DNA repair

1. DNA repair Mutación y reparación del DNA

2. Why repair DNA? Replication error OH. H+ • 1. Errors in DNA replication • 2. Endogenous DNA damage and mutagens • 3. Environmental insults to DNA • 4. Un-repaired damage leads to: • -mistakes in RNA/protein synthesis • -inherited as genetic alteration-a mutation • -death dUTP UV hn mC 8-oxoG TT Depur- ination P/P U All of these events are rare, but the number of bp in each nucleus is very large so the total frequency is significant.

3. Types of DNA damage • Base Loss • Base modification & Deamination • Chemical Modification • Photodamage • Inter-strand crosslinks • DNA-protein crosslinks • Strand breakage

4. Example of the product of a very small and a very large number yielding a significant effect • Number of bp in the nucleus of a human cell=~3109. • Rate of breakage of purine glycosidic bonds in neutral solution predicts =~104 depurinations/day/cell. • 1013 cells/human = 1017 depurinations/day/organism! • If these are not repaired, it would lead to massive errors in the synthesis of proteins. • Mutations in the germ line would transmitted to offspring, leading to genetic disease.

5. Base loss Abasic site -loss of a nucleobase (apurinic or apyrimidinic) Deamination

6. Potential Sites of modification/damage

7. Chemical Damage Alkylation Oxidative damage

8. UV-induced damage

9. Types of mutations, causes , consequences. • Substitution: G/C>A/T (transition); G/C>C/G (tranversion). Consequence: usually change the amino acid sequence if in ORF. Causes: errors in replication; deamination; oxidation. • Deletion: GCTAAAAAGCT>GCTAAAAGCT. • Addition: GCTAAAAAGCT>GCTAAAAAAGCT. • Consequences: termination of protein synthesis due to frame-shifting the genetic code. Causes: intercalating agents; slipped-strand replication errors.

10. Consequences of mutations (cont) Accumulation of mutations: • Cancer, (Loeb’s hypothesis): cancer is a genetic “disease” caused by an elevated mutation rate-as by an error-prone polymerase or faulty repair machinery. “Two-hit” model. Somatic versus germ line mutations. • Aging (error catastrophe hypothesis); failure of normal cell death (apoptosis) due to accumulation of mutations in genes responsible for the normal operation of these processes. • Single mutations and genetic disease. How many genes, when inactivated, would cause a “disease”? • Multiple polymorphisms (remember SNPs) and pre-disposition to susceptibility to endogenous agents (oxidizing agents) and environmental insults.

11. Mutation avoidance First line of defense: • Preventing the accumulation of mutation- generating agents: • metabolism of active oxygen species by reducing agents and enzymatic mechanisms (superoxide dismutase); • dUTPase to prevent mis-incorporation of dUMP into DNA; • shielding from harmful irradiation (melanin in skin).

12. Types of Damage Repair • Photolyase • De-alkylation proteins (not catalytic) • Base Excision Repair • Nucleotide Excision Repair (GG and TC) • Recombination Repair • Error-prone Repair • Double strand Break Repair (if time permits)

13. Mutation prevention: DNA repair Evidence for repair mechanisms: Evelyn Witkin: UV light, death, mutagenesis, and survival. 1.0 0.1 S/So 0.01 Mutation frequency 0.001 0.0001 UV dose Note: the dose-response curve is not linear: at low doses there is high survival; at higher doses survival drops off rapidly; at very high doses there is more resistance. Mutations occur at increasing frequency, and then decline. Why the decline?

14. Interpretation of the UV survival curve ~100 UV damage is repaired efficiently but some damage is mutagenic More than one “hit” is required to kill an organism Repair can’t keep up with damage; mutants too are killed 1.0 0.1 0.01 Mutation frequency S/So 0.001 0.0001 Rare mutants are UV- resistant. S=survival; So=survival before UV treatment UV dose polA (DNA polI mutants)

15. Could low does protect against cancer-the hormesis hypothesis: data for and against 100 Linear response cancers extrapolated to zero dose-is this an appropriate extrapolation? 0 0 10 100 1000 dose

16. Radiation-sensitive mutants are easy to identify UV light-individual cells on a petri plate to induce mutations • E. coli~ 30 genes are involved in DNA repair. Yeast~ 50 genes. • Humans? Grow to colonies Replica plate using a piece of velvet to pick-up and transfer colonies to a new petri plate. +UV -UV One colony is missing because the cells are more UV-sensitive than those in other colonies

17. Redundancy of repair mechanisms • 1. Proof-reading or editing by DNA polymerase • 2. Direct reversal of damage. • 3. Base excision repair • 4. Nucleotide excision repair* • 5. Mismatch repair. • 6. Error-prone (SOS) repair* • 7. Recombination repair * Induced by DNA damaging agents

18. Editing: Frequency of errors in replication depends on the polymerase

19. DNA repair polymerases DNA polymerase Eta (XP-V) - addition of two dA residues across pyrimidine dimers DNA polymerase Zeta - addition of random residues across pyrimidine dimers

20. Direct removal of damage: T-T dimers A common photoproduct of UV treatment of DNA in vivo and in vitro is an intra-strand dimer formed beween adjacent thymines. Note that formation of the cyclobutane ring destroys the aromatic nature of the pyrimidine ring and distorts the helix.

21. Enzymatic reversal of T-T dimer formation DNA photolyase is present in all organisms. Which cells in your body do you think would have the most photolyase? 200-300 nm light AGCATTCTGA TCGTAAGACT AGCA{T/T}CTGA TCGT {AA} GACT 300-500 nm light DNA photolyase AGCATTCTGA TCGTAAGACT Direct reversal of DNA damage: No excision of bases or nucleotides

22. Alkyltransferase detoxification of alkylated DNA by the Ada protein • A second type of direct reversal of DNA damage removes offending akyl groups from O6-alkyl guanine and methylated phosphate triesters. Alkylation of Cys321 inactivates the protein-the protein “commits suicide”. Buried active site cysteine321 covalently binds methyl group of O6mG.

23. Base Excision Repair

24. Base Excision Repair

25. Nucleotide Excision Repair (E.coli)

26. Nucleotide Excision Repair (Global Genome Repair -Humans)

27. Nucleotide Excision Repair(Transcription Coupled -Humans)

28. Common features of GGR & TCR

29. 4. DNA glycosylases remove altered bases Deamination of cytosine, particularly 5-methyl cytosine leaves uracil (thymine if 5-methyl cytosine) in the DNA. Uracil would pair as thymine during replication and thus cause a mutation. Uracil-N-glycosylase removes uracil from DNA. An endonuclease then cleaves the backbone at that site, creating a substrate for NER

30. 5. DNA methylation and repair The DNA methyl transferases lag several thousand bases behind the replication fork. This “marks” the parental DNA strand. Methylation occurs on cytosine and adenine residues in DNA. N6 of A is methylated in the sequence GmATC; C is methylated in the sequence CmC(A/T)GG = methyl group

31. N6-methyl-adenine Mismatch repair corrects errors occurring during DNA replication Mismatch correction accounts for the “discrepancy” between the error rate of polIII in vitro and error rates measured in vivo.

32. Mismatch repair corrects the unmethylated strand Mis-paired bases What happens if the “old” strand needs repair? eg 5-methyl cytosine>deamination to 5-methyl uracil (=thymine!). In E. coli a small fraction of C is 5-methylated and these are “hot-spots” for spontaneous mutation. This implies that 5-methyl cytosine is frequently either not repaired or is mistakenly repaired on the “wrong” strand.

33. Mismatch repair-the finale

34. 6. Error-prone or induced DNA repair- the SOS reponse UV’d T4 phage UV’d T4 phage UV • Irradiation of bacteria before virus infection enhanced repair of damaged viral genes but led to mutations. This has an evolutionary advantage for the viral population since it increases the probability that some members will survive albeit in altered form E. coli E. coli Higher frequency of surviving phage, but many mutants. Few surviving phage

35. Error-prone repair is due to a novel damage-induced DNA polymerase activity • Two genes induced by cleavage of LexA (a DNA binding repressor protein), umuC and umuC, encode a DNA polymerase activity that is active on damaged DNA templates-ie templates lacking a proper DNA sequence. It allows replication past the damaged site, often inserting (incorrectly) one or a few A’s. AGCTAGTCAT/TCAGTC Replication stops at T/T dimer SOS response: AGCTAGTCAT/TCAGTC TCGATCANNNNGTCAG Error-prone polymerase allows replication to proceed, albeit inaccurately

36. Error Prone Bypass (E. coli)

37. Experimental evidence for Error prone repair(E.coli) UV-responsive activation of the umuC gene Revertant in His- genes (umuC mutated strain)

38. Human mismatch repair genes and cancer • Yeast mutants defective in mismatch repair genes have unstable microsatellite sequences (repetitive tracts of mono- and dinucleotides). • Some human colon cancers also display microsatellite instability. • Are these due to defective mismatch repair genes? ANSWER: Yes: Genetic mapping of human non- polyposis colon cancer genes identifies these genes as defective human mismatch repair genes. Can these be corrected? …..

39. Model for activation of DNA damage repair

40. Damage & Repair • Multiple forms of DNA damage occur • These are repaired constantly by several mechanisms • Failure to repair damage leads to mutations • Often defects in damage sensing machinery or DNA repair processes can be correlated with increased incidence of diseases such as cancer

41. Factors involved in Damage Sensing

45. DNA Repair-summary • DNA repair mechanisms exist in all organisms to maintain the fidelity of the DNA sequence. • DNA is repaired during and after replication, and by constitutive and damage-inducible enzyme systems • Multiple repair mechanisms are necessary to correct errors arising during replication and to repair DNA damage by intrinsic and extrinsic agents. • Failure of DNA repair leads to mutations and cancer.