At which two points can the cell cycle be stopped if genome is damaged?
Restriciton point: Blocks passage into S phase
Intra-S phase: Blocks DNA replication
What three routes can a cell take after checks for DNA damage at restriciton point?
Divide - enter S phase
Arrest - pause to rest
Die - undergo apoptosis
Which gene product is commonly compromised in cancer cells and hence cause the cell to loss ability to deal with DNA damage 'decisions'?
Which type of mutations ofP53 are commonly seen?
Alterations in arganine248, which binds the DNA backbone, and other DNA binding residues
Mutations in the zinc finger motif, which is vital for the interaction between loop 2 and 3 of the p53 to allow DNA binding.
Which Cdki is regulated by P53?
p21 is upregulated by P53
What role does p21 play?
Inhibits Cdk2 (entry into S phase)
Inhibits PCNA (halts ongoing DNA replication)
What are the roles of PCNA?
DNA polymerase 'sliding clamp'
Holds polymerase onto the DNA and increases possible length of product by several orders of magnitude
Also attracts DNA repair factors to arrested DNA replication forks (not inhibited by P21)
What is the other way a cell can deal with damage if it has lost its p53?
The DNA damage response in S1
Why is the DNA damage response being targeted as a possible cancer therapy?
If cancer cell has lost p53 dependent cell cycle arrest then knocking out DNA damage response should cause cell death. But healthy cells will be unaffected due to still having p53 pathway available
What are the four broad classes of molecule involved in DNA damage response?
What is the main role of the transducer molecules?
Amplify the once local signal to spread across the entire genome
Outline the general cascade for recruiting repair proteins to sites of damage
A PI3 Kinase (PIK) family kinase is recruited to the site of damage by partner proteins where they phosphorylate signal proteins to recruit repair proteins
Outline an example cascade for recruiting repair proteins to sites of DS break damage
Nbs1, part of the MRN complex, recruits ATM which phosporylates CHK2 and causes signalling.
CHK2 can then phosphorylate cdc25A leading to its destruction and inhibiting its ability to remove the inhibitory phosphate group from cdk2, causing cell cycle arrest.
Additionally AMT and the MRN complex can then cuase DNA repair through a variety of mechanisms
How will ATM 'choose' between homologous repair or non-homologous end joining?
NHEJ is the default pathway but HR will be used if in S phase - measures the level of CDK which are higher in S phase
Why does CHK2 offer a quick damage response?
Constantly available in the genome and does not need to be made when damage discovered
What is activated by replicative stress?
CHK1 via ATR
Germ line muations of what protein are found in familial breast and ovarian cancers?
50% for breast
70% for ovarian
What is the role of BRCA1?
Scaffolding protein for all proteins involved in damage repair
How is damaged flagged by ATM/ATR?
Phosphorylation of which causes ubiquitination
What non-repair protein is attracted by this H2AX flag?
Cohesin - which acts ot hold the chromatids togther
What is the outcome of chronic activation of DDR?
Deactivation and then hyper mutation
What can cause replication forks to slow down or stall?
- Disruption of nucleotide pools
- DNA damage/obstructions
- DNA secondary structures stop unwinding of DNA
Why would a stalled replication fork be dangerous?
Transiently in single stranded form