DNA damage and repair

1. DNA damage and repair •Types of damage •Direct reversal of damage •Excision repair in prokaryotes and eukaryotes base excision nucleotide excision •Nonhomologous end-joining in eukaryotes •Mismatch repair •Recombination repair, error-prone bypass and error-free bypass

2. DNA damage vs. mutation •DNA damage refers to a chemical alteration of the DNA (e.g. G-C bp to methyl-G-C is DNA damage) •Mutation refers to a change in a base-pair (e.g. G-C bp to A-T bp is a mutation) •Problems arise when DNA damage is converted to mutation

3. Most inherited syndromes in humans are due to mutations •Whether a syndrome occurs or not depends on where the mutation occurs and how a protein altered by the mutation is affected •Mutation may cause a protein: -to be non-functional -to have an altered function -to act less efficiently -to function as the wild type

4. Causes of gene mutations •Spontaneous -errors by DNA Polymerases during replication can lead to base changes -slipped strand mispairing can occur at homopolymeric runs (mono, di, or trinucleotide repeats) -chemical modification of bases followed by mispairing •Exposure to mutagens -ionizing radiation -UV radiation

5. Slipped Strand Mispairing Normal replication Backwards slippage causes insertion Forwards slippage causes deletion Levinson and Gutman, Nature 322: 652-656, 1987

6. Spontaneous deamination of C gives rise to U, and spontaenous deamination of 5-methylC gives rise to T.

7. Sponaneous deamination of A gives rise to hypoxanthine which can base-pair with C (but with 2 H-bonds instead of 3). Cytosine Hypoxanthine deamination

8. Alkyation highly mutagenic (forms a “noncoding base”) Alkyation “harmless” Electron rich centers in DNA susceptible to electrophilic attack

9. Alklyation of guanine by EMS leads to base-pairing with thymine

10. Pyrimidine dimers

11. Model for Photoreactivation

12. Mechanism of O6-methylguanine methyl transferase activity O6-methylguanine methyl transferase is a “suicide enzyme.” It is irreversibly inactivated after activity.

13. Base excision repair in E. coli

14. The human BER pathway DNA pol b APE1 DNA pol b APE1 APE1=apurinic/apyrimidinic endonuclease

15. Nucleotide excision repair in E. coli

16. Human global genome NER Xeroderma pigementosum

17. Model for nonhomologous end-joining Ku heterodimer (Ku70 and Ku80) DNA-PKcs

19. Mismatch Repair in Prokaryotes •Occurs when DNA Polymerase puts in the wrong nucleotide during replication and the proofreading activity does not correct it. •Repair should occur on the correct strand, the newly synthesized strand. •E. coli methylates A of GATC sequence. •There is a time lapse before newly synthesized strand is methylated. •Repair occurs on unmethylated (newly synthesized) strand during this window of time.

20. Mismatch repair in E. coli

21. Mismatch Repair in Eukaryotes •Eukaryotes are also capable of mismatch repair. •Less well understood than prokaryotes. •Homologues of mutS and mutL genes exist so enzymes involved in eukaryotic mismatch repair likely to be similar to prokaryotic enzymes. •BUT, no homologue of MutH (protein that recognizes unmethylated newly synthesized strand) so recognition of newly synthesized strand does not appear to occur via a methylation signal. •Failure of mismatch repair in humans can lead to hereditary nonpolyposis colon cancer (HNPCC)

22. Recombination repair in E. coli

23. Error prone SOS bypass in E. coli

25. Reversion of ochre his- mutation in E. coli umuC- + muc+ umuC+ umuC-

26. Error-prone and error-free bypass in humans •Error-prone repair: DNA Pol z (zeta) inserts bases at random to get by pyrimidine dimers •Relatively error-free bypass: DNA Pol h (eta) inserts two dAMPs across from pyrimidine dimers which are often (but not always) T-T dimers •The two A’s cannot base pair though because the two T’s are still joined together •DNA Pol h cannot synthesize more DNA after adding the two dAMPs •Another polymerase continues….

27. Activities of DNA polymerases alpha and eta on damaged and undamaged bases