Checkpoint Regulation of the Cell Cycle

Question 1

Structure

Answer 1

1. The Mutator Hypothesis and familial cancers
2. Studying checkpoint mutants
3. DSB checkpoints
4. Fork stalling checkpoints
5. p53

Question 2

The Mutator Hypothesis

Answer 2

1) normal cell gets a mutation in a repair/replication checkpoint gene
2) increased mutation rare
3) second hit on oncogenes/tumour suppressors
4) tumourigenesis

Question 3

Familial cancer syndromes affecting genome stability

Answer 3

• Xeroderma pigmentosum: NER
• Fanconi anaemia: ICL
• Lynch syndrome: MMR
• PPAP: proofreading
• Li-Frameni syndrome: p53
• Ataxia telangiectasia: ATM

Question 4

Studying checkpoint mutants

Answer 4

• cells cannot arrest
• inviable micro colonies

Question 5

Checkpoint proteins at DSBs

Answer 5

1) MRN
2) ATM
3) phospho-H2AX
4) Mdc1

Question 6

ATM

Answer 6

• interacts with MRN
• phosphorylates H2AX

Question 7

phospho-H2AX

Answer 7

• recruits Mdc1

Question 8

Mdc1

Answer 8

• recruits MRN

Question 9

MRN

Answer 9

recruits ATM

Question 10

replication fork stalling

Answer 10

• helicase and polymerase uncoupling
• ssDNA
• block to G2-M

Question 11

What causes replication fork stalling?

Answer 11

• insufficient dNTP
• template damage
• template barriers (bulky adducts)

Question 12

How is the G2-M checkpoint regulated under fork stalling?

Answer 12

• RPA binds ssDNA
• recruits ATR
• phosphorylated Chk1
• phosphorylates cdc25

Question 13

Fork blockage

Answer 13

1) ATR activated (phosphorylates Chk1)
2) Rad51 binds are recruits fork protectors

Question 14

Fork protectors

Answer 14

prevent collapse, from which it is difficult to recover

Question 15

p53 - the basics

Answer 15

• tumour suppressor
• v. frequently inactivated in cancers
• key TF in apoptotic induction

Question 16

p53 activity

Answer 16

1) unstressed: constant degradation by Mdm2
2) stress: stabilisation (Mdm2 Ub-ligase is inactivated)
3) induces apoptotic TFs (tumour suppressors)