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from Marnett and Plastaras, Trends Genet . 17 , 214 (2001)

Endogenous DNA Damage. from Marnett and Plastaras, Trends Genet . 17 , 214 (2001). Biological Molecules are Labile. RNA is susceptible to hydrolysis. Reduction of ribose to deoxyribose gives DNA greater stability. N-glycosyl bond of DNA is more labile.

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from Marnett and Plastaras, Trends Genet . 17 , 214 (2001)

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  1. Endogenous DNA Damage from Marnett and Plastaras, Trends Genet. 17, 214 (2001)

  2. Biological Molecules are Labile RNA is susceptible to hydrolysis Reduction of ribose to deoxyribose gives DNA greater stability N-glycosyl bond of DNA is more labile DNA damage occurs from normal cellular operations and random interactions with the environment

  3. Spontaneous Changes that Alter DNA Structure deamination oxidation depurination from Alberts et al., Molecular Biology of the Cell,4th ed., Fig 5-46

  4. Hydrolysis of the N-glycosyl Bond of DNA from Alberts et al., Molecular Biology of the Cell,4th ed., Fig 5-47 Spontaneous depurination results in loss of 10,000 bases/cell/day Causes formation of an AP site – not mutagenic

  5. Deamination of Cytosine to Uracil from Alberts et al., Molecular Biology of the Cell,4th ed., Fig 5-47 Cytosine is deaminated to uracil at a rate of 100-500/cell/day Uracil is excised by uracil-DNA-glycosylase to form AP site

  6. 5-Methyl Cytosine Deamination is Highly Mutagenic Deamination of 5-methyl cytosine to T occurs rapidly - base pairs with A 5-me-C is a target for spontaneous mutations from Alberts et al., Molecular Biology of the Cell,4th ed., Fig 5-52

  7. Deamination of A and G Occur Less Frequently A is deaminated to HX – base pairs with C G is deaminated to X – base pairs with C from Alberts et al., Molecular Biology of the Cell,4th ed., Fig 5-52

  8. Oxidative Damage of DNA Oxidative damage results from aerobic metabolism, environmental toxins, activated macrophages, and signaling molecules (NO) Compartmentation limits oxidative DNA damage

  9. Oxidation of Guanine Forms 8-Oxoguanine The most common mutagenic base lesion is 8-oxoguanine guanine 8-oxoguanine from Banerjee et al., Nature 434, 612 (2005)

  10. Repair of 8-oxoG Replication of the 8-oxoG strand preferentially mispairs with A and mimics a normal base pair and results in a G-to-T transversion 8-oxoguanine DNA glycosylase/ b-lyase (OGG1) removes 8-oxo-G and creates an AP site MUTYH removes the A opposite 8-oxoG

  11. Oxidation of dNTPs are Mutagenic cGTP is oxidized to 8-OH-dGTP and is misincorporated opposite A MutT converts 8-OH-dGTP to 8-OH-dGMP

  12. UV-Irradiation Causes Formation of Thymine Dimers from Lodish et al., Molecular Cell Biology, 6th ed. Fig 4-38

  13. Nonenzymatic Methylation of DNA Formation of 600 3-me-A residues/cell/day are caused by S-andnosylmethionine 3-me-A is cytotoxic and is repaired by 3-me-A-DNA glycosylase 7-me-G is the main aberrant base present in DNA and is repaired by nonenzymatic cleavage of the glycosyl bond

  14. Effect of Chemical Mutagens Nitrous acid causes deamination of C to U and A to HX U base pairs with A HX base pairs with C

  15. Spores Use Strategies to Overcome Intrinsic Instability of DNA DNA exists in an A-like conformation and is bound to proteins that reduce the rate of depurination Lack of dNTPs in spores prevents DNA repair before germination Extensive DNA repair occurs upon spore germination

  16. Repair Pathways for Altered DNA Bases from Lindahl and Wood, Science 286, 1897 (1999)

  17. Direct Repair of DNA Photoreactivation of pyrimidine dimers by photolyase restores the original DNA structure O6-methylguanine is repaired by removal of methyl group by MGMT 1-methyladenine and 3-methylcytosine are repaired by oxidative demethylation

  18. Base Excision Repair of a G-T Mismatch BER works primarily on modifications caused by endogenous agents At least 8 DNA glcosylases are present in mammalian cells DNA glycosylases remove mismatched or abnormal bases AP endonuclease cleaves 5’ to AP site AP lyase cleaves 3’ to AP site from Lodish et al., Molecular Cell Biology, 6th ed. Fig 4-36

  19. Mechanism of hOGG1 Action from David, Nature434, 569 (2005) hOGG1 binds nonspecifically to DNA Contacts with C results in the extrusion of corresponding base in the opposite strand G is extruded into the G-specific pocket, but is denied access to the oxoG pocket oxoG moves out of the G-specific pocket, enters the oxoG-specific pocket, and excised from the DNA

  20. Nucleotide Excision Repair in Human Cells NER works mainly on helix-distorting damage caused by environmental mutagens The only pathway to repair thymine dimers in humans is nucleotide excision repair Mutations in at least seven XP genes inactivate nucleotide excision repair and cause xeroderma pigmentosum XPC recognizes damaged DNA Helicase activities of XPB and XPD of TFIIH create sites for XPF and XPG cleavage An oligonucleotide containing the lesion is released and the gap is filled by POL d or e and sealed by LIG1 from Lindahl and Wood, Science286, 1897 (1999)

  21. Transcription-coupled Repair Repair of the transcribed strand of active genes is corrected 5-10-fold as fast as the nontranscribed strand All the factors required for NER are required for transcription-coupled repair except XPC The arrest of POL II progression at a lesion served as a damage recognition signal Recruitment of NER factors also involves CS-A and CS-B

  22. Nucleotide Excision Repair Pathway in Mammals Cockayne’s Syndrome and Trichothiodystrophy are multisystem disorders defective in transcription-coupled DNA repair

  23. Mismatch Repair in E. coli Newly replicated DNA is hemimethylated MutS binds to mismatch and recruits MutL Activates endonuclease activity of MutH and nicks the nearest unmethylated GATC Recruits MutU (helicase) and exonucleases DNA pol III fills in the gap

  24. Mismatch Repair in Human Cells MSH2 and MSH6 bind to mismatch- containing DNA and distinguish between the template and newly synthesized strand MMR complex identifies newly synthesized strand by the presence of a 3’-terminus MutLa introduces random nicks at distal sites on the same strand EXO1 at 5’-side of the mismatch activates a 5’-3’ exonuclease and removes mismatch The gap is filled in by DNA polymerase and DNA ligase Defective mismatch repair is the primary cause of certain types of human cancers from Lodish et al., Molecular Cell Biology, 6th ed. Fig 4-37

  25. Causes of and Responses to ds Breaks DSBs result from exogenous insults or normal cellular processes DSBs result in cell cycle arrest, cell death, or repair Repair of DSBs is by homologous recombination or nonhomologous end joining from van Gent et al., Nature Rev.Genet. 2, 196 (2001)

  26. ATM Mediates the Cell’s Response to DSBs DSBs activate ATM ATM phosphorylation of p53, NBS1 and H2AX influence cell cycle progression and DNA repatr from van Gent et al., Nature Rev.Genet. 2, 196 (2001)

  27. Repair of ds Breaks by Homologous Recombination ssDNAs with 3’ends are formed and coated with Rad51, the RecA homolog Rad51-coated ssDNA invades the homologous dsDNA in the sister chromatid The 3’-end is elongated by DNA polymerase, and base pairs with ss 3-end of the other broken DNA DNA polymerase and DNA ligase fills in gaps from Lodish et al., Molecular Cell Biology, 5th ed. Fig 23-31

  28. Repair of ds Breaks by Nonhomologous End Joining KU heterodimer recognizes DSBs and recruits DNA-PK Mre11 complex tethers ends together and processes DNA ends DNA ligase IV and XRCC4 ligates DNA ends from van Gent et al., Nature Rev.Genet. 2, 196 (2001)

  29. Structure of Rad50 and the Mre11 Complex Mre11 complex maintains genome integrity and participates in HR and NHEJ AT-like syndrome and Nijmegen breakage syndrome are caused by mutation in MRE11 or NBS1 Dimerization domain of Rad50 keeps DNA fragments within close proximity from de Jager and Kanaar, Genes Dev.16, 2173 (2002)

  30. XRCC4 X-ray-repair-cross-complementing defective repair in Chinese hamster mutant 4

  31. The SCID Mouse is Defective in DSB Repair The SCID mouse carries a mutation preventing the production of mature B and T cells The phenotype is a defect in the development of the immune system and hypersensitivity to ionizing radiation The phenotype is caused by a mutation in DNA-PK

  32. Translesion Replication by DNA Polymerase V Translesion DNA synthesis occurs in the absence of Pol III Translesion DNA polymerases are error prone and exhibit weak processivity Most of the mutations caused by DNA damaging agents are caused by TLR TLR protects the genome from gross rearrangements Pol V is regulated by LexA and the SOS response from Livneh, J.Biol.Chem. 276, 25639 (2001)

  33. Expandable Repeats Form Unusual DNA Structures One of the complementary strands is more structure-prone than the other Unusual structural features predispose them to instability Stalling of lagging strand synthesis disrupts coordination with leading strand synthesis from Mirkin, Nature447, 932 (2007)

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