1. DNA Repair

2. DNA Damage Tolerance and Repair 1-Dealing with Problems occurring during DNA replication Mutations resulting from errors made during DNA replication  Mismatch Repair Pathway Ribonucleotides incorporated during DNA replication 2- Dealing with Problems coming after DNA replication resulting from DNA damage Non exhaustive list of Damages Not treated: -Double-stranded break repair -Transcription coupled DNA repair Strategies and mechanisms of DNA damage tolerance and repair:  Bypass/Translesion DNA polymerases  Direct Reversal  Base Excision Repair  Nucleotide Excision Repair

3. 10 -2 10 -6 10 -8 -10 -9 Editing ? Increasing Replication Fidelity by mismatch repair template New strand How to differentiate between right and wrong Parental and new strand ? ! ? NH 2 N3N3 N1N1 N7N7 N9N9 dR O OH S+S+ CH 3 COO - + 3 HN : A S-adenosyl methionine New strand ! CH 3 DNA methylation is delayed after replication 2) DNA methylation 1) GATC CTAG Dam methylase CH 3 3 HC CTAG GATC

4. CH 3 ATP ADP+Pi ATP ADP+Pi MutL/MutSMutH Cleavage by MutH CH 3 DNA sliding through MutL/MutS CH 3 Ligation DNA unwinding by Helicase II (MutU/UvrD) Exonuclease VII (5’-->3’) or Exonuclease I (3’-->5’) CH 3 Methyl-directed mismatch repair in prokaryotes Uvr genes = genes that promote UV Resistance Mut genes = When these genes are mutated, bacteria show increased rates of mutations Filling the gap

5. The Problem of ribonucleotide incorporation by DNA polymerases DNA polymerases discriminate deoxy vs riboNTP, but not at 100% [rNTPs] >> [ dNTPs] in vivo -> this leads to riboNTP incorporation in newly synthesized DNA Enzymes that deal with removal of RNA in Okazaki fragments also remove riboNTPs mis-incorporated into DNA : Rnase H, Fen1, DNA Pol.I             (PNAS 107, p4950, 2010)

6. Post-replicative DNA Damages that need to be repaired or dealt with - Hydrolysis of glycosidic bond (Depurination) - Alkylation of bases Methylation of guanine N6 G-> O 6 meG -Pyrimidine dimers (UV light) - Deamination of bases Spontaneous Chemically induced C->U 5 meC -> T A->HX - Oxidative damages G -> 8 oxoguanine Strand Break - Bulky DNA adducts

7. Induction of Pyrimidine Dimers by UV light PDB ID = 1SM5

8. Spontaneous Deaminations A --> H 10 -9 /24 hours G --> X 10 -9 /24 hours C --> U 10 -7 /24 hours: 100 events/day for a mammalian cell

9. If Uracil were a natural base, the DNA repair machinery would not know whether these Uracil are “normal” uracil that come from incorporation by polymerase, or “non-natural” uracil coming from deamination of cytosines. Since there is no way to discri- minate between these, the genetic systems did not select uracil as a natural base (exceptions) Why Uracil was not selected as a natural base in DNA: A C A T G G T G U A U C The problem of Uracil in DNA Due to incorporation of dUTP by polymerase Due to spontaneous Deamination of C->U 5’3’ 5’

10. Chemical Sources and Genetic Consequences of Deaminations =

11. Spontaneous Depurinations 1/10 5 in 24 hours: 10,000 events/day for a mammalian cell

12. Chemical Sources and mechanism of alkylations

13. Consequences of O 6 -meG For Replication :

14. O2O2 O2-O2- 1e - 2H + H2O2H2O2 1e - - OH +. OH 2H 2 O CytC oxidase Oxidative damage of DNA - Source of oxidative agents 2O 2 - +2H + H 2 O 2 +O 2 2H 2 O 2 superoxide dismutase catalase 2H 2 O+ O 2. OH No cellular Neutralization -> Main source of Oxidative agent - Neutralization of reactive species N N N NH 2 O NH H2O2H2O2 - OH Guanine 5-formyl Uracil Thymine Consequences for Nucleotides: Consequences for Nucleic Acids: Strand Breaks (bad for DNA replication) Deoxyribose Ribose N N N NH 2 O NH O H 8-oxo Guanine Fenton Chemistry (Metals) Respiratory Chain:

15. G-C DNA replication H 2 O 2 - OH 8-oxo - guanine generates replication block or G-C -> T:A transversions after DNA Replication J Am Chem Soc. 2005 Oct 12;127(40):13906-18 8xoGC 8xoGA T-A 8xoGA DNA Polymerases tend to incorporate A opposite to 8-oxoG because of the tendency of 8-oxoG to switch in the syn conformation; A is the only nt that can form a base pair with syn8-oxoG whose geometry resembles that of a Watson-Crick base pair

16. Bulky DNA adducts caused by: cigarette smoke diesel engine exhaust cooking/broiling of food block DNA Replication

17. DNA repair and tolerance Strategies & Enzymes 3- Base excision repair (deamination, alkylation, oxidation of bases) Uracil-N glycosylase 8-oxoG glycosylase 2- Direct Reversal of Damage (alkylation of bases, pyrimidine dimers) Photolyase reversion of Y dimers Dealkylation of guanines by suicidal MGMTase Dealkylation of 1mA and 3mC by AlkB (not shown) 4- Nucleotide excision repair (pyrimidine dimers, bulky DNA adducts) Bacteria: UvrA, UvrB, UvrC, Helicase II (UvrD) DNA pol. I, DNA ligase Eukaryotes : Xeroderma pigmentosum proteins, TFIIH 1- Bypass of lesions: avoids DNA replication stalls bypass of DNA damage by translesion DNA Polymerases -> not a “repair” but is used to prevent DNA replication blocks

18. Primer- -X* 75nt- X*X* X X X*X* Primer (41nt) 5’-AGG Template (75nt) 3’-TCCGTAXAATG--5’ Pol  Pol  Bypass of 8-oxoG lesions by a specialized eukaryotic DNA polymerase (Pol  X = Guanosine X * = 8-Oxo Guanosine <- Bypass product Block of polymerization at 8-oxoG Primer Extension Assay to map template replication by the two DNA polymerases Bacterial and Eukaryotic cells possess multiple translesion polymerases that are capable of bypassing DNA lesions

19. http://www.pathology.unc.edu/faculty_labs/vaziri_lab/image012.jpg (4) Pol.  binds to mono-ubiquitinated PCNA and performs replicative bypass of damaged DNA, preserving replication fork movement. 1)A DNA lesion (red) causes stalling of the replicative DNA Pol. . 3) Rad18 monoubiquitinates PCNA at stalled replication forks. Switch between Replicative and Translesion DNA polymerases involves PCNA Ubiquitination and prevents stalling of replication at the sites of DNA damage 2) The E3 ubiquitin ligase Rad18 guides Pol.  (aTLS DNA polymerase) to stalled replication forks. PCNA

20. Photoreactivation Repair of pyrimidine dimers h  damage DNA photolyase Photoreactivating enzyme with 2 chromophores e.g. 5-deazaflavin (or N 5 N 10 methyleneTHF) FADH - h  300-500nm Direct Reversal:

21. The “inactivated” enzyme serves as a transcription factor to induce expression of DNA repair genes -> amplifies the cellular response to DNA damage Mutations of Human Homologues of O6MGMT linked to cancer :  maintaining DNA information is required for tumor suppression Dealkylation of guanines by Methyl Guanine Methyl Transferase (MGMT)

22. Base Excision Repair: General strategy damaged base + DNA glycosylase/ glycosidase apurinic or apyrimidinic site AP endonuclease Cuts strand at AP site (APurinic or Apyrimidic) leaves 5’ terminal deoxyribose P moiety Second cleavage (1nt further) by another enzyme DNA Pol + DNA ligase OH P P Probably removes several nts by nick translation Cleaves glycosidic bond at damaged base

23. 1) Two sequential enzymatic activities are involved on two different enzymes: 2) The same polypeptide carries both glycosylase & AP endonuclease activities Ex: 8-OxoG DNA glycosylase (hOGG1) Ex: Uracil N-glycosylase cleaves glycosidic bonds of deoxyuridine but does not have AP endonuclease activity – needs another enzyme Two types of base excision repair mechanisms A) Pure glycosylase/glycosidase then B) AP endonuclease Typically one enzyme cleaves the glycosidic bond of the damaged base, then the phosphodiester backbone is cleaved by an endonuclease specific for sites lacking a base (AP sites)

24. Structure and activity of uracil N-glycosylase PDB ID = 2OXM

25. DNA Repair Strategies for 8-oxoG damage OGG1 = glycosidase for 8-oxoG MUTYH = glycosidase for A’s misincorporated in front of the 8-oxoG’s David et al., Nature 447, 941 - 950 (2007)-Figure 1

26. The oxoG base is stacked between Phe 319 and Cys 253. Residues Gly 42, Gln 315 and two water molecules hydrogen bond to the Watson–Crick and Hoogsteen faces of the lesion base. The cytosine paired opposite oxoG is recognized by H-bonding Interactions with Arg 154 and Arg 204, and an additional H bond with Asn 149. How does hOGG1 recognize 8-OxoG damages ? Bruner et al., Nature 403, 859 - 866 (24 February 2000)-Figure 6 recognition of 8-OxoGrecognition of unpaired C PDB ID = 1EBM

27. Nucleotide Excision Repair in Prokaryotes Uvr = UV Resistance When mutated, genes coding for Uvr proteins show an decrease in UV resistance

28. Nucleotide Excision Repair in Eukaryotes (Y dimer, bulky adducts) XP = Xeroderma pigmentosum TF II H = basal RNA Polymerase II transcription factor H; contains XPB & XPD subunits important because it links DNA repair to transcription