Lecture 8 Flashcards
What happens if DNA damage is not repaired before replication?
Mismatch base pair - Usual replication undergone - Mutation
DNA lesions not always repaired before replication
Normal polymerases can’t replicate damaged DNA - leads to stalled forks
DNA damage response induced
Specialised translesion synthesis - DNA pols replicate some DNA with damaged template
DNA damage tolerance - translesion DNA synthesis
- TLS DNA pols have more open and flexible active site - some DNA replication, but low fidelity synthesis
- TLS DNA pols lack 3’ to 5’ proofreading activity
- Error rates 10-2-10-4 (10-5-10-6 in DNA pols)
- Continued DNA rep at unpaired lesion - increased risk of incorrect insertion - Mutation
- Error prone replication better than no replication
TLS vs Replicative polymerases
TLS polymerase active site more open and flexible, allowing them to bypass problem DNA and continue synthesis, albeit with lower fidelity
What happens if damaged strands or bases not repaired before replication?
- Polymerases encounter legions and stall, leads to large regions of ssDNA
- Single-stranded nicks can lead to dsDNA breakages - could form lethal chromosomal rearrangements
DNA damage response induces…
- More DNA repair proteins
- Delay cell cycle
- Apoptosis
DNA damage response in bacteria
SOS response
Induces expression of >40 proteins
RecA and LexA central to cotnrol
RecA is a DNA binding protein and damage sensor - inactive in absence of DNA
LexA protein - Repressor - Prevents transcription of SOS genes by binding operators
LexA binds as dimer at sequences similar to consensus sequence - block transcription
What happens when replication fork stalls?
RecA binds to form filament on ssDNA - Activates and cleaves LexA
Cleaved LexA can’t bind DNA - SOS genes transcribed including RecA and DinI
- DNA damaged repaired
- ssDNA decreases as DNA repaired - reducing RecA filament assembly and LexA cleavage
DinI - DNA mimic - RecA binds and sequestered
- Newly synthesised LexA repressor binds to SOS boxes, SOS genes repressed
What DNA repair proteins can be encoded for under SOS regulation
UvrA, B, D proteins - NER
RecA, RuvA, RuvB proteins - Recombination repair of strand breaks
TLS pols - polIV (DinB) amd polV (UmuC)
SulA protein - cell division inhibitor
DNA damage response in eukaryotes
DNA damaged sensors recognise damaged DNA
RPA binds to ssDNA and MRN
KU binds to DSBs
Downstream proteins activated by transducer regulatory kinases
Downstream proteins phosphorylated to recruit effector proteins to repair damage and halt cell cycle
RPA with ssDNA
- RPA binds ssDNA at fork
- RPA normally remove when lagging strand template replicated
- DNA damage causes DNA pol to stall at damage - DNA helicase continues
- RPA remains bound to ssDNA
What does accumulation of RPA lead to?
- RPA recruits ATR by adapter (ATRIP) - binds to both RPA and ATR
RPA recruits repair specific sliding clamp-clamp loader complex
Damage specific sliding clamp known as 9-1-1 (RAD9-RAD1-HUS1)
9-1-1 recuits TOPBP1 and these activate ATR
