REPLICATION OF DAMAGED DNA Flashcards
(46 cards)
What happens if we don’t respond to DNA damage repair?
It will lead to genome instability, leading to DNA repair disorders and cancer.
How does E.coli avoid damage?
Recombination
How are mutations generated?
By transletion synthesis using Y family DNA polymerases.
Name 2 Y family DNA polymerases in E.coli and what are they for?
Pol 4 and pol 5 for TLS
What characterizes XPV?
Patients are defective in polymerase eta
What is polymerase switching mediated by?
Ubiquitination of PCNA
What is the role of polymerase eta?
It can replicate over DNA damage and is needed when cells enter S phase.
What are the effects of DNA damage on replication?
- Stops initiation
- No effect e.g. N7MA
- Obstructs fork progression
- Misreplication e.g. 06MG
- Aressets cell cycle
SNOMA
Describe the model for recombination repair of daughter strand gaps in E.coli
- Replication of a strand with CPD = replicated strand with gap due to damage (CPD)
- Initiation of strand exchange by RecA helicase = filling in gap using the other strand (it will cross over ygm)
- RecAFRO forms holiday junction to fill in the new gap left from the migration of the other strand moving up
- RecA, RuvAB and RecG mediates branch migration and repair synthesis
- Cleavage of holiday junction by RuvC
OR
Reverse branch migration by RecG and RuvAB
How many CPD’s can E.coli UVRA strains tolerate?
50 CPDs per genome
What makes E.coli sensitive to UV?
- UVRA mutation
- RecA mutation
- Double mutation (uvra-reca) = extra sensitive, so just the formation of a CPD is enough to kill the cell
What is required for mutations?
umuDC - it isn’t sensitive to UV so its proper evil
What is LexA?
It is a repressor of RecA, umuDC and other NER genes
What is the SOS response in E.coli?
- During replication of damaged DNA, it gets locked at site of damage = exposure of SS DNA at replication fork
- This leads to the formation of SS DNA RecA filaments
- RecA catalyses cleavage/inactivation of LexA = increased RecA and umuDC
- RecA also cleaves 24 amino acids at the N terminus of umuD = activate umuD
- umuD’2C complex forms
What is umuD’2C?
It is a DNA polymerase 5 which can synthesize past DNA damage (but can make mistakes)
Name a polymerase that cannot synthesize past DNA damage?
Pol 3
What is transletion synthesis?
Synthesis past damage
Describe the TLS pathway in E.coli
- Pol 3 moves along DNA using the B clamp
- When it reaches the damage it is blocked so it changes to Pol 5
- Pol 5 passes the damage, incorporating either correct or incorrect nucleotide
- But pol 5 is not effective at replication so pol 3 eventually comes back to continue its job
What happens as a result of a umuDC (pol5) mutation?
= No pol 5 = less mutations because normally pol 5 would add the wrong base as it wouldn’t recognize the damage
If pol 5 can add the wrong base, what is the point of it?
It happens so replication doesn’t just stop if there is some damage
List the Y family polymerases in mammalian cells
- Rev1
- Pol eta
- Pol kappa
- Pol iota
What are the properties of Y family polymerases?
- No proofreading of the DNA strands
- Low processivity
- Low fidelity (except Rev1)
What is pol eta good for?
CPD
What is the structure of Y family polymerases?
- Finger, palm and thumb domains
- Extra little finger domain
- Conserved catalytic domain at N terminus
- C terminus is for protein-protein interactions
