MB - DNA Repair II Flashcards
What are the two options for repairing DNA damage?
Damage can be directly repaired
Damage can’t be directly repaired
- Remove and replace the affected DNA
When is it best to repair DNA damage?
Before the next round of replication, or mutations will be locked in
What are the types of Sequence repair (4) and Molecule repair (2)?
Sequence repair
1) Direct reversal/repair
2) Base Excision Repair (BER)
3) Nucleotide Excision Repair (NER)
4) Mismatch Repair (MMR)
Molecule repair
1) Homologous recombination
2) Non-homologous end repair
How does Direct Reversal repair work?
Where the damage has converted A,C,G,T to something else, it may be possible to convert back to the original nt
- e.g. Demethylation following alkylation
- e.g. Removal of crosslinks following UV light damage
How does demethylation and photolysis of dimers result in direct repair?
Demethylation
Specific reversal proteins
- e.g. O6-methylguanine-DNA methyltransferase (MGMT)
Transfers methyl/ethyl group from G to a Cys residue on itself
- G restored
Photolysis of dimers (photoreactivation)
DNA photolyase
Absorbs blue light and breaks T-T internucleotide bonds, using FADH → 2 Ts restored
- Mammals have to use another system for repairing thymine dimers,as they don’t have this enzyme
How does Base Excision Repair (BER) work?
Involves the removal of individual bases (local correction)
- Group of >6 DNA glycosylases which recognise abnormal bases and cleave them from the deoxyribose, creating an abasic site
Glycosylases flip out bases for closer checking
After base is cleaved, UDGase remains attached to DNA
How does UDGase discriminate between U and T?
Steric clash of methyl group on T with Tyr residue
How is uracil removed from DNA? (5)
- Base removed by Uracil-N-glycosylase (ung)
- Baseless nt recognised and phosphodiester backbone cleaved by Ap (apyrimidinic) endonuclease
- Nicked DNA
- Pol 1 nick translation restores T
- DNA ligase seals nick
How does Nucleotide Excision Repair (NER) work?
Removal of oligonucleotide fragments from one strand
- Triggered by changes in the physical structure of the duplex as a result of damage
Achieved by a protein complex called UvrABC exinuclease in E.coli (mechanism goes into greater detail)
How does Mismatch Repair (MMR) work?
Detection and removal of incorrect base pairs
- A mismatched pair will distort the helix
- This can be detected by specialist proteins
- Incorrect base removed
→ Must somehow know which one of the two is the wrong one: strand-directed mismatch repair
How does Methyl-directed Mismatch Repair work in E.coli? (7)
Hemi-methylation provides the information on which strand is parent (correct) and daughter
- MutH binds to unmethylated GATC at OriC, identifying the daughter strand.
- MutS binds to a distorted site on the duplex
- MutL binds to MutS
- MutL/MutS complex travels back to the origin and activates MutH
- MutH cleaves daughter strand (nicked)
- Specialized helicase and exonucleases remove nt until past the distortion
- Pol III fills in missing nt. DNA ligase seals nick
How does Multi-pronged approach work in E.coli? (3)
- MutT recognises 8-oxo-GTP and hydrolyses it
- MutM recognises 8-oxo-G in DNA and removes it, via BER
- MutY recognises 8-oxo-G opposite A in DNA and removes the A, via BER
What is homologous recombination and non-homologous end-joining (NHEJ)?
1) Homologous recombination
- Following replication, while the sister chromatids are still joined one can be used as a template to repair the other
2) Non-homologous end-joining (NHEJ)
- A protein complex binds the naked ends of duplex fragments and recruits DNA ligase IV, which can ligate both strands – but it does it blindly, to any two pieces of DNA, with loss of some nt
NER Mechanism (6)
- UvrA and UvrB bind to thymine dimer
- UvrA subunits dissociate
- UvrC subunit binds
- UvrB and UvrC migrate ~5 nts away and cleave DNA
- UvrB and UvrC dissociate
- UvrD helicase displaces the damaged DNA
