DNA Repair Lecture 9 PDF

BIOL2010 DNA repair Lecture 9 Dr M.L.Bellamy Overview 1) Sources and types of DNA damage 2) Repair mechanisms Repair mechanisms Errors in sequence must first be detected by scanning the DNA Checking if it’s: → Not a standard DNA nt → The wrong DNA nt for the base pair Specia...

BIOL2010 DNA repair Lecture 9 Dr M.L.Bellamy Overview 1) Sources and types of DNA damage 2) Repair mechanisms Repair mechanisms Errors in sequence must first be detected by scanning the DNA Checking if it’s: → Not a standard DNA nt → The wrong DNA nt for the base pair Specialised proteins either directly reverse the error, or edit the DNA to reset that section of sequence Repair mechanisms Deamination, Replication Alkylation Oxidative damage UV light errors X-rays O6-MeG, U, 8-oxo-G, Pyrimidine Mismatches Double-strand 3-MeA inosine dimers breaks Direct BER Direct MMR Non- reversal reversal, homologous NER end joining → Ideally, repair occurs before the next round of replication, or mutations will be locked in  Repair mechanisms p53 P53 Tumour suppressor Responds to/detects DNA damage → Arrests the cell cycle at the end of G1, which allows time for repair → Activates DNA repair proteins If the damage is too severe, it triggers apoptosis or senescence  Repair mechanisms Sequence repair 1) Direct reversal/repair 2) Base Excision Repair (BER) 3) Nucleotide Excision Repair (NER) 4) Mismatch Repair (MMR) Sequence repair 1 Direct 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 Sequence repair 1 Direct 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 Sequence repair 1 Direct Demethylation 6 1 Sequence repair 1 Direct Photolysis of dimers (photoreactivation) DNA photolyase Absorbs blue light and breaks T-T internucleotide bonds, using FADH → 2 Ts restored T T T T Mammals have to use another system for repairing thymine dimers, as they don’t have this enzyme Sequence repair 2 BER Base excision repair 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 Deaminated A Deaminated C (now U) Deaminated methyl-C (now T) Oxidised Alkylated Opened ring Double bond loss Sequence repair 2 BER Base excision repair Glycosylases flip out bases for closer checking If the base is found to be incorrect, the sugar-base bond is cleaved, but UDGase remains attached to DNA Also responsible for leading (Uracil-N-glycosylase) strand fragments Sequence repair 2 BER Base excision repair How does UDGase discriminate between U and T? → Steric clash of methyl group on T with Tyr residue Asp145 Asp145 Tyr147 Tyr147 U T Phe158 Phe158 There is a separateTDGase for GT pairs Sequence repair 2 BER Base excision repair How does UDGase discriminate between U and T? → Steric clash of methyl group on T with Tyr residue If Tyr147 is mutated to Ala, both T and U will be removed by the enzyme Sequence repair 2 BER Base excision repair U in DNA U Base removed by Uracil-N-glycosylase Baseless nt recognised and phosphodiester backbone cleaved by Also works following AP(apyrimidinic) endonuclease depurination events Nicked DNA Pol I nick translation restores T Pol β in + DNA ligase seals nick eukaryotes Sequence repair 3 NER Nucleotide excision repair Removal of oligonucleotide fragments from one strand (bulk correction) Triggered by changes in the physical structure of the duplex as a result of damage Corrects any of the types ofbenzopyrene Adduct of sequencewith damage Adenine e.g. thymine dimer removal Achieved by a protein complex called UvrABC exinuclease in E.coli (many more proteins in eukaryotes) Sequence repair 3 NER Nucleotide excision repair Heterotrimer Sequence repair 3 NER Nucleotide excision repair (a fragment of ~12 nt) Sequence repair 4 Mismatch repair Detection and removal of incorrect base pairs Principle:  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 Sequence repair 4 MMR Methyl-directed Mismatch Repair (E.coli) Hemi-methylation provides the information on which strand is parent (correct) and daughter 1. MutH binds to unmethylated GATC at OriC, identifying the daughter strand. MutH -Me Sequence repair 4 MMR Methyl-directed Mismatch Repair (E.coli) 2. MutS binds to a distorted site on the duplex 3. MutL binds to MutS MutS MutH MutL -Me Sequence repair 4 MMR Methyl-directed Mismatch Repair (E.coli) 4. MutL/MutS complex travels back to the origin and activates MutH 5. MutH cleaves daughter strand (nicked) MutS 6. Specialized helicase (UvrD) MutH MutL and exonucleases remove nt until -Me past the distortion 7. Pol III fills in missing nt. DNA ligase seals nick. Sequence repair 4 MMR Mismatch Repair (Eukaryotes) Eukaryotes have several homologues of MutL and MutS e.g. MLH1-5 and MSH1-6 No homologues of MutH, but they don’t use hemimethylation replication tags either Sequence repair 8-oxo-G Multi-pronged approach (E.coli) Additional Mut proteins: 1. MutT recognises 8-oxo-GTP and hydrolyses it 2. MutM recognises 8-oxo-G in DNA and removes it, via BER 3. MutY recognises 8-oxo-G opposite A in DNA and removes the A, via BER Sequence repair Summary Error rate of polymerase: 10-5 Error rate with proofreading: 10-7 Error rate with repair: 10-10 →There are 1000x fewer mutations because of repair systems Sometimes a risk of mutation is better than the alternative, so there are polymerases that can add nt where processive, proof-reading polymerases cannot →Translesion synthesis e.g. Pol IV&V in E.coli and at least 8 Pols in eukaryotes → Blockage is bypassed, but the nt added may not be correct Insights from disease Xeroderma Pigmentosum (XP) Individuals show dry, parchment-like skin (xeroderma) and many freckles (pigmentosum) Increased sensitivity to UV light 1000-fold increased risk of skin cancer → Due to inherited defects in one of eight distinct genes responsible for components of the NER complex Insights from disease Hereditary non-polyposis colon cancer (HNPCC) Individuals exhibit a predisposition to colon cancer (2-3% of all colon cancer cases) Leads to the accumulation of mutations throughout the genome → Due to defects in the human equivalents of the MutS/L MMR system (MSH2 and MLH1) Lung cancer genome Comparison of lung cancer genome to normal genome Rearrangements: 58 Point mutations: 22,910 ~1 mutation per 15 cigarettes Nature 463, 184-190