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DNA Damage, Mutations, and Repair

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

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DNA Damage, Mutations, and Repair

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  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 • Frameshift mutations • Deletion of one or more base pairs • Insertion of one or more base pairs

  3. NH 2 Spontaneous mutations due to DNA polymerase errors • Very low rate of misincorporation (1 per 108 - 1 per 1010) • Errors can occur due to the presence of minor tautomers of nucleobases. NH N N N HN O N N C Rare imino tautomer of A amino 10-4 Normal base pairing Mispairing

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

  5. Induced mutations result from DNA damage • Sources of DNA damage: endogenous • 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. Normal base pairing in DNA and an example of mispairing via chemically modified nucleobase A G T T G A G C

  7. DNA oxidation Reactive oxygen species: HO•, H2O2, 1O2, LOO• • 10,000 oxidative lesions/cell/day in humans

  8. O N H H N G A N N H N N N O Deamination N H O 2 N N N N H H y p o x a n t h i n e N N N N A O O N N N H N H X a n t h i n e N N N O N N H G 2 H N H O 2 N N H U r a c i l N O N O 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. Direct repair: O6-alkylguanine DNA alkyltransferase (AGT) • Directly repaires O6-alkylguanines(e.g. O6-Me-dG, O6-Bz-dG) • In a stoichiometric reaction, the O6 alkyl group is transferred to a Cys residue in the active site. The protein is inactivated and degraded.

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

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

  17. O N N H X a n t h i n e N N O H 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

  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: • Recognize parental DNA strand (correct base) and daughter • strand (incorrectbase) • 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 Pigmentosum NER Hereditary nonpolyposis MMR colorectal cancer Cockrayne’s syndrome NER Falconi’s anemia DNA ligase Bloom’s syndrome BER, 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. Direct repair: O6-alkylguanine DNA alkyltransferase (AGT) • Directly repaires O6-alkylguanines(e.g. O6-Me-dG, O6-Bz-dG) • In a stoichiometric reaction, the O6 alkyl group is transferred to a Cys residue in the active site. The protein is inactivated and degraded.

  23. AGT inhibitor O6-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 O6-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: • Recognize damage. • Remove damage by excising part of one DNA strand. • The resulting gap is filled using the intact strand as the template. • Ligate the nick.

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

  28. X a n t h i n e Base excision repair (BER) • 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 O N N H N N O H

  29. Uracil DNA glycosylase removes deaminated C No Me group N H O 2 BER C N N H N O N O Cytosine Not normally present in DNA U r a c i l 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: • Recognize parental DNA strand (correct base) and daughter • strand (incorrectbase) • 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 Pigmentosum NER Hereditary nonpolyposis MMR colorectal cancer Cockrayne’s syndrome NER Falconi’s anemia DNA ligase Bloom’s syndrome BER, ligase Lung cancer (?) BER

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