetic information both by arresting cell cycle
progression and facilitating DNA repair pathways. Studies on many different species have uncovered a
network of proteins that form the DNA damage checkpoints. Central to this network are protein
kinases of ATM/ATR family known as Tel1/Mec1 in budding yeast and Tel1/Rad3 in fission yeast 4.
These kinases sense DNA damages and phosphorylate number of proteins that regulate cell cycle
progression and DNA repair pathways 3.
 
(b)  DNA replication checkpoint
 Accurate replication of the millions or billions of DNA base pairs in a eukaryotic genome is a remarkable
achievement. This accomplishment is even more astonishing when one considers for DNA synthesis are
rarely ideal. Damaged template, protein complexes bound to DNA, and poor supply of dNTPs are among
the many obstacles that must be overcome to replicate genome. All of these situations can stall replication
forks. Stalled forks pose grave threats to genome integrity because they can rearrange, break, or collapse
through disassembly of the replication complex 5. The pathways that respond to replication stress are signal
transduction pathways that are conserved across evolution 6 7. Atop the pathways are also ATM/ATR
family kinases. These kinases together with a trimeric checkpoint clamp (termed 9-1-1 complex) and five-
subunit checkpoint clamp loader (Rad17-RFC2-RFC3-RFC4-RFC5) senses stalled replication forks and
transmit a checkpoint signal 3. One of major functions of replication checkpoint is to prevent the onset of
mitosis by regulating mitotic control proteins such as Cdc25. But perhaps the most important activity of
replication checkpoint is to stabilize and protect replication forks 8. The protein
kinase Cds1 (human Chk2 homolog; in human, Chk1 is a functional Cds1 homolog) is a critical effector
of the replication checkpoint in the fission yeast Schizosaccharomyces pombe 9, 10. Cds1 is required to
prevent stabilization of replication fork in cells treated with hydroxyurea (HU), a ribonucleotide reductase
inhibitor that stalls replication by depleting dNTPs 11. In the budding yeast Saccharomyces cerevisiae, a
failure to activate Rad53 (Chk2 homolog) is associated with collapse and regression of replication forks
and gross chromosomal rearrangements in cells treated with HU 12-15.

Replication fork protection complex (FPC)

The DNA replication checkpoint stabilizes replication forks that have stalled at DNA adducts and other lesions
that block DNA polymerases. In the absence of DNA replication checkpoint, stalled forks are thought to
collapse, creating strand break that threatens genome stability and cell viability 5. Therefore, discovering how
cells cope with aberrant replication forks is essential for understanding mechanisms of genome maintenance.
The Chk1 and Chk2/Cds1 checkpoint kinases, which are key mediators of DNA damage and DNA replication
checkpoints, are thought to be involved in cancer development 16. We found the Swi1 protein is required for
survival of replication fork arrest and effective activation of Chk2 kinase in fission yeast. Swi1 forms tight
complex with Swi3 protein and moves with replication forks. Swi1-Swi3 complex is also important for
proficient DNA replication even in the absence of agents that cause genotoxic stress, creating single-strand
DNA gaps at replication forks 11, 17. These results led us to propose Swi1-Swi3 define a replication fork
protection complex (FPC) that stabilizes replication forks in a configuration that is recognized by replication
checkpoint sensors11, 17. Interestingly, Tof1 protein (Budding yeast Swi1 homolog) has been reported to have
similar functions. Tof1 is also involved in Rad53 (Chk2 homolog) activation and travels with replication
fork 18, 19. Tof1 is needed to restrain fork progression when DNA synthesis is inhibited by HU indicating that
Tof1 is required for coordination of DNA synthesis and replisome (replication machinery) movement 19.

KEY POINTS
o A checkpoint is one of several points in the eukaryotic cell cycle at which the progression of a
cell to the next stage in the cycle can be halted until conditions are favorable.
o Damage to DNA and other external factors are evaluated at the G1 checkpoint; if conditions are
inadequate, the cell will not be allowed to continue to the S phase of interphase.
o The G2 checkpoint ensures all of the chromosomes have been replicated and that the replicated
DNA is not damaged before cell enters mitosis.
o The M checkpoint determines whether all the sister chromatids are correctly attached to the
spindle microtubules before the cell enters the irreversible anaphase stage.

TERMS
 cyclin
any of a group of proteins that regulates the cell cycle by forming a complex with kinases
 G2 checkpoint
ensures all of the chromosomes have been replicated and that the replicated DNA is not damaged
 restriction point
(G1 checkpoint) a point in the animal cell cycle at which the cell becomes "committed" to the cell cycle,
which is determined by external factors and signals
 spindle checkpoint
(M checkpoint) prevents separation of the duplicated chromosomes until each chromosome is properly
attached to the spindle apparatus
FULL TEXT
Regulation at Internal Checkpoints
It is essential that the daughter cells are exact duplicates of the parent cell. Mistakes in the duplication or
distribution of the chromosomes lead to mutations that may be passed forward to every new cell produced from
an abnormal cell. To prevent a compromised cell from continuing to divide, internal control mechanisms
operate at three main cell cycle checkpoints. A checkpoint is one of several points in the eukaryotic cell cycle at
which the progression of a cell to the next stage in the cycle can be halted until conditions are favorable (e.g. the
DNA is repaired). These checkpoints occur near the end of G1, at the G2/M transition, and during metaphase .

Internal Checkpoints During the Cell Cycle

The cell cycle is controlled at three checkpoints. The integrity of the DNA is assessed at the G1 checkpoint.
Proper chromosome duplication is assessed at the G2 checkpoint. Attachment of each kinetochore to a spindle
fiber is assessed at the M checkpoint.

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 energyreserves. 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 (inactive) phase and await further signals when conditions improve.
If a cell meets the requirements for the G1 checkpoint, the cell will enter S phase and begin DNA replication.
This transition, as with all of the major checkpoint transitions in the cell cycle, is signaled by cyclins and cyclin
dependent kinases (CDKs). Cyclins are cell-signaling molecules that regulate the cell cycle.
The G2 Checkpoint
The G2 checkpoint bars entry into the mitotic phase if certain conditions are not met. As with the G 1 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 DNA has been correctly replicated, cyclin dependent
kinases (CDKs) signal the beginning of mitotic cell division.
The M 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.