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

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 from Alberts et al., Molecular Biology of the Cell, 4 th ed., Fig 5-46 depurination deamination oxidation

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

5. Cytosine is deaminated to uracil at a rate of 100-500/cell/day Uracil is excised by uracil-DNA-glycosylase to form AP site Deamination of Cytosine to Uracil

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

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, 4 th 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. guanine8-oxoguanine The most common mutagenic base lesion is 8-oxoguanine from Banerjee et al., Nature 434, 612 (2005) Oxidation of Guanine Forms 8-Oxoguanine

10. Repair of 8-oxoG 8-oxoguanine DNA glycosylase/  -lyase (OGG1) removes 8-oxo-G and creates an AP site Replication of the 8-oxoG strand preferentially mispairs with A and mimics a normal base pair and results in a G-to-T transversion 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, 6 th ed. Fig 4-38

13. Nonenzymatic Methylation of DNA Formation of 600 3-me-A residues/cell/day are caused by S-adnosylmethionine 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. Repair Pathways for Altered DNA Bases from Lindahl and Wood, Science 286, 1897 (1999)

16. Direct Repair of DNA Photoreactivation of pyrimidine dimers by photolyase restores the original DNA structure O 6 -methylguanine is repaired by removal of methyl group by MGMT 1-methyladenine and 3-methylcytosine are repaired by oxidative demethylation

17. Base Excision Repair of a G-T Mismatch 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, 6 th ed. Fig 4-36 BER works primarily on modifications caused by endogenous agents

18. Each glycosylase has limited substrate specificity, but there is redundancy in damage recognition DNA Glycosylases from Xu et al., Mech.Ageing Dev. 129, 366 (2008)

19. Mechanism of hOGG1 Action 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 from David, Nature 434, 569 (2005)

20. Nucleotide Excision Repair in Human Cells Mutations in at least seven XP genes inactivate nucleotide excision repair and cause xeroderma pigmentosum The only pathway to repair thymine dimers in humans is nucleotide excision repair XPC recognizes damaged DNA Helicase activities of XPB and XPD of TFIIH create sites for XPF and XPG cleavage NER works mainly on helix-distorting damage caused by environmental mutagens An oligonucleotide containing the lesion is released and the gap is filled by POL  or  and sealed by LIG1 from Lindahl and Wood, Science 286, 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. GATC sequences are methylated by dam methylase Newly replicated DNA is transiently hemimethylated MutS recognizes a mismatch of small IDL MutS bends DNA, recruits MutL and forms a small dsDNA loop MutH nicks the unmethylated GATC Helicase unwinds the nicked DNA which is degraded past the mismatch Gap is repaired by Pol III and ligase from Marra and Schar, Biochem.J. 338, 1 (1998) Mismatch repair in E. coli

24. Mismatch Repair in Eukaryotes from Hsieh and Yamane, Mech.Ageing Dev. 129, 391 (2008) MutS homologs recognize mismatch and form a ternary complex with MulL homologs and the mismatch PMS2 is a mismatch-activated strand- specific nuclease, and the break is directed to the strand contain the preexisting nick EXO1 excises the mismatch The gap is filled in by Pol  and DNA ligase Defective mismatch repair is the primary cause of certain types of human cancers

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

26. Initiation of Double-stranded Break Repair from van Attikum and Gasser, Trends Cell Biol. 19, 204 (2009) MRN complex recognizes DSB ends and recruits ATM ATM phosphorylates H2A.X and recruits MDC1 to spread  H2A.X TIP60 and UBC13 modify H2A.X MDC1 recruits RNF8 which ubiquitylates H2A.X RNF168 forms ubiquitin conjugates and recruits BRCA1

27. ATM Mediates the Cell’s Response to DSBs from van Gent et al., Nature Rev.Genet. 2, 196 (2001) DSBs activate ATM ATM phosphorylation of p53, NBS1 and H2A.X influence cell cycle progression and DNA repatr

28. 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, 5 th ed. Fig 23-31 Repair of ds Breaks by Homologous Recombination

29. Role of BRCA2 in Double-stranded Break Repair BRCA2 mediates binding of RAD51 to ssDNA RAD51-ssDNA filaments mediate invasion of ssDNA to homologous dsDNA from Zou, Nature 467, 667 (2010)

30. from van Gent et al., Nature Rev.Genet. 2, 196 (2001) 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

31. Translesion Replication by DNA Polymerase V From Livneh, J.Biol.Chem. 276, 25639 (2001) 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