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

2. 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

3. Repair of 8-oxo-G 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 from David et al., Nature 447, 941 (2007)

4. Free dNTPs are much more susceptible to oxidative damage than bases in duplex DNA Oxidized precursors are misincorporated and are mutagenic MTH1 removes oxidized nucleotides from the pool from Dominissini and He, Nature 508, 191 (2014) MTH1 Prevents Incorporation of Oxidized dNTPs into DNA

5. MTH1 is not essential in normal cells Higher levels of ROS in cancer cells causes a non-oncogene addiction to MTH1 from Gad et al., Nature 508, 215 (2014) Inhibition of MTH1 Selectively Kills Cancer Cells

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

7. Nonenzymatic Methylation of DNA Formation of 600 3-me-A residues/cell/day are caused by S-adenosylmethionine 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

8. 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

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

10. 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

11. 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

12. 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)

13. 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)

14. UV-induced pyrimidine dimers Nucleotide Excision Repair Bulky adducts Repairs helix-distorting lesions Intrastrand crosslinks ROS-generated cyclopurines

15. Global Genome NER – Damage Recognition Probes for helix distorting lesions XPC is the damage sensor which finds the ssDNA gap caused by disrupted pairing UV-DDB (DDB1 and DDB2) can stimulate XPC binding by extruding the lesion to create ssDNA from Marteijn et al., Nature Rev.Mol.Cell Biol. 15, 465 (2014)

16. Transcription-coupled NER – Damage Recognition Repairs transcription-blocking lesions CSB, UVSSA and USP7 interact with Pol II With CSA, promotes backtracking of Pol II to expose lesion from Marteijn et al., Nature Rev.Mol.Cell Biol. 15, 465 (2014)

17. TFIIH complex is recruited to the lesion XPB and XPD are helicases with opposite polarity XPD verifies the existence of lesions and XPA binds to altered nucleotides XPG nuclease binds to the complex RPA protects the undamaged strand from nucleases NER – Lesion Verification from Marteijn et al., Nature Rev.Mol.Cell Biol. 15, 465 (2014)

18. NER – Strand Excision XPF nuclease is recruited by XPA and directed to the damaged strand by RPA XPF and XPG excises the lesion from Marteijn et al., Nature Rev.Mol.Cell Biol. 15, 465 (2014)

19. PCNA recruits DNA polymerase to fill ss gap Nick is sealed by DNA ligase NER – Gap Filling and Ligation from Marteijn et al., Nature Rev.Mol.Cell Biol. 15, 465 (2014)

20. NER is stimulated by an open chromatin environment UV-DDB ubiquitylates core histones and associates with PARP1 which PARylates chromatin Histone acetylation stimulates NER Chromatin remodelling complexes displace nucleosomes Chromatin Dynamics in GG-NER from Marteijn et al., Nature Rev.Mol.Cell Biol. 15, 465 (2014)

21. Clinical Implications of Defective NER GG-NER is elevated in germ cells to maintain the entire genome to prevent mutagenesis TC-NER is elevated in somatic cells to repair expressed genes to prevent cell death Defective GG-NER increases cancer predisposition Defective TC-NER causes premature cell death, neurodegeration and accelerates aging Xeroderma pigmentosum Cockayne Syndrome

22. Mismatch Repair Repairs DNA replication errors and insertion-deletion loops Decreases mutation frequency by 10 2 - 10 3 Plays a role in triplet repeat expansion, somatic hypermutation and class switch recombination

23. GATC sequences are methylated by dam methylase Newly replicated DNA is transiently hemimethylated MutS recognizes a mismatch or 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 PCNA, 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 DNA Synthesis from Sale et al., Nature Rev.Mol.Cell Biol. 13, 141 (2012) Replicative polymerase encounters DNA damage on template strand Replicative polymerase is replaced by TLS polymerase which inserts a base opposite lesion Base pairing is restored beyond the lesion and replicative polymerase replaces TLS polymerase TLS can occur in S or G2 Catalytic site of replicative polymerases is intolerant of misalignment between template and incoming nucleotide

32. TLS polymerases are recruited by interactions with the sliding clamp There are multiple TLS polymerases TLS polymerases have low processivity and low fidelity, and lack 3’-5’ exonucleases TLS polymerases are selective for certain lesions Most mutations caused by DNA lesions are caused by TLS polymerases from Sale et al., Nature Rev.Mol.Cell Biol. 13, 141 (2012) There are Multiple TLS Polymerases

33. TLS Polymerases Can Be Accurate or Error-prone Pol  bypasses an abasic site and often causes a -1 frameshift Pol  bypasses a thymine dimer and inserts AA Pol  is accurate with dA template and error-prone with dT template Replicative polymerases insert dC or dA opposite 8-oxo-G, Pol  inserts dC The likelihood that TLS polymerases are error-prone depends on the nature of the lesion and the TLS polymerase that is utilized

34. Somatic Hypermutation of Ig Genes Depends on TLS Polymerases from Sale et al., Nature Rev.Mol.Cell Biol. 13, 141 (2012) Uracil DNA glycosylase forms an abasic site, and REV1 incorporates dC opposite the site AID deaminates dC to dU MMR proteins lead to the formation of a ss gap, PCNA is ubiquitylated, and Pol  is recruited, generating mutations at A-T