Cell cycle and checkpoints in Human cell, role of CDK and Cyclin, Regulation of Cell Cycle.

1. CELL CYCLE AND ITS REGULATION IN
HUMAN CELL
Submitted by: Sejal, Swati Gaur, Bhanu
Krishan, Diksha Kanwar, Tanvir Khehra
GGDSD COLLEGE, SECTOR 32C,
CHANDIGARH
1

2. Cell cycle and its phases
Cell cycle is the ordered
series of events that lead to
cell division and the
production of two daughter
cells, each containing
chromosomes identical to
that of the parent cell.
Two main processes that
take place during cell cycle
are:-
1. Chromosome
replication- S phase
2. chromosome
segregation- M phase
2

3. Stages of cell cycle
A. G0 PHASE
B. INTERPHASE
1. G1 Phase
2. S Phase
3. G2 Phase
C. MITOTIC PHASE
1. KARYOKINESIS
1. Prophase
2. Metaphase
3. Anaphase
4. Telophase
2. CYTOKINESIS
1. Cell plate formation
2. Cell furrow formation
3

4. G0 PHASE
• Also known as QUISCENT STAGE or INACTIVE STAGE
• Quiescent state represents a reversible resting stage – cells in
this stage remain metabolically active and do not proliferate
unless depending on the environment.
4

5. INTERPHASE
• Interphase is a period
when the cell carries out its
normal metabolic activities
and grows.
• The DNA- containing
material is in the form of a
chromatin. The nuclear
envelope and one or more
nucleoli are intact and
visible.
• It is a longer and a
metabolically active phase
• Synthesize the enzymes
and the proteins required
for DNA replication in S
phase.
5

6. G1 PHASE
• Period of growth
• It is the period between the end of M phase and the start of
DNA replication.
• Cell doubles its organelles
• Cell grows in size
• Longest phase. It lasts even for years.
• Synthesis of proteins, amino acids and enzymes required for
DNA replication in S phase.
• RNA synthesis (transcription)
6

7. S (Synthesis)PHASE
• DNA synthesis occurs, and all of the chromosomes are
replicated, leading to a doubling of the DNA content of a cell.
Amount of DNA doubles per cell but there is no increase in
chromosome number.
•
• Replication of DNA
7

8. G2 PHASE
• Gap period between the S phase and Mphase of cell cycle.
• During the G2 phase, the cell prepares for nuclear and cell
division, by synthesizing the proteins (like tubulin needed to
construct microtubules which is used to make spindle fibres)
that will be required to drive this process.
• Metabolic activities essential for cell division occur during this
phase.
8

9. M (MITOTIC) PHASE
•Division phase i.e.
the cell divides
•It is the process of
distribution of two
sets of chromosomes
into two separate and
equal nuclei.
•Equational cell
division.
• this phase lasts for
about an hour in the
24 hr duration of cell
cycle.
9

10. PROPHASE
• Longest phase
• Chromatin begin to condense and become visible in light
microscope as chromosomes
• Centrioles move towards opposite poles
• Spindle apparatus begin to appear
10

11. LATE PROPHASE
• Starts with the breakdown of nuclear membrane
• Chromosomes can now attach to spindle microtubules via
kintechores and undergo active movement.
• Nucleolus disappear
• Chromosomes are set free in cytoplasm
11

12. METAPHASE
• Spindle fibers are completely formed.
• Chromosomes become short and thick with two distinct
chromatids each.
• They move towards centre and arrange in equatorial plane to
form metaphasic plate

13. ANAPHASE
• the shortest phase of mitosis, anaphase begins abruptly as
the centromeres of the chromosomes split simultaneously.
Each chromatoid now becomes a chromosome in its own
right.
• Kinetochore microtubules get shorter and the spindle poles
also move apart. Both processes contribute to chromosome
segregation.
13

14. TELOPHASE
• Also called as reverse prophase because the events that occur during this
phase are direct reversal of prophase.
• Chromatids reach the opposite poles and decondense.
• Nucleolus and nuclear membrane reappear.
14

15. CYTOKINESIS
• Division of cytoplasm
• Division of cell into two identical halves called daughter cells
• In plant cells, cell plate form at equator to divide cell
• In animal cells, cleavage furrow forms to split cell.
• Starts in LATE ANAPHASE
15

16. Significance of mitosis
• Used for growth and repair.
• The only way to increase cell no. without change in genetic material.
• Helpful in the replacement of dead cells by new daughter cells.
• Takes place in somatic cells.
• Mitotic divisions in meristematic tissues result in continuous growth of
plants throughout their life.
• Development of an organism occurs by mitosis only.
• Cell growth results in disturbing the ratio b/w nucleus and
cytoplasm(nucleo- cytoplasmic). So it essential for the cell to restore this
ratio via mitosis.
16

17. Cyclins and CDK in human cell cycle
• The cell-cycle process is highly conserved and precisely controlled to govern the
genome duplication and cell cycle.
• The cell cycle is regulated by many cyclins and cyclin-dependent kinases (CDKs)
that are a group of serine/threonine kinases
• CDKs form complexes with cyclins to stabilize, activate, and phosphorylate CDKs
in the specific phases
• The formation of cyclin/CDKs controls the cell-cycle progression via
phosphorylation of the target genes, such as tumor suppressor protein
retinoblastoma (Rb).
• The activation of cyclins/CDKs is induced by mitogenic signals and inhibited by
the activation of cell-cycle checkpoints in response to DNA damage
17

18. CDKs in Cell cycle
• CDKs respond to the extracellular and intracellular signals to regulate cell
division.
• Act as the catalytic subunits by forming a heterodimer
complex with the cyclins, which function as
the regulatory subunits.
• In human cells, there are 20 CDKs and 29 cyclins, where
CDK1, CDK2, CDK3, CDK4, CDK6, and CDK7 directly regulate
cell-cycle transitions and cell division, whereas CDK7–11 mediate
gene transcription (Ding, et.al., 2020).
• The expression of CDKs fluctuates in a cyclical fashion throughout the cell
cycle.
Figure: CDK6 of Human cell
18

19. CDKs in Cell cycle
• CDKs range in size from approximately 250 amino acid residue
• Like all kinases, CDKs have a two-lobed structure, with amino- and/or
carboxy-terminal extensions of variable lengths.
• In the cyclin-free monomeric form the CDK catalytic cleft is closed by the
T-loop, preventing enzymatic activity (Malumbres, 2014)
19

20. FIGURE: CDK-cyclin complexes. A comparison of the CDK-cyclin
complexes, for which structures are available, highlights the
differences in the CDK response to cyclin association. (a) CDK6-viral
cyclin (PDB 1JOW, CDK6, cyan with activation loop (residues 163-
189) shown in red; viral cyclin, grey). (b) CDK4-cyclin D1 (PDB
2W96, CDK4, orange; cyclin D1, light purple, RXL-binding site
shown as a red translucent surface (residues 54-61) and partially
resolved LXCXE motif shown in cyan (residues 6-9)). (c) CDK4-
cyclin D3 (PDB 3G33, CDK4, orange; cyclin D3, purple, RXL-
binding site shown as a red translucent surface (residues 56-61).
(d) CDK5p25 (PDB 1H4 L, CDK5, light blue with activation loop
(residues 144-171) shown in red; p25, gold). (e) CDK8-cyclin C
(CDK8, green with C-terminal residues 343-353 in orange; cyclin C,
purple). ( f ) CDK9-cyclin T1 (PDB 3BLH, CDK9 lilac with C-
terminal residues 317-325 in orange; cyclin T, pale yellow). (g)
CDK12cyclin K (PDB 4UN0, CDK12, light grey, C-terminal rail
residues 1025-1036 in orange; cyclin K, green). (h) CDK13-cyclin K
(PDB 5EFQ, CDK13, gold, C-terminal tail residues 1011-1025 in
orange; cyclin K, green).
The activation segment sequences are shown in red where resolved in
the structures.
20

21. CDKs in Cell cycle
• CDKs that regulate the cell cycle (CDKs 1, 2, 4 and 6).
• A substantial sub-branch of the family (CDKs 7, 8, 9, 12 and 13) regulates
transcription through phosphorylation of the heptad repeats that
comprise the C-terminal tail of RNA polymerase II.
• CDK7 is unusual in that it also indirectly regulates the cell cycle by
activating CDKs 1, 2, 4 and 6.
• CDK3 phosphorylates retinoblastoma protein (pRB) to promote the
transition from quiescence (G0) into G1.
21

22. Cyclins in Cell cycle
• Cyclins are the key components of the cell cycle regulation machinery.
• Cyclins are a family of regulatory proteins that control the
progression of the cell cycle. E.C No: 2.7.11.22
• In combination with their respective cyclin-dependent
protein kinases (CDKs), cyclins form the
holoenzymes that phosphorylate different sets of proteins at
consecutive stages of the cell cycle.
• In human cells there are 20 cyclins which interact with different CDKs. Many
cyclins also regulate gene transcription and mRNA processing.
• In cell cycle, cyclins A, B, D, E and C are more prominent.
Figure: Structure of Human cell
Cyclin
22

23. Cyclin CDKs Complexes and their functions
CDKs Cyclins Cell Cycle phase Function
CDK 4 Cyclin D G1 phase Drives the cell cycle
transition from G1 to
S phase
CDK 2, CDK 6 Cyclin E Late G1 / S phase Cyclin binds to CDK
at the end of G1 to
initiate the cell to
DNA replication
CDK 2 Cyclin A Late S phase/ G2
phase
Cyclins bind the CDK
during S phase and
are necessary to
initiate the DNA
replication
CDK 1 Cyclin B M phase Cyclins promote the
events of mitosis
CDK 3 Cyclin C G0 phase Mediated
inactivation of pRb
that controls the
G1/S transition 23

24. Source: Biology Online. (2019, October 7). D-glucose Definition and Examples -
Biology Online Dictionary. Biology Articles, Tutorials & Dictionary Online.
24

25. Cyclin binding and activation of CDK
• In the G1 state CDK level is low as the cyclins are missing because Cyclin
mRNA synthesis is inhibited and cyclin protein is rapidly degraded.
• As the cell cycle initiates, cyclin synthesis is induced and cyclin
degradation is inhibited with increase in the levels of CDKs.
• CDKs consists of an active site or ATP binding
site which is a cleft between a small amino terminal
lobe and a large carbonyl terminal lobe.
• Thus, cyclin regulates the ATP binding site and determines
the specificity of CDK- Cyclin complex for particular substrate. Figure: Structure of CAK
25

26. Activation of CDKs
• Activation of CDKs requires 2 steps:
• Cyclin binding to the CDK
• In second step Cyclin Activating Kinase (CAK) must phosphorylate
Cyclin- CDK complex on the Threonin160 residue, which is located in the
CDK activating segment.
• Also, CAK activity is indirectly regulated by Cyclins.
• https://youtu.be/nEMMKzYQf9A
26

27. 27

28. Regulation of cell cycle
• Cell cycle transitions are regulated by cyclin-CDK protein kinases, protein
phosphatases and ubiquitin protein ligases.
• Cyclins regulate the cell cycle only when they are tightly bound to
CDKs. To be fully active, the CDK-cyclin complex must also be
phosphorylated in specific locations. Like all kinases, CDKs are
enzymes (kinases) that phosphorylate other proteins.
Phosphorylation activates the protein by changing its shape. The
proteins phosphorylated by CDKs are involved in advancing the
cell to the next phase. The levels of CDK proteins are relatively
stable throughout the cell cycle; however, the concentrations of
cyclin fluctuate and determine when CDK-cyclin complexes form.
The different cyclins and CDKs bind at specific points in the cell
cycle and thus regulate different checkpoints.
• CDKs are regulated not only by cyclin binding, but also by both activating
and inhibitory phosphorylation. Together, these regulatory events ensure
that CDKs are active only at the appropriate cell cycle stage.
28

29. 29

30. How CDKs regulate cell cycle progression?
Cells harbour different types of CDKs that initiate different events
of the cell cycle. Importantly, the CDKs are active only in the stages
of the cell cycle they trigger.
• G1/S phase CDKs are active at the G1–S phase transition to
trigger entry into the cell cycle.
• S phase CDKs are active during S phase and trigger S phase.
• Mitotic CDKs are active during mitosis and trigger mitosis.
• The anaphase-promoting complex or cyclosome (APC/C)
ubiquitin-protein ligase catalyses two key cell cycle transitions by
ubiquitinylating proteins, hence targeting them for degradation.
• APC/C initiates anaphase and exit from mitosis.
30

31. Regulator molecules of cell cycle
Regulatory molecules either promote progress of the cell
to the next phase (positive regulation) or halt the cycle
(negative regulation). Regulator molecules may act
individually, or they can influence the activity or
production of other regulatory proteins.
Two types of regulators are:
1. Positive regulators
2. Negative regulators
31

32. POSITIVE REGULATORS
1. Cyclin-dependent kinases
Cyclin-dependent kinases are a family of small (30–40 kD)
serine/threonine kinases. They are not active in the monomeric form, but
require an activating subunit (cyclins) to be active as protein kinases.
Key features responsible for CDK activity are:
• Cyclin dependent kinases (CDKs) are active only when bound to a
regulatory cyclin subunit.
• Different types of cyclin–CDK complexes initiate different events. G1
CDKs and G1/S phase CDKs promote entry into the cell cycle , S phase
CDKs trigger S phase , and mitotic CDKs initiate the events of mitosis.
• Multiple mechanisms are in place to ensure that the different CDKs are
active only in the stages of the cell cycle they trigger.
32

33. 2 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.
• A typical cyclin is present at low levels for most of the cycle, but
increases strongly at the stage where it's needed.
33

34. NEGATIVE REGULATORS
The second group of cell cycle regulatory molecules are
negative regulators. Negative regulators halt the cell cycle.
The best understood negative regulatory molecules are
retinoblastoma protein (Rb), p53, and p21. All three of these
regulatory proteins were discovered to be damaged or non-
functional in cells that had begun to replicate uncontrollably
(became cancerous).
1.Retinoblastoma protein(Rb).
• Retinoblastoma proteins are a group of tumor-suppressor
proteins common in many cells.
• The products of tumor-suppressor genes function in various ways to
inhibit progression through the cell cycle (loss-of-function mutations
in RB are associated with the disease hereditary retinoblastoma. )
34

35. Rb exerts its regulatory influence on other positive regulator proteins. Chiefly,
Rb monitors cell size. In the active, dephosphorylated state, Rb binds to
proteins called transcription factors, E2F. E2F activate specific genes,
allowing the production of proteins encoded by that gene. When Rb is bound
to transcription factors, production of proteins necessary for the G1/S
transition is blocked. As the cell increases in size, Rb is slowly phosphorylated
until it becomes inactivated. Rb releases the transcription factors, which can
now turn on the gene that produces the transition protein, and this particular
block is removed.
35

36. 2. p53 and p21
• p53 is a multi-functional protein that has a major impact on the
commitment of a cell to division because it acts when there is
damaged DNA in cells that are undergoing the preparatory
processes during G1.
• If damaged DNA is detected, p53 halts the cell cycle and recruits
enzymes to repair the DNA.
• If the DNA cannot be repaired, p53 can trigger apoptosis, or cell
suicide, to prevent the duplication of damaged chromosomes.
• As p53 levels rise, the production of p21 is triggered. p21
enforces the halt in the cycle dictated by p53 by binding to and
inhibiting the activity of the CDK/cyclin complexes in G1 phase.
• As a cell is exposed to more stress, higher levels of p53 and p21
accumulate, making it less likely that the cell will move into the S
phase.
36

37. FUNDAMENTAL PROCESSES IN
THE EUKARYOTIC CELL CYCLE
• There are different cyclin-CDK complexes that control passage
through the cell cycle: the G1, S-phase, and mitotic cyclin-CDK
complexes.
• The catalytic subunits of kinases, called Cyclin-dependent kinases
(CDKs) have no kinase activity unless they are associated with
cyclins.
• Each CDK can associate with different cyclins, and the associated
cyclin determines which proteins are phosphorylated by a
particular cyclin-CDK complex.
37

38. FIGURE: Fundamental Processes in
eukaryotic cell cycle
38

39. G1 cyclin-CDKs
• G1 cyclin-CDKs (Cyclin dependent kinase) inactivate Cdh1
Cdh1 is inhibited by Cdk activity, Cdk phosphorylate Cdh1. When APC is not
associated with Cdh1 then it is inactive. For cell re-entry, APC-cdh1 must be
turned off to allow for the re-accumulation of S-phase and mitotic cyclins
during cycle.
• G1 cyclin-CDKs activate expression of S phase cyclin CDKs components
• G1 cyclin-CDKs phosphorylate S-phase inhibitor
39

40. S phase cyclin-CDKs
• SCF/proteasome degrades phosphorylated S-phase cyclin-CDK inhibitor
SCF complexes are named for their constituent protein components, Skp,
Cullin, F-box containing complex containing is a multi protein ubiquitin ligase
complex that catalyzes the ubiquitination of protein.
SCF has important roles in the ubiquitination of proteins involved in the cell
cycle.
• S-phase cyclin-CDK activates pre-replication complexes.
40

41. Mitotic cyclin-CDKs
• Cdc25 phosphatase activates mitotic CDKs, which activate early mitotic
events.
Cdc25 (cell division cycle 25) phosphatase removes inhibitory phosphate
residues from target cyclin dependent kinase active site.
• APC-Cdc20/ proteasome degrades securin.
APC-Cdc20 initiates the metaphase-anaphase transition through mediating the
ubiquitination and degradation of securin.
41

42. .
• Securin is protein involved in the regulation of accurate cell cycle
by preventing premature sister-chromatid separation during
mitosis (metaphase-anaphase transition and anaphase onset).
• Proteolysis of securin results in degradation of protein complexes
that connect sister chromatids at metaphase, thereby initiating
anaphase, the mitotic period in which sister chromatids are
separated and moved to the opposite poles.
• APC-Cdh1/ proteasome degrades mitotic cyclins.
42

43. .
Cdh1 activation can be induced by dephosphorylation through
phosphatase which leads to binding of Cdh1 to APC/C. APC-Cdh1
degrade S/M cyclin until they are needed in the next cycle.
• Reduction in the activity of mitotic cyclin-CDK complexes
caused by proteolysis of mitotic cyclin permits the late mitotic
events and cytokinesis to occur.
• Passage through three critical cell cycle transitions-
G1 S phase, metaphase anaphase, anaphase telophase
and cytokinesis is irreversible because these transitions are triggered
by the regulated degradations of proteins.
43

44. Checkpoints in cell cycle
• Checkpoints:- These are the cell cycle control mechanisms in eukaryotic cells.
These checkpoints verify whether the processes at each phase of cell cycle have
been accurately completed before progression into the next phase .
• 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 parent cell. Two main molecular processes take place during the cell
cycle, with resting intervals in between: during the S phase of the cycle, each parent
chromosome is duplicated to form two identical sister chromatids; and in mitosis
(M phase), the resulting sister chromatids are distributed to each daughter cell.
Chromosome replication and segregation to daughter cells must occur in the proper
order in every cell division. If a cell undergoes chromosome segregation before the
replication of all chromosomes has been completed, at least one daughter cell will
lose genetic 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 accuracy and fidelity are required to ensure that DNA replication is carried
out correctly and that each daughter cell inherits the correct number of each
chromosome. To achieve this, cell division is controlled by surveillance
mechanisms is known as checkpoint pathways which prevent initiation of each step
in cell division until the earlier steps on which it depends have been completed and
any mistakes that occurred during the process have been corrected.
44

45. There are three main checkpoints that control the
cell cycle in eukaryotic cells:-
They are:-
• G1 checkpoint (G1
restriction point)
• G2 checkpoint
• Metaphase checkpoint
( Spindle checkpoint)
45

46. G1 checkpoint (G1 restriction
point)
• This checkpoint is present at the end of the G1 phase and before S phase. This
checkpoint helps in taking the decision of whether the cell should divide, delay
division or enter a resting stage (G0 phase). If there are unfavorable conditions for
the cell division, then this restriction point restrict the progression to the next phase
by passing the cell to G0 phase for an extended period of time.
• 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 cell large enough to divide?
• Nutrients :- does the cell have enough energy reserves or available
nutrients to divide?
• DNA integrity :- is any of the DNA damaged?
46

47. • This restriction point is mainly controlled by the action of the CKI-p16
(CDK inhibitor p16). The inhibited CDK not bind with cyclin D1 , hence
there is no cell progression.
• Active cyclin D-cdk complexes phosphorylate retinoblastoma protein
(pRb) in the nucleus .
• Unphosphorylated pRb acts as an inhibitor of G1 by preventing E2F-
mediated transcription.
• Once pRb gets phosphorylated E2F activates the transcription of cyclins
E and A which then interacts with CDK2 to allow for G1-S phase
transitions.
• This bring the cell to the end of the first checkpoint.
47

48. G2 checkpoint
• This restriction point is located at the end of the G2 phase.
• To make sure that the cell division goes smoothly the cell has an additional
checkpoint before M phase, called G2 checkpoint.
• At this stage , the cell will check is any of the DNA damaged, was DNA replication
completed.
• If errors and damage are detected , the cell will pause at the G2 checkpoint to allow
for repairs.
• If the damage is irreparable, the cell may undergo apoptosis or programmed cell
death.
• Maturation promoting factor or mitosis promoting factor or M- phase promoting
factor (MPF) is a protein composed of cyclin B and CDK1. This protein promotes
the G2 phase into the entrance of M phase . MPF is activated at the end of G2 phase
by a phosphatase (Chk) which removes an inhibitory phosphate group added earlier.
• The main function of MPF in the restriction point are:- triggers the formation of
mitotic spindle, promotes chromosome condensation, causes nuclear envelope
breakdown.
• If any damages are noticed in this restriction point, then the phosphatase not activate
the MPF, resulting in the arrest of cell cycle in G2 phase till the repair of the
damaged DNA. This prevents the transfer of defected DNA into the daughter cells.
48

49. Metaphase checkpoint
• The M checkpoint is also known as the spindle checkpoint.
• Anaphase promoting factor regulates this checkpoint. If
there are mistakes then it delay the cell in entering into
anaphase .
• 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.
• It seems that cell don’t actually scan the metaphase plate to
confirm that all the chromosomes are there. Instead , they
look for straggler chromosomes that are in wrong place.
• If a chromosome is misplaced, the cell will pause mitosis,
allowing time for the spindle to capture the stray
chromosome.
49

50. Overview of checkpoint controls
in the cell cycle
• The Growth Checkpoint Pathway Ensures That Cells Enter the Cell Cycle Only
After Sufficient Macromolecule Biosynthesis:- Cell proliferation requires that
cells multiply through the process of cell division and that individual cells grow
through macromolecule biosynthesis. Cell growth and cell division are separate
processes, but for cells to maintain a constant size as they multiply, cell growth
and cell division must be tightly coordinated. 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 surveillance mechanisms exist that adjust cell division rate
according to growth rate.
• The DNA Damage Response System Halts Cell Cycle Progression When DNA Is
Compromised:- The complete and accurate duplication of the genetic material is
essential for cell division. If cells enter mitosis when DNA is incompletely
replicated or otherwise damaged, genetic changes occur. In many instances,
those changes will lead to cell death, they can also lead to genetic alterations
that result in loss of control over cell growth and division and, eventually,
cancer.
50

51. • The enzymes that replicate DNA are highly accurate, but their 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
mitosis. 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 progression until
the damage has been repaired. Cell cycle arrest can occur in G1, S phase, or G2, depending on
whether DNA damage occurred before cell cycle entry or during DNA replication.
• DNA damage exists in many different forms and degrees of severity. A break of the DNA helix,
known as a double strand break, is perhaps the most severe form of damage because such a lesion
would almost certainly lead to DNA loss if mitosis ensued in its presence. More subtle defects
include single-strand breaks, structural changes in nucleotides, and DNA mismatches.
• Central to the detection of these different types of lesions is a pair of homologous protein kinases
called ATM (for ataxia telangiectasia mutated) and ATR (for ataxia telangiectasia and Rad3-related
protein). These proteins were identified and characterized.
51

52. • Both protein kinases are recruited to sites
of DNA damage. They then initiate the
sequential recruitment of adapter proteins
and another set of protein kinases called
Chk1 and Chk2. Those kinases then activate
repair mechanisms and cause cell cycle
arrest or apoptosis in animals.
• ATR and ATM recognize different types of
DNA damage. ATM is very specialized in that
it responds only to double-strand breaks.
ATR is able to recognize more diverse types
of DNA damage, such as stalled replication
forks, DNA mismatches, 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 the
repair process.
• The association of ATR with single-stranded
DNA is thought to activate its protein kinase
activity, leading to the recruitment of
adapter proteins whose function is to
recruit and help activate the Chk1 kinase.
Active Chk1 then induces repair pathways
and inhibits cell cycle progression. Chk1 and
Chk2 halt the cell cycle. These protein
kinases inhibit Cdc25 by phosphorylating
that phosphatase on sites that are distinct
from the CDK-activating phosphorylation
sites.
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 part of the repair process, they also,
albeit indirectly (as depicted by a dashed line), activate ATR.
ATM and ATR, once activated by DNA damage, activate
another pair of related protein kinases, Chk1 and Chk2. These
kinases then induce the DNA repair machinery and cause cell
cycle arrest by inhibiting Cdc25. In metazoan cells, Chk1 and
Chk2 also activate the transcription factor p53, which induces
cell cycle arrest by inducing transcription of the CKI p21. When
the DNA damage is severe, p53 induces apoptosis.
52

53. • When the DNA damage occurs during G1, Cdc25A inhibition results in inhibition of
G1/S phase CDKs and S phase CDKs . As a result, these kinases cannot initiate DNA
replication.
• When the DNA damage occurs during S phase or in G2, Cdc25C inhibition by Chk1/2
results in the inhibition of mitotic CDKs and hence arrest in G2. Active DNA replication
also inhibits entry into mitosis. ATR continues to inhibit Cdc25C via Chk1 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 stalling or slowing of
replication forks. Such stress triggers activation of the ATR-Chk1 checkpoint pathway
and results in down regulation of S phase CDK activity and prevents the firing of late-
replicating origins.
• Chk1-mediated inhibition of the Cdc25 family of phosphatases 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 p53, a
transcription factor that induces the expression of the gene encoding the CDK
inhibitor p21. The p21 binds to and inhibits all metazoan cyclin-CDK complexes. As a
result, cells are arrested in G1 and G2.
• ATR, activated ATM also halts cell cycle progression by Chk2-mediated inhibition of
Cdc25, thus preventing activation of CDKs. This inhibition can occur in G1 or G2.
• p53 in response to DNA damage greatly increase its ability to activate transcription of
specific genes that help the cell cope with DNA damage. One of these genes encodes
the CKI p21. Under some circumstances, such as when DNA damage is extensive, p53
also activates expression of genes that lead to apoptosis, the process of programmed
cell death that normally occurs in specific cells during the development of
multicellular animals.
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54. 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 properly attached
to spindle microtubules.
If even a single kinetochore is unattached or not under tension, anaphase
is inhibited, as such a defect would almost certainly lead to chromosome
loss if mitosis ensued in its presence. To achieve this, cells harbor a
surveillance mechanism that prevents anaphase entry in the presence of
tensionless or unattached kinetochores. The spindle assembly checkpoint
pathway recognizes unattached kinetochores.
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55. Overview of DNA damage checkpoint controls in the cell cycle:- During G1, the p53-p21 pathway inhibits
G1 CDKs. During ongoing DNA replication and in response to replication stress (slow DNA replication fork
movement or DNA replication fork collapse), the ATR-Chk1 protein kinase cascade phosphorylates and
inactivates Cdc25C, thereby preventing the activation of mitotic CDKs and inhibiting entry into mitosis. In
response to DNA damage, the ATM or ATM protein kinases (ATM/R) inhibit Cdc25 via the Chk1/2 protein
kinases. They also activate p53, which induces production of the CKI p21. During G1, the DNA damage
checkpoint pathway inhibits Cdc25A, inhibiting G1/S phase CDKs and S phase CDKs, and thereby blocking
entry into or passage through S phase. During G2, ATM/R and Chk1/2 inhibit Cdc25C. The p53-p21 pathway
is also activated. Red symbols indicate pathways that inhibit progression through the cell cycle.
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56. https://www.youtube.com/watch?v=VLJF8Pf8s
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57. References
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https://doi.org/10.1186/gb4184
• Casimiro, M. C., Crosariol, M., Loro, E., Li, Z., & Pestell, R. G. (2012). Cyclins and cell
cycle control in cancer and disease. Genes & cancer, 3(11-12), 649–657.
https://doi.org/10.1177/1947601913479022
• Nigg, E. A. (1995). Cyclin‐dependent protein kinases: key regulators of the eukaryotic
cell cycle. Bioessays, 17(6), 471-480.
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