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DNA REPAIR

DNA REPAIR. DNA is the only biological molecule that is repaired DNA damage Alteration to the chemical structure of DNA Mutation Change in the sequence of DNA. DNA REPAIR. NATURE OF DNA DAMAGE. Loss of bases Modification of bases Inter/intra-strand crosslinks

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DNA REPAIR

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  1. DNA REPAIR

  2. DNA is the only biological molecule that is repaired DNA damage Alteration to the chemical structure of DNA Mutation Change in the sequence of DNA DNA REPAIR

  3. NATURE OF DNA DAMAGE • Loss of bases • Modification of bases • Inter/intra-strand crosslinks • DNA strand breakage (single and double strand)

  4. CAUSES OF DNA DAMAGE • Endogenous factors Spontaneous - Errors in proofreading - Deamination of bases - Depurination/Depyrimidination Induced - Byproducts of normal cellular processes (reactive oxygen species etc ) • Exogenous factors - UV irradiation (sunlight) - High energy irradiation (x-rays) - Mutagenic chemicals (Mustard gas, cigarette smoke, food additives)

  5.  ERRORS IN PROOFREADING Incorporation of the wrong base/s resulting in mismatches Approximate error rate = 10-9

  6.  DEAMINATION May be spontaneous or induced by chemicals Cytosine  Adenine  Guanine  Thymine  Uracil Hypoxanthine Xanthine ??

  7.  DEAMINATION • Deamination leads to unusual base pairing in DNA - Uracil pairs with adenine - Hypoxanthine pairs with cytosine

  8. Parental strand C T U T C G A A A G DNA Replication C T C T C G A G A G Parental strand FAILURE TO REPAIR A DEAMINATED BASE = A POINT MUTATION Mutation Deamination New strand C T U C T C G A G A G New strand Unchanged

  9. C C T T C G A G G  DEPURINATION/DEPYRIMIDINATION • Cleavage of the glycosidic bond removes bases • Abasic (Apurinic/apyrimidinic, AP sites) • ~2000-10,000 purines lost per mammalian cell/24 hr

  10. FAILURE TO REPAIR ABASIC SITES = DELETIONS Parental strand C T C T C G A G A G New strand C C T T C DNA Replication G A G G New strand C T C C G G A G Parental strand Unchanged AP site Mutation

  11. REACTIVE OXYGEN SPECIES Generated during normal aerobic respiration • Superoxides, O2-, • Hydroxyl ions (OH.) • H2O2 Most biological damage by OH. Guanine 8-oxodG

  12.  Exogenous – UV IRRADIATION Dimerizes adjacent thymine residues. The dimer creates a kink in the DNA that blocks the progression DNA polymerase

  13.  HIGH-ENERGY RADIATION X-rays and gamma rays may directly break DNA strands and/or generate reactive oxygen species

  14.  Exogenous – CHEMICALS • Alkylating agents (e.g., mustard gas) • Add CH3/CH2CH3 groups to N and O groups of bases. • O6 of guanine particularly susceptible. 6-ethyl guanine acts as an analogue of adenine and pairs with thymine. • Polycyclic Hydrocarbons (cigarette smoke, exhaust fumes etc)

  15.  Exogenous – CHEMICALS • Food Additives • Nitrates and Nitrites • Metabolized to Nitronium ion/Nitrous acid • Chemotherapeutic drugs • Base Analogues (e.g. 5-bromouracil, 5BU) • Intercalating Agents • Acridine dyes (e.g., proflavin) • Interfere with DNA replication

  16. THE CELL CYCLE

  17. ☺ METHODS OF REPAIR ☺ Excision repair - Base excision -Nucleotide excision ☺ Mismatch repair ☺ Recombination repair

  18. EXCISION REPAIR Recognition of damage Removal of damage Resynthesis of gap Ligation

  19. EXCISION REPAIR • Two types of excision repairs • Base Excision Repair • Repair of methylated, deaminated, oxidized bases and AP sites. • Nucleotide Excision Repair • Repair of large adducts or distortion in the double helical structure of DNA (pyrimidine dimers, benzo(a)pyrene)

  20. BASE EXCISION REPAIR Glycosylase AP endonuclease AP Lyase DNA polymerase DNA ligase

  21. NUCLEOTIDE EXCISION REPAIR A A B Thymine dimer urvAB excinuclease urvC excinuclease A A B DNA polymerase DNA ligase No thymine dimer

  22. NER ASSOCIATED DISEASES • Xeroderma pigmentosum • Cockayne Syndrome • PIBIDS (photosensitivity, ichthyosis, brittle hair, impaired intelligence, decreased fertility, short stature) - Characterized by an increased sensitivity to sunlight

  23. Vignette 12 A 3-year-old boy, was referred to the dermatology clinic for evaluation of severe sun sensitivity and freckling. On physical examination, he was photophobic and had conjunctivitis and prominent freckled hyperpigmentation in sun-exposed areas; his development and physical examination were otherwise normal. The parents of the child revealed that they were first cousins; no one else in the family was similarly affected. The dermatologist explained that the boy had classic features of xeroderma pigmentosum (XP), that is, "parchment-like pigmented skin". To confirm the diagnosis, he had a skin biopsy to evaluate DNA repair and ultraviolet (UV) radiation sensitivity in his skin fibroblasts. The results of this testing confirmed the diagnosis of XP. Despite appropriate preventive measures, the boy developed metastatic melanoma at 15 years of age and died 2 years later. His parents had two other children; neither was affected with XP.

  24. XERODERMA PIGMENTOSUM • Can be caused by defects in any one of seven different NER genes • Predisposition to skin cancer • Pigmentation abnormalities • Premalignant lesions • Degeneration of the nervous system

  25. Base excision repair Repair of modified bases Glycosylase removes base, leaves backbone intact AP endonuclease cut backbone, AP lyase removes sugar Nucleotide excision repair Repair of adducts and large distortions in DNA double helix Double excision removes damage as an oligonucleotide (12-13 nt in E. Coli, 27-29 nt in humans) ☺ EXCISION REPAIR • DNA polymerase fills gap • DNA ligase seals nick

  26. ☺ Mismatch repair • Repair of replication (proofreading) errors • Recognition of bases that do not form normal Watson-Crick pairs

  27. ☺ Mismatch repair • How do the repair enzymes recognize which strand to fix??? ? CH3 CH3 A G A T C T C T T C G A T C x T C T A G A G C A G C T A G ?

  28. ☺ Mismatch repair CH3 CH3 CH3 CH3 CH3 CH3 MutS/MutL MutH CH3 CH3 DNA Polymerase DNA Ligase

  29. HEREDITARY NONPOLYPOSIS COLORECTAL CANCER (HNPCC) • Lynch syndrome • Accounts for 2 -10% of all colon cancers • Caused by defects in mismatch repair genes MSH2, MSH6, MLH1, PMS1 or PMS2

  30. DNA STRAND BREAKS

  31. DNA STRAND BREAKS • 10 -100 naturally occurring double-strand breaks per cell per day • Two mechanisms for repair • Homologous recombination repair (HRR) • Nonhomologous recombination repair (NHRR)

  32. Homologous recombination repair (HRR)

  33. Nonhomologous recombination repair (NHRR)

  34. HRR Identical copies made Only possible in the S and G2 phase of the cell cycle NHRR Small deletions occur Any time in the cell cycle HRR Vs. NHRR

  35. Defects in DNA-repair systems associated with certain cancers

  36. The End!

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