Week 5 L4: ds DNA repair Flashcards
What are the 3 mechanisms for ds repair?
non-homologous end-joining
Single-strand annealing
Homologous recombination
What is the best pathway for repair?
homologous recombination
What are the flaws with non-homologous recombination and single-stranded annealing?
Can result with deletion in that region.
local change sin DNA.
What are the oncogenic markers in homologous recombination?
BRACA-1 BRACA-2
What is a feature of BRACA-1?
Has an affinity for various branch DNA species.
Why are the changes to DNA not too important?
happens in somatic cells not germline cells.
What is the key protein in NHEJ?
Ku protein
What are the features of Ku?
heterodimer of 2 subunits
70/80 kD
Mediates the interaction between the 2 broken ends, acts like a splint.
What is the enzyme that carries out the ligation in NHEJ?
DNA ligase IV with XRCC4
Does Ku dimer make specific contacts to DNA?
NO
How does Ku bind to DNA?
non-specific contacts to DNA. acts like a basket to hold DNA. DNA held in splint to align broken ends
How can a mismatch occur in DNA bases?
- Errors in DNA repair
- From homologous recombination (2 seq being recombined are not 100% identical) in heteroduplex location there will be non-watson crick pairs.
What is confusing about mismatch pairs?
How do we know what base is the original???
How do we tell parental vs newly-replicated DNA?
The dam methyltransferase methylate’s Bases.
when the DNA is replicated, the new bases transiently are not subject to Dam methylation. transiently a hemi-methylatd site
What enzyme methylates DNA?
dam methyltransferase
What can you call the newly synthesised stand?
hemi-methylated site
What do we call the mismatch correction mechanism and what strand does it act upon?
methyl-directed mismatch correction.
on unmethylated strand
Where are the dam sites located?
usually up to a kbp away from the mismatch.
What are the first 3 proteins involved in methyl-directed mismatch correction?
MutS, MutH, MutL
What is the function of MutS?
mismatch recognition acts as a MUTS2 dimer
What is the function of MutH?
latent endonuclease, binds at dam site
What is the function of MutL?
latent ATPase
mediates MutS-MutL interaction
What activates the endonuclease activity of MutH?
the communication of S+L to H at the dam site
What does H do when activated?
cleaves the unmethylated strand
What is the role of the exonuclease?
removes DNA to allow DNA synthesis of strand to allow Pol III an ligase to repair DNA
What are the protein’s which bind to Mismatch in DNA?
HUMANS
recognition: complex of MSH2 and GTPB (MSH6 in yeast)
Followed by of MLH1-PMS2 (PMS1 in yeast)
What do mutations in the mismatch recognition site lead to?
HUMAN CANCER
HPNCC syndrome
germline mutations in hMSG2 and hMLH1
What is the key protein (regulator) for upregulation of genes for DNA repair?
LexA
What happens to LexA in the presence of RecA?
LexA cuts itself so it is no longer a repressor but a non-repressor and the repair genes are no longer repressed. upregulated
What is the coordinated response to DNA damage called?
SOS response of E.coli
What is indicative that DNA damage has occurred?
RecA filament
What genes does LexA repress? (regulate)
umuC, UvrA, UvrB, ruvA, runB
What are the whole cell responses to DNA damage in EUK?
activate repair
transcriptional regulation
cell cycle control
apoptosis or senescene
What is the key tumour suppressor protein which coordinates the response to DNA damage?
p53
coordinated within cell cycle
What is the last resort of p53?
APOPTOSIS
