DNA Repair Lectures (BIOL2010) - PDF

BIOL2010 DNA repair Lectures 8-9 Dr M.L.Bellamy Overview 1) Sources and types of DNA damage 2) Repair mechanisms DNA damage Types DNA damage Sources Spontaneou s Induced Replication errors Intercalating agents Tautomerisation...

BIOL2010 DNA repair Lectures 8-9 Dr M.L.Bellamy Overview 1) Sources and types of DNA damage 2) Repair mechanisms DNA damage Types DNA damage Sources Spontaneou s Induced Replication errors Intercalating agents Tautomerisation Base analogues Deamination Deaminating agents Depurination Alkylating agents Oxidising agents Radiation, UV light DNA damage Spontaneous 1. Replication errors Normal replication introduces the wrong base once every 107 bp, even with proofreading → Good chance it will get repaired Repair systems reduce error rate to 10-10 Some repetitive regions cause slippage of the growing strand and insertion of more repeats → Can't be repaired  DNA damage Spontaneous 1. Replication errors Insertions due to strand slippage 5’T A C G G A T G A A A A 3’A T G C C T A C T T T T T C G C G A Strand slips out Strand re-anneals incorrectly A 5’T A C G G A T G A A A 3’A T G C C T A C T T T T T C G C G A Extra A added→frame shift A 5’T A C G G A T G A A A A A G C G C T 3’A T G C C T A C T T T T T C G C G A Particularly prevalent with trinucleotide repeats e.g. CAG in polyglutamine diseases like Huntington’s disease DNA damage Spontaneous 2. Tautomerisation (tautomeric shifts) O O CH3 CH3 HN HN T T O N O N KETO ENOL KETO 99.99% 0.01% Shifted (enol) form binds G instead of A DNA damage Spontaneous 2. Tautomerisation (tautomeric shifts) AMINO A KETO G KETO T AMINO C T C A G IMINO A ENOL G ENOL T IMINO C C T G A Mutation consolidated on next replication DNA damage Spontaneous 3. Deamination Loss of base amino group NH2 H2O O N HN C Hydrolysis Deamination U + NH3 O N O N ~100 C→U/cell/day U pairs with A A and G ~1/cell/day DNA damage Spontaneous 3. Deamination Loss of base amino group C Uracil (pairs A) A Inosine/Hypoxanthine (pairs C) G Xanthine (pairs C less strongly) C, A and G deaminations can be repaired 5-Methyl-C → T which can't be repaired DNA damage Spontaneous 4. Depurination (A or G)Cleavage of base-sugar bond Forms an abasic site (apurinic) A =T ~10,000 purine glycosidic bonds G A hydrolyse/cell/day T= A ~600 pyrimidine glycosidic bonds CG hydrolyse/cell/day A= T 4x more likely in ssDNA Mutation consolidated on next replication DNA damage Induced Mutagens – Ames test DNA damage Induced Mutagens Chemical #1 - Intercalating agents Insert themselves between bases e.g. ethidium bromide  NH2 N+ H2N DNA damage Induced Mutagens Chemical #1 - Intercalating agents Insert themselves between bases e.g. ethidium bromide  Insertion Deletion...GATTACA......GATTACA......CTAGATGT......CTATGT...  ...CTAGATGT......CTATGT......GATCTACA......GATACA... FRAMESHIFT MUTATIONS DNA damage Induced Mutagens Chemical #2 – Base analogues e.g. bromouracil (T analogue) O O Br CH3 HN HN BrU T O N O N Incorporated into DNA More prone to tautomeric shifts DNA damage Induced Mutagens Chemical #3 - Alkylating agents Add alkyl groups to nucleobases e.g. Nitrosamines Methyl bromide (former grain fumigant) e.g. G→O6-methyl G (pairs T) CH3 G Alkylation can also speed up depurination DNA damage Induced Mutagens Chemical #4 - Deaminating agents Remove amino groups e.g. Nitrous acid (HNO2) Nitrosamines Nitrite, Nitrate Far quicker than spontaneous deamination DNA damage Induced Mutagens Chemical #5 - Oxidising agents e.g. superoxide ion (O2-), H2O2 Cause of most mutations Many possible nucleotide alterations OH + G 8-oxo G Base pairs A H H O O N H H H N O H N H N H N N N N N H G H O N A N N N N N N N N H N N dR H dR H dR dR O DNA damage Guanine OXIDATION ALKYLATION DEAMINATION DNA damage Induced Mutagens Physical - UV light UV-induced formation of dimers between adjacent thymines O O NH NH R N T O R N T O O O CH3 CH3 NH NH R N T O R N T O CH3 CH3 Stops correct base-pairing in polymerases DNA damage Induced Mutagens Physical - Radiation, γ & X rays, UV Break bonds & create free radicals Single or double-strand breaks Bases chemically altered, linked or detached Leading source of mutation Number of mutations α radiation dose DNA damage Summary Common DNA lesions and pathways Deamination, Oxidative Replication UV light errors γ/X-rays Alkylation damage O6-MeG, U, 8-oxo-G, Pyrimidine Mismatches Double- 3-MeA inosine dimers strand breaks DNA damage Rates Estimated rates of DNA damage per human cell per day: Depurination 10,000 Deamination 600 Oxidative base damage 2000 Alkylated bases 5000 Intra-strand cross links 10 Single strand breaks 50,000 DNA double-strand break 10 (caused by ionising radiation) Total DNA damaging events per cell per day: 60,000 Total DNA damaging events per cell per hour: 2,500 Estimate 1013 - 1014 cells in human body ~ 2.5 x 1016 DNA damaging events during this lecture! Repair systems Deamination, Replication X-rays Alkylation Oxidative damage UV light errors O6-MeG, U, 8-oxo-G, Pyrimidine Mismatches Double-strand 3-MeA inosine dimers breaks Direct BER Direct MMR Non- reversal reversal, homologous end joining NER Molecule repair Double-strand breaks Two repair methods: 1) Homologous recombination Fragments of a damaged Holliday junction chromosome can be aligned to, and cross-over with, its homologue Two Holliday junctions form (one for each end of the break) DNA synthesis and ligation corrects the break Molecule repair Double-strand breaks Two repair methods: 2) Non-Homologous end-joining (NHEJ) Ku proteins bind the two broken ends of duplex fragments, and then to each other. DNA ligase IV is recruited to the join. It can ligate both strands at once – but it does it blindly, with loss of some nt. → Risk of deleterious mutation Repair systems Deamination, Replication X-rays Alkylation Oxidative damage UV light errors O6-MeG, U, 8-oxo-G, Pyrimidine Mismatches Double-strand 3-MeA inosine dimers breaks Direct BER Direct MMR Non- reversal reversal, homologous end joining NER