Flashcards in REPLICATION OF DAMAGED DNA Deck (46)
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?
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
Describe the model for recombination repair of daughter strand gaps in E.coli
1. Replication of a strand with CPD = replicated strand with gap due to damage (CPD)
2. Initiation of strand exchange by RecA helicase = filling in gap using the other strand (it will cross over ygm)
3. RecAFRO forms holiday junction to fill in the new gap left from the migration of the other strand moving up
4. RecA, RuvAB and RecG mediates branch migration and repair synthesis
5. Cleavage of holiday junction by RuvC
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?
1. During replication of damaged DNA, it gets locked at site of damage = exposure of SS DNA at replication fork
2. This leads to the formation of SS DNA RecA filaments
3. RecA catalyses cleavage/inactivation of LexA = increased RecA and umuDC
4. RecA also cleaves 24 amino acids at the N terminus of umuD = activate umuD
5. 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?
What is transletion synthesis?
Synthesis past damage
Describe the TLS pathway in E.coli
1. Pol 3 moves along DNA using the B clamp
2. When it reaches the damage it is blocked so it changes to Pol 5
3. Pol 5 passes the damage, incorporating either correct or incorrect nucleotide
4. 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
- 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?
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
What is the difference between the structure of Y family polymerases and other polymerases?
The catalytic domain has more of an open structure in the Y family
How can we increase sensitivity to UV in XPVs?
What is the effect of UV on XPV cells?
- Mildly sensitive to killing by UV but it is hyper-mutable
What are XPV cells normal in and what are they defective in?
- Normal NER
- Defective in replication of UV damaged DNA (post replication repair)
What is a symptom of XPV?
- Hypersensitive to sun induced pigmentation changes
- Skin cancer
What do diagnostic tests of XPV patients show?
- Normal UDS
- Normal cell survival after UV
- Reduced cell survival after caffeine and UV
Describe the relationship between pol eta and zeta
- Pol zeta is a good extender for replication for most lesions but it isn't good for CPDs
- Therefore, pol eta is needed because it can do both insertion and extension
Describe the ubiquitin pathway?
1. Ub is activated by E1 = UbE1
2. E1 gets swapped for E2 = UbE2
3. E3 ligase catalyses transfer of Ub to target protein (PCNA)
What genes are required for post replication repair?
Rad 6 and Rad 18
What genes are involved in the error free branch?
MMS2, UBC13 and RAD5
Name 3 E2 ubiquitin conjugating enzymes
RAD6 and UBC13MMS2
What are RAD18 and RAD5?
E3 ubiquitin ligases
Describe the polymerase switch pathway?
1. Pol eta has a tail with PIP box and UBZ = weakly bound to PCNA via PIP (Pol delta is still attached)
2. Pol delta will come across damage and get blocked which will activate Rad6 and Rad18
3. Rad6 and Rad18 ubiquitinates PCNA on lys164 on the UBZ of pol eta = now tightly bound via both domains and is able to displace pol delta
What is PCNA and what does it do?
It is the ukaryotic sliding clamp which holds DNA pol onto the DNA.
Describe the structure of PCNA
- It is a homotrimer.
- Has an interdomain connecting loop which PIP box proteins bind to.
- It has a whole in the middle for DNA
Describe the structure of Y family pols
All have a tail with PIP box and UBZ
Describe 1 outcome after polymerase switch
Translesion synthesis will happen, pol eta will replicate a few bases then pol will switch back to delta
Describe the other outcome after polymerase switch
Template switch damage avoidance process via poly-ubiquitination on lys63 via E2 and E3 enzymes (MMS2UBC13 and RAD5)
Why do you get more mutations in XPVs?
Because XPV patients don't have pol eta so other Y family polymerases comes in which make more errors, leading to mutations
What are the 3 possible outcomes of UV mutagenesis in E.coli?
2. Homologous recombination
3. Inaccurate TLS polymerase
What is the fate of UVRABC- cells in E.coli?
They cannot do NER, therefore they generate more mutations via TLS to try and correct the damage