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DNA Damage and Repair

DNA Damage and Repair. Why do we care? Genetic diseases Cancer. Cellular Responses to DNA Damage. Reversal of DNA Damage Enzymatic photoreactivation Ligation of DNA strands Repair of photoproduct Tolerance of DNA Damage

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DNA Damage and Repair

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  1. DNA Damage and Repair • Why do we care? • Genetic diseases • Cancer

  2. Cellular Responses to DNA Damage • Reversal of DNA Damage • Enzymatic photoreactivation • Ligation of DNA strands • Repair of photoproduct • Tolerance of DNA Damage • Replicative bypass of template damage with gap formation and recombination (gap repair) • Excision of DNA Damage • Base excision repair • Nucleotide excision repair • Mismatch repair

  3. Mutagens and Carcinogens • Essentially all mutagens are carcinogens • Most carcinogens are mutagens

  4. Somatic vs. germ line mutations • Somatic mutations can lead to cancer • Germ line mutations can lead to birth defects • Most mutations cause neither • Some fall in non-coding DNA • Others are silent

  5. Types of mutations

  6. Types of substitutions • Missense • Results in an amino acid substitution • Nonsense • Results in a stop codon (TAG, TAA, TGA) • Same sense • No effect (silent mutation)

  7. Slippage

  8. Types of mutations • Multisite • Point mutations

  9. Multisite mutations • Cause gross chromosome abnormalities • Involve large regions of DNA • Arise during meiosis

  10. Types of multisite mutations • Inversions: ACBDEF • Duplications ABCDEEF • Deletions: ABCDF • Insertions: ABCDSEF • Substitutions: ATCDEF

  11. Point mutations • Involve only one or a few nucleotides • Arise during DNA replication • Require two errors • An error during DNA replication • Failure to correct that error

  12. Types of point mutations • Substitutions: GATC CATC • Insertion: GATC GGATC • Deletion: GATC GTC • Duplication: GATC GAGATC • Inversion: GATC GTAC

  13. What is the first defense against mutations? • 3’ to 5’ exonuclease activity of the polymerases

  14. Natural causes of mutations • Base tautomerization • UV damage • Spontaneous deamination

  15. Adenine tautomer

  16. Generation of a mutation by the adenine tautomer- About every 1 in 104 bases

  17. UV damage

  18. Spontaneous deamination • Three of the four bases have exocyclic amino groups • Adenosine produces hypoxanthine • Guanine produces xanthine • Cytosine produces uracil

  19. Hypoxanthine

  20. Deamination of cytosine produces uracil

  21. If replication occurs a mutation will result

  22. Removal of uracil from DNA

  23. Why do cells use thymine rather than uracil?

  24. Answer • The reason cells use thymine in their DNA • Is to allow recognition of uracil formed from cytosine • But what about RNA? • RNA is short lived and in many copies.

  25. Chemical mutagens • Chemicals that accelerate the deamination reaction • Base analogues • Alkylating agents • Intercalation agents

  26. Base analogues • 5-bromouracil • Goes in as T • Can base pair with A but also G to a smaller degree

  27. Generation of a mutation by 5-bromouracil

  28. Intercalation • Flat aromatic compounds • Acridine dyes • Ethidium bromide • Cause frame-shifting

  29. Repair mechanisms • We are exposed to mutagens all the time • you would expect repair mechanisms to exist • A number of different repair mechanisms do exist

  30. Repair Mechanisms • In mismatch repair • Incorrect base is identified • On short section of a newly synthesized DNA • Removed, and replaced • by DNA synthesis directed by the correct template. • In excision repair • bulky lesions in DNA • exposure to UV light • removed by specialized nuclease systems • DNA polymerase fills gap • DNA ligase joins the free ends.

  31. DNA Mismatch Repair

  32. Intro to DNA Mismatch Repair • Mismatch Repair Genes • recognition and repair of certain types of DNA damage or replication errors • Function to help preserve the fidelity of the genome • through successive cycles of cell division

  33. Mismatch repair • Occurs just after replication • Improves accuracy 102 - 103 fold • Must distinguish the parent from the daughter strand

  34. History of MMR • System first discovered in bacteria • Partially homologous system in yeast • Marked homology between yeast and higher order organisms • Human MMR genes first described 1993.

  35. DNA Mismatches • Damage to nucleotides in ds-DNA • Misincorporation of nucleotide • Missed or added nucleotides

  36. Acquired DNA Damage M -C-A- -T-A- -G-T- -G-T- Demethylation

  37. Nucleotide Misincorporation -C-A-G-C-T- -G-T-C-C-A- CT substitution -C-A-G-C-T- -G-T-T-C-A- -C-A-G-C-T- -G-T-C-C-A- -C-A-G-C-T- -G-T-C-C-A- correctly copied

  38. nucleotide added -C-A-G-C-T- -G-T-C C-A- -C-A-G-C-T- -G-T-C-C-A- A correctly copied Added Nucleotides -C-A-G-C-T- -G-T-C-C-A- -C-A-G-C-T- -G-T-C-C-A-

  39. Mismatch Repair Genes • Recognition and repair of mismatches • Other functions • Repair of branched DNA structures • Prevent recombination of divergent sequences • Direct non-MMR proteins in nucleotide excision and other forms of DNA repair • MSH4 & MSH5 involved (with MLH1) in meiotic crossover

  40. Human Mismatch Repair Genes • MLH1 (3p21) • PMS1 (2q31-33) • PMS2 (7p22) • MSH2 (2p16) • MSH3 (5q3) • MSH6 (2p16) (=GT Binding Protein)

  41. Mismatch Repair Function • MMR Proteins combine as heterodimers • Recognise and bind mismatches • ATP consumption • Recruit other proteins • Separate, destroy and resynthesise new DNA strand • Mechanism works for up to 20 base pairs

  42. MSH Protein Complexes • MutS (MSH2-MSH6) • GT mispairs and short (1 base pair) loops/deletions • MutS (MSH2-MSH3) • Larger mispair loops and deletions • Some overlap in function • MSH2 loss is greater cancer risk

  43. MLH Protein Complexes • MutL (MLH1-PMS2) • MutL (MLH1-PMS1) • No established function • Can bind other MMR proteins, MSH heterodimers and replication factors • As for MSH2, overlap means loss of MLH1 confers the greater cancer risk

  44. Other MMR Proteins • DNA ligase • Replication protein A • Replication factor C • Proliferating Cell Nuclear Antigen • Exonucleases • DNA polymerase 

  45. Defective Mismatch Repair • Defects in MMR Genes and Function • Microsatellite Instability • Cancer development

  46. Defects in MMR Genes • Control sequences  Nonexpression • Premature stop codon  Truncated protein • Point mutations  Altered sequence • Insertions/Deletions  Frameshift effects • Somatic loss of second allele

  47. Microsatellite Instability • Simple nucleotide repeat sequences • Length should be stable at any one locus • Poly-A and poly-CA repeat sequences particularly prone to mismatch errors • Alterations in length are a sign of deficient mismatch repair • Also called RER (Replication ERror)

  48. Microsatellite Instability -C-A-C-A-C-A-C-A- -G-T-G-T-G-T-G-T- shortened repeat -C-A-C-A-C-A- -G-T-G-T-G-T -C-A-C-A-C-A-C-A -G-T-G-T-G-T-G-T -C-A-C-A-C-A- -G-T-G-T G-T- CA skipped G-T heteroduplex results

  49. MI Positive Tumours • 90% of HNPCC colorectal cancers • 20% of sporadic colorectal cancers • 30% of sporadic uterine cancers

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