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Cell Cycle or Cell-Division Cycle – Part 3

https://www
...
com/watch?v=eqJqhA8HSJ0

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Cell Cycle Control System contd
...
REGULATION
OF CDK 2 BY PHOSPHORYLATION AND DEPHOSPHORYLATION; 2
...
GROWTH FACTORS STIMULATE CDK AND CYCLIN
SYNTHESIS – ROLE OF RETINOBLASTOMA PROTEIN (pRB); 4
...
g
...
g
...
e
...
g
...
e
...
g
...
– Role of cyclin dependent kinase (CdKs) Regulation of CdKs at the time of DNA damage

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Regulation of CdK at the time of DNA damage – Role of pRb and p53
• When DNA damage is detected, pRb participates in a mechanism that arrests cell division in G1
• Braking action is regulated by phosphorylation and dephosphorylation

• Cell cycle arrest due to DNA damage at G1
• Damage (DNA breaks) to the cell’s DNA triggers a series of events that inactivate CdK2, blocking
cell division
• MRN complex (MRE11-RAD50-NBS1) detects damage to the DNA (double-strand break site)
• Binding of MRN to DNA damage activates two protein kinases, ATM and ATR
• ATM and ATR phosphorylate and activate the transcription factor p53
• Active p53 promotes the synthesis of another protein, p21, an inhibitor of CdK2
• Inhibition of CdK2 stops pRb phosphorylation
Ataxia-telangiectasia
• Phosphorylated pRb continues to bind and inhibit E2F
mutated
and
ataxia
telangiectasia and Rad3
• With E2F inactivated, genes essential to cell division are not transcribed
related proteins are key
• Cell division is arrested at G1

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regulators of DNA damage
response
(DDR)
and
maintain genome integrity in
25
eukaryotic cells

• This gives the cell time to repair its DNA
before entering the S phase, thereby
avoiding the potentially disastrous transfer
of a defective genome to one or both
daughter cells
• When DNA has been repaired by repair
enzymes, this inhibition is released, and
the cell divides
• When the damage is too severe to allow
effective repair, this same machinery
triggers apoptosis, a process that leads to
the death of the cell, preventing the
possible development of a cancer

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Cell cycle arrest due to DNA damage at G2
• The presence of a DNA breaks in DNA signals arrest of the cell cycle in G2 by activating two proteins
(ATM and ATR)
• These proteins trigger a cascade of responses that include inactivation of the PTP (protein tyrosine
phosphatase) that dephosphorylates P-Tyr15 of the CdK1
• With the CdK1 inactivated, the cell is arrested in G2, unable to divide until the DNA is repaired and
the effects of the cascade are reversed

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p53 functions (not under normal conditions)
•
•
•
•
•
•

TSG
Enable cell to cope safely with DNA damage
Keep check on cell proliferation in stressful circumstances
Protects organism against cellular damage and disorder
Blocks cell cycle progression
Emergency brake on cell division in cells that have suffered genetic change

• Role
• In cell cycle control
• In apoptosis
• In maintenance of genetic stability

• Whether p53 causes cell cycle arrest or apoptosis depends on the type of post transcriptional
modifications to which it is subjected
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Low concentration of p53 in normal cells
• The level of p53 in a healthy G1 cell is very low, which keeps its potentially lethal action under
control
• Present in lower amount in cells than pRb
• Not required for normal development of cell
• Required occasionally or in special circumstances as DNA damage / stress / cancer
• In unstressed / undamaged cells, p53
• Has a half-life of a few minutes
• Is highly unstable
• Is present at very low concentrations
• This is because
• p53 interacts with MDM2 (Mouse double minute 2 homolog)
• MDM2
• Is ubiquitin ligase
• Escorts p53 out of the nucleus and into the cytosol
• Once in the cytosol, MDM2 adds ubiquitin molecules to the p53, leading to its
destruction by a proteasome
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Rise in p53 concentration during stress, DNA damage (e
...
, ss DNA), oxygen deprivation,
exposure to radiations and cancer
• Concentration rises rapidly
• If a G1 cell sustains genetic damage, for example, by UV light or chemical carcinogens
• A similar response can be elicited simply by injecting a cell with DNA containing broken strands
• Under stressful conditions, during DNA damage or cancer
• When normal cells are deprived of oxygen
• Exposed to treatments that damage DNA, e
...
, UV or -rays
• Concentration rises by reducing their degradation rate
• The increase in p53 levels is not due to increased expression of the gene but to an increase in
the stability of the protein

• DNA damage lead to stabilization of p53
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Role of ATM or ATR in p53 stabilization

Stress, DNA damage, oxygen deprivation and cancer
↓
ATM or ATR protein kinase is activated following DNA damage
Persons suffering from ataxia telangiectasia lack a protein
kinase called ATM and are unable to respond properly to
DNA-damaging radiation

↓
Stabilized p53 then
• Activates expression of Bax, Bid →
leads to apoptosis
• Stimulates gene transcription of p21
→ cell cycle arrest at G1

↓
ATM or ATR phosphorylate p53
↓
Phosphorylated p53 does not interact with MDM2
↓
Decreased degradation, increased concentration and
stabilization of existing p53 in the nucleus

↓

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p53 promotes cell cycle arrest at G1 (action at G1 DNA damage checkpoint)
Action of p53 under stress / DNA damage / cancer conditions in cell cycle arrest (in G1 phase)
Stabilized p53 (level of p53 rises in the damaged G1 cell)
↓

Binds to DNA
↓

Induces transcription of p21
(p21 is mammalian CKI - a regulatory gene product that binds to Cdk complexes required for entry into S phase)

(p21 binds to the complexes of G1 cyclin with Cdk2 that normally serve to drive the cell past G1 checkpoint)
↓

Blocks CdK2
↓

Blocks entry of cell into S phase and replicating its DNA

[Activated CKI protein called p21 → binds G1/S-Cdk and S-Cdk and inhibit them →
pRb remains unphosphorylated → E2F blocked → Cell cycle arrest in G1 → Time for DNA
damage repair → If not repaired, cell undergoes apoptosis]
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G2 DNA damage checkpoint
Cell cycle regulation under stress / DNA damage / cancer conditions in the regulation of cell
cycle in G2 phase
When cells in G2 are exposed to damaging radiations
↓
Damaged DNA sends a signal to a series of protein kinases that phosphorylate and inactivate
phosphatase Cdc25
↓
Blocks dephosphorylation and hence activation of M-Cdk
[M-CdK remains inactive]
↓
Blocks entry into M phase

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p53 promotes apoptosis
• Cell cycle arrest is not the only way that p53 protects an organism from developing cancer
• Alternatively, p53 can direct a genetically damaged cell along a pathway that leads to death by
apoptosis, thereby ridding the body of cells with a malignant potential
Stabilized p53 (in high concentration)
↓
Binds to DNA
↓
Activation of the expression of Bax gene
or
Binds directly to several members of the Bcl-2 family including Bax in outer mitochondrial membrane
and directly triggers membrane permeabilization and release of apoptotic factors
[Not all actions of p53 are dependent on its activation of transcription]
↓
Stimulates apoptosis
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Cell Cycle Control System contd
...
– Cell Cycle Checkpoints

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Checkpoints
• In the standard cell cycle the control system is regulated by brakes that can stop the cycle at
specific checkpoints
• Periods immediately before entry into S and M phases are looked upon as key cell cycle
checkpoints
• It is at one of these checkpoints that the cycle becomes arrested if
• Critical genes involved in cell cycle control are mutated
• The cell undergoes trauma such as extensive DNA damage
• Replication process itself can be arrested under certain circumstances, for e
...
, if DNA is
damaged and must be repaired before copying can be completed
• Three checkpoints in cell cycle
• G1 checkpoint
• G2 checkpoint
• M checkpoint
• Besides, there is Start or Restriction point
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• Key regulator is itself regulated at certain critical points of the cycle by feedback from the processes
that are being performed
• Feedback signals conveying information about the downstream processes can delay progress of the
control system itself
• To prevent it from triggering the next downstream process before the previous one has finished
• Allow the cell cycle control system to be regulated by signals from the environment

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Or Quiescent
state

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• Start or Restriction point
• In higher eukaryotic cells signals that arrest the cycle usually act at the G1 control point
• In mammalian cells, restriction point is at the same position at G1 checkpoint
• When circumstances forbid cell division, it is at this point in the cycle that many cells halt

• G1 checkpoint and G2 checkpoint
• Environmental controls generally act on the control system at one or other of two major
checkpoints in its cycle
• G1 checkpoint in G1, just before entry into S phase
• G2 checkpoint in G2, at the entry to M phase
• In a continuously cycling cell
• G1 checkpoint is the point where the cell cycle control system triggers a process that will
initiate S phase
• G2 checkpoint is where cell cycle control system triggers a process that will initiate M
phase
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G1 checkpoint (just before entry into S phase)
• Check for
• Cell size - is cell large enough to divide?
• Nutrients - does the cell have enough energy reserves or available nutrients to divide?
• Growth factors - is the cell receiving positive cues (such as growth factors) from neighbors
(some cells also require mechanical cues such as being attached to ECM in order to divide)
• DNA integrity - is any of the DNA damaged?
• If a cell does not get the go ahead cues it needs at G1 checkpoint it may leave the cell cycle
and enter a resting state G0
• Blocks progression into S phase by inhibiting activation of G1/S-Cdk and S-Cdk

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G2 checkpoint (before M phase)
• Checks for
• DNA damage - is any of the DNA damaged?
• DNA replication completeness - was the DNA completely copied during S phase?

• If errors or damage are detected, cell pauses at checkpoints to allow for repairs
• If the damage is irreparable, the cell may undergo apoptosis

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M checkpoint (spindle checkpoint)
• Checks for
• Chromosome attachment to spindle at metaphase plate - whether all the sister chromatids are
correctly attached to spindle microtubules
• Because the separation of 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
fibres from opposite poles of cell
• Metaphase plate is not scanned to confirm that all chromosomes are there
• Rather ‘straggler’ chromosomes are looked into as being in wrong place (example floating in
cytoplasm)
• If a chromosome is misplaced, the cell will pause mitosis, allowing time for the spindle to
capture the stray chromosome

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• The mechanism operating at the G1 checkpoint is much less well understood than that at the G2
checkpoint, but the principles are believed to be similar
• Just as the assembly of MPF ultimately triggers the events of mitosis, so the assembly of a related
complex comprising a Cdk protein and G1 cyclin is thought to drive the cell past the G1 checkpoint,
triggering the events that lead to DNA replication

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