1. DNA Damage, Mutations, and Repair See Stryer p. 768-773

2. DNA Mutations 1. Substitution mutations: one base pair for another, e.g. T for G the most common form of mutation transitions; purine to purine and pyrimidine to pyrimidine transversions; purine to pyrimidine or pyrimidine to purine 2.Frameshift mutations Deletion of one or more base pairs Insertion of one or more base pairs

3. Rare imino tautomer of A N N NH 2 O HN N N N NH C Very low rate of misincorporation (1 per 10 8 - 1 per 10 10 ) Errors can occur due to the presence of minor tautomers of nucleobases. Spontaneous mutations due to DNA polymerase errors Normal base pairingMispairing 10 -4 amino

4. A(imino) T ATAT A(imino) C ATAT GCGC Final result: A  G transition (same as T  C in the other strand) Consider misincorporation due to a rare tautomer of A ATAT 1 st replication 5’ 3’ Normal replication 2 nd replication

5. Induced mutations result from DNA damage Sources of DNA damage: endogenous 1.Deamination 2.Depurination: 2,000 - 10,000 lesions/cell/day 3. Oxidative stress: 10,000 lesions/cell/day Sources of DNA damage: environmental 1. Alkylating agents 2. X-ray 3. Dietary carcinogens 4. UV light 5. Smoking

6. G  A G C G T A T Normal base pairing in DNA and an example of mispairing via chemically modified nucleobase

7. DNA oxidation Reactive oxygen species: HO, H 2 O 2, 1 O 2, LOO 10,000 oxidative lesions/cell/day in humans

8. N N N N NH 2 N NH N N O NH 2 N N NH 2 O N NH N N O N NH N H N O O NH N O O Hypoxanthine Xanthine Uracil N NH N N O N N NH O H A G Deamination A G C C Rates increased by the presence of NO (nitric oxide)

9. Depurination to abasic sites 2,000 – 10,000/cell/day

10. UV light-induced DNA Damage …CC… Pyrimidine dimer Easily bypassed by Pol  (eta) in an error-free manner

11. Deletions and insertions can be caused by intercalating agents Stryer Fig. 27.44

12. Importance of DNA Repair DNA is the only biological macromolecule that is repaired. All others are replaced. More than 100 genes are required for DNA repair, even in organisms with very small genomes. Cancer is a consequence of inadequate DNA repair.

13. DNA Repair Types Direct repair –Alkylguanine transferase –Photolyase Excision repair –Base excision repair –Nucleotide excision repair –Mismatch repair Recombination repair

14. Directly repaires O 6 -alkylguanines (e.g. O 6 -Me-dG, O 6 -Bz-dG) n In a stoichiometric reaction, the O 6 alkyl group is transferred to a Cys residue in the active site. The protein is inactivated and degraded. Direct repair: O 6 -alkylguanine DNA alkyltransferase (AGT)

15. Excision Repair Takes advantage of the double-stranded (double information) nature of the DNA molecule. Four major steps: 1.Recognize damage. 2.Remove damage by excising part of one DNA strand. 3.The resulting gap is filled using the intact strand as the template. 4.Ligate the nick.

16. Antiparallel DNA Strands contain the same genetic information A :: G ::: T :: T C A 3' 5' A :: G T :: T A 3' 5' A :: G ::: T :: T C A 3' 5' Original DNA duplexDNA duplex with one of the nucleotides removed Repaired DNA duplex

17. Base excision repair (BER) Used for repair of small damaged bases in DNA (AP sites, methylated bases, oxidized bases…) Human BER gene hogg1 is frequently deleted in lung cancer N NH N H N O O Xanthine

18. Nucleotide Excision Repair Corrects any damage that both distorts the DNA molecule and alters the chemistry of the DNA molecule (pyrimidine dimers, benzo[a]pyrene-dG adducts, cisplatin-DNA cross-links). Xeroderma pigmentosum is a genetic disorder resulting in defective NER

19. Mismatch Repair Enzymes Nucleotide mismatches can be corrected after DNA synthesis! Repair of nucleotide mismatches: 1.Recognize parental DNA strand (correct base) and daughter strand (incorrect base) Parental strand is methylated: 2. Replace a portion of the strand containing erroneous nucleotide (between the mismatch and a nearby methylated site –up to 1000 nt)

20. Genetic diseases associated with defective DNA repair Xeroderma PigmentosumNER Hereditary nonpolyposis MMR colorectal cancer Cockrayne’s syndromeNER Falconi’s anemiaDNA ligase Bloom’s syndromeBER, ligase Lung cancer (?)BER

21. DNA Repair Types Direct repair –Alkylguanine transferase –Photolyase Excision repair –Base excision repair –Nucleotide excision repair –Mismatch repair Recombination repair

22. Directly repaires O 6 -alkylguanines (e.g. O 6 -Me-dG, O 6 -Bz-dG) n In a stoichiometric reaction, the O 6 alkyl group is transferred to a Cys residue in the active site. The protein is inactivated and degraded. Direct repair: O 6 -alkylguanine DNA alkyltransferase (AGT)

23. AGT inhibitor O 6 -benzylguanine is in clinical trials to be used in conjunction with antitumor alkylnitrosoureas

24. AGT overexpression in tumors makes them resistant to alkylnitrosoureas

25. Combination therapy with O 6 -benzylguanine overcomes tumor resistance to alkylnitrosoureas

26. Excision Repair Takes advantage of the double-stranded (double information) nature of the DNA molecule. Four major steps: 1.Recognize damage. 2.Remove damage by excising part of one DNA strand. 3.The resulting gap is filled using the intact strand as the template. 4.Ligate the nick.

27. Antiparallel DNA Strands contain the same genetic information A :: G ::: T :: T C A 3' 5' A :: G T :: T A 3' 5' A :: G ::: T :: T C A 3' 5' Original DNA duplexDNA duplex with one of the nucleotides removed Repaired DNA duplex

28. Used for repair of small damaged bases in DNA (AP sites, methylated bases, deaminated bases, oxidized bases…) Human BER gene hogg1 is frequently deleted in lung cancer Base excision repair (BER) N NH N H N O O Xanthine

29. Uracil DNA glycosylase removes deaminated C N N NH 2 O NH N O O Uracil BER C Not normally present in DNA No Me group Cytosine Normal DNA base Not recognized by BER

30. Nucleotide Excision Repair Corrects any damage that both distorts the DNA molecule and alters the chemistry of the DNA molecule (pyrimidine dimers, benzo[a]pyrene-dG adducts, cisplatin-DNA cross-links). Xeroderma pigmentosum is a genetic disorder resulting in defective NER

31. Mismatch Repair Enzymes Nucleotide mismatches can be corrected after DNA synthesis! Repair of nucleotide mismatches: 1.Recognize parental DNA strand (correct base) and daughter strand (incorrect base) Parental strand is methylated: 2. Replace a portion of the strand containing erroneous nucleotide (between the mismatch and a nearby methylated site –up to 1000 nt) G T

32. Genetic diseases associated with defective DNA repair Xeroderma PigmentosumNER Hereditary nonpolyposis MMR colorectal cancer Cockrayne’s syndromeNER Falconi’s anemiaDNA ligase Bloom’s syndromeBER, ligase Lung cancer (?)BER