L4 - Early molecule events II Flashcards
How is DNA damage repair integrated into the cell cycle?
Cells run safety check in G1 to look for damage
What are the three things a cell can decide to do after safety check?
Divide (enter S phase)
Arrest (pause to repair)
Apoptosis
What is usually mutated so that the safety check is compromised?
p53 ‘the gate keeper’
Features of p53
- DNA binding domain into the major groove
- Beta barrell
- loop 2 positions loop 3 that has arginine 248
- zinc finger
- transcription factor
What does p53 do?
Increases p21^cip1 which causes cell cycle arrest
What does p21 inhibit?
Cyclin E and A/Cdk2 (prevents entry to S)
PCNA (halts DNA replication)
Possibly Ciz1
What is PCNA?
Multimeric ring structure, ‘sliding clamp’, accessory factor of DNAPol
What does PCNA do other than its role in DNA synthesis?
Attracts DNA repair factors to arrested DNA replication forks
What is selection inhibition?
p21 is able to inhibit DNA replication activity of PCNA but leave its repair activity intact
What a few things responders can do when told there is DNA damage?
- stop txn
- block cell cycle
- change energy metabolism
- repair
- chromatin remodelling
What do the PIK family kinases do?
An example?
Recruited by partner proteins where they phosphorylate signalling proteins.
ATM (ataxia talengectasia mutated)
How do ATM end up inhibiting DNA synthesis?
NBS1 binds leading to Chk2.
Chk2 inhibits the phosphorylation of cdc25A so Cyclin E/A-Cdk2 is not phosphorylated and DNA synthesis is halted
What do ATM and ATR signalling do?
Inhibition of CDKs which arrests cell cycle at G1-S, intra-S or G2-M checkpoints.
Also activates repair enzymes by inducing their txn, recruiting them or modulating RNA processing
Why can homologous recombination only occur in S/G2?
Has to have a sister chromatid for the information to be copied from
What does ATM initiate?
Homologous recombination or non-homologous end joining
How does the cell choose whether to use HR or NHEJ?
By concentration of CDK.
In G1 cells CDK is low so goes through ATM to Chk2-p53-G1 arrest and to MRN-resection-NHEJ
In S/G2 cells CDK is high - leads to HR
What is Chk2/
Serine/threonine kinase that transduces the signal from PIKs to decide if cell is arrested or not
What happens to Chk2 when damage occurs?
- Sits everywhere around chromatin
- Phosphorylated by ATM causing Chk2 to homodimerise and autophosphorylate itself
- Activated Chk2 can now phosphorylate other targets so responds quickly and spreads signal throughout nucleus
What is Chk1 activated by and when is it activated?
Activated by ATR in response to replicative stress
What was shown by staining of Chk2 in breast cancer?
Expression is lost in the tumour
What was shown by staining phosphorylated Chk2 in breast cancer?
Normal cells - not much activation
Breast cancer - loads of activation
What are BRCA1 and BRCA2?
Platform proteins with mutations found in 50% of breast cancers and 70% ovarian cancers
Support assembly of large multiprotein complexes involved in detection, signalling and attracting repair factors
What is H2AX?
A histone who’s phosphorylation is mediated by ATM/ATR and is incorporated into chromatin at low frequency all over the genome
What is H2AX used for and how?
To visualise the DNA damage repair.
- UV light creates a stripe of damage on a cell
- H2AX is phosphorylated locally at damage
- Phosphorylated H2AX helps attracts repair factors
What was shown by using H2AX?
BRCA1 was recruited to the strip of UV damage
Who’s paper is referenced when answering ‘what drives the loss of repair functions?’
Bartkova et al 2005
What did Bartkova show with one of their fluorescences?
Precursor lesions of breast, lung, colon and bladder cancers express markers of an activated DDR
What was Bartkova’s hypothesis?
- At an early stage incipient tumour cells experience ‘oncogenic stress’
- respond to activating pathways (arrest or die)
- mutations compromising pathways might allow proliferation
- compromises checkpoints and increased genomic instability and tumour progression
- chronic activation leads to deactivation and hyper mutation
What can cause replication forks to slow down or stall?
inefficient DNA replication
What can cause inefficient DNA replication?
- disruption of nucleotide pools
- DNA damage or protein obstructions
- DNA secondary structure
