1. Chapter 5
DNA Repair

2. TABLE 5-2: Inherited Syndromes with Defects in DNA Repair
NAME PHENOTYPE ENZYME OR PROCESS AFFECTED
HNPCC colon cancer mismatch repair
MSH2, 3, 6, MLH1, PMS2
Xeroderma pigmentosum (XP) skin cancer, cellular UV sensitivity, nucleotide excision-rep
groups A-G neurological abnormalities
XP variant cellular UV sensitivity translesion synthesis by DNA
polymerase 
Ataxia-telangiectasia (AT) leukemia, lymphoma, cellular -ray ATM protein, a protein kinase activated sensitivity, genome instability by double-strand breaks
BRCA-2 breast and ovarian cancer repair by homologous recombination
Werner syndrome premature aging, cancers accessory 3’-exonuclease and DNA at several sites, genome instability helicase
Bloom syndrome cancer at several sites, accessory DNA helicase for replication stunted growth, genome instability
Fanconi anemia groups A-G congenital abnormalities, leukemia, DNA interstrand cross-link repair genome instability
46 BR patient hypersensitivity to DNA-damaging DNA ligase I
agents, genome instability

3. Spontaneous mutations that require DNA repair

4. Sites of DNA base damage

5. DNA intercalating agents

6. Depurination and deamination

7. Pyrimidine dimers

8. How do chemical modifications cause mutations?

9. Mutations caused by DNA modifications

10. General strategies for DNA repair
Direct Reversal of DNA damage:
Photolyases
Alkyltransferases
Excision-Repair:
Base Excision Repair
Nucleotide Excision Repair (NER)
Mismatch repair (MMR)
Transcription-coupled Repair (NER)
Double-Strand Break
Repair:
Non-homologous end-joining (NHEJ)
Homologous recombination (HEJ)
Error-prone Repair:
Trans-lesion replication (TLS) (SOS)

11. DNA photolyase

12. DNA methyltransferates

14. Glycosylases use base flipping to identify modified bases

15. Glycosylases use base flipping to identify modified bases

16. Nucleotide deamination

17. The cell can use multiple mechanisms to remove damaged bases

19. DNA mismatch repair

20. DAM methylation

21. Removal of mismatched DNA

22. Bacterial Nucleotide Excision Repair

23. Transcription-Coupled NER
RNAP
DNA lesion
Recognition of Stalled RNA Pol.
Recruitment of NER factors
Removal of 24 to 32 bp oligonucleotide
Fill-in of Gap

24. Methods for repairing double stranded breaks
Rad51

25. DSB -> homologous recombination in mitosis
Formulated to account for several experimental observations
in budding yeast… DNA transformation experiments
Linearized plasmids, cut in a region of similarity (“homology”) with a chromosome, transformed into yeast are repaired and integrated into the chromosome efficiently
Plasmids containing a gap in the homologous region are repaired by filling-in of all of the information present on the template but missing on the plasmid
chromosome
gapped plasmid
chromosome
repaired plasmid
Damaged molecule gains information from the donor

26. Non-homologous End Joining (NHEJ) KU: 2 proteins (Ku70p/Ku80p)
Recognizes ends
DNA-dependent protein kinase
(DNA-PKCS)
Rad50p-Mre11p-NBS1p:
exonuclease complex to process ends for ligation; checkpoint signaling
Ligase IV rejoins ends

27. Non-homologous End Joining (NHEJ)
One function of the MRE11/RAD50/NBS1 (MRN) complex may be to bring the ends together
(Xsr2)
Rad50 has structure similar to SMC proteins

28. Human syndromes associated with defects in DSB repair
Ataxia telangiectasia
Nijmegen breakage syndrome
Breast and Ovarian Cancer (BRCA1-2)

29. General strategies for DNA repair
Direct Reversal of DNA damage:
Photolyases
Alkyltransferases
Excision-Repair:
Base Excision Repair
Nucleotide Excision Repair (NER)
Mismatch repair (MMR)
Transcription-coupled Repair (NER)
Double-Strand Break
Repair:
Non-homologous end-joining (NHEJ)
Homologous recombination (HEJ)
Error-prone Repair:
Trans-lesion replication (TLS) (SOS)

30. E. coli DNA polymerases Enzyme Gene Function I polA repair
II polB replication start
III polC replicase
IV dinB translesion replication
V umuD’2C translesion replication

31. DNA pol Function Structure
 (alpha) Nuclear rep / primer syn 350 kD tetramer
 (delta) Nuclear replication / BER 250 kD tetramer
 (epsilon) Nuclear replication / BER 350 kD tetramer
 (gamma) Mitochondrial replication 200 kD dimer
high fidelity repair
 (beta) Base excision repair 39 kD monomer
low fidelity repair
 (zeta) Thymine dimer bypass heteromer
 (eta) Base damage repair monomer
 (iota) Meiosis/ TLS / somatic hyper monomer
(kappa) Deletion and base substitution monomer
 (theta) DNA repair of crosslinks monomer
 (lambda) Meiosis-assoc DNA repair monomer
 (mu) Somatic hypermutation monomer
Rev1 Translesion synthesis monomer

32. Specialized DNA polymerases
Have error rate 2-4 orders of magnitude higher than replicative DNA polymerases on undamaged templates
Lack 3’-5’ proofreading exonuclease activity
Not very processive
They support TLS of damaged DNA
Induced in bacteria; constitutively expressed in eukaryotes

33. Translesion synthesis
DNA Gene infidelity on
undamaged DNA
(relative to pol  = ~1)
 POLB ~50
 REV3L ~70
 POLK ~580
 POLH ~2000
 POLI ~20,000
 POLL ?
 POLM ?
 POLQ ?
Rev1 REV1L ?
Errol C. Friedberg, Robert Wagner, Miroslav Radman Specialized DNA Polymerases, Cellular Survival, and the Genesis of Mutations SCIENCE VOL MAY 2002

34. Translesion synthesis
TLS N M
Primer Extension N M
Resumption of Replication N M
Repair of Lesion N M N M
Errol C. Friedberg, Robert Wagner, Miroslav Radman Specialized DNA Polymerases, Cellular Survival, and the Genesis of Mutations SCIENCE VOL MAY 2002

35. } TABLE 1 Lesion bypass by one or two DNA polymerases
Two Pols Error-free or
DNA lesion One Pol Inserter Extender mutagenic
8-oxoG Polη Error-free
CPDs at TT, TC, CC sites Polη Error-free
(6-4) photoproducts:
at TT Polη Polζ Mutagenic
at TC and CC Polη Polζ Error-free
at TT, TC, and CC Polι Polζ Mutagenic
Abasic sites Polδ Polζ Mutagenic
Polη
Rev1
Polι
Tg Polδ Polζ Error-free
AAF- or AF-adducted G Polη Polζ Error-free
γ -HOPdG Rev1 Polζ Error-free
Polι Polκ Error-free }
CPDs = cyclobutane pyrimidine dimers
6-4 = another UV photo damage
Tg = Thymine glycol
N-2-acetyl aminofluorene (AAF)
γ -hydroxy-1,N2-propano-deoxyguanosine (γ -HOPdG) (
Satya Prakash, Robert E. Johnson, and Louise Prakash EUKARYOTIC TRANSLESION SYNTHESIS DNA POLYMERASES: Specificity of Structure and Function Annu. Rev. Biochem :317–53

36. Eukaryotic DNA polymerases

37. MicroReview
Evolution of mutation rates in bacteria. E. Denamur and I. Matic
Molecular Microbiology (2006) 60(4), 820–827
Fig. 3. Diffusion test for antibiotic susceptibility of E. coli natural isolates using commercial fosfomycin discs. The mutator strain (natural mutS– mutant), which generates mutations conferring resistance to the antibiotic at a high rate, is clearly differentiated from the nonmutator natural isolate by the presence of squatter colonies inside growth inhibition zone.