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DNA Damage and Repair PowerPoint Presentation
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DNA Damage and Repair

DNA Damage and Repair

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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