Chapter 5

•DNA Repair

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

Spontaneous mutations that require DNA repair

Sites of DNA base damage

DNA intercalating agents

Depurination and deamination

Pyrimidine dimers

How do chemical modifications cause mutations?

Mutations caused by DNA modifications

General strategies for DNA repair

Direct Reversal of DNA damage:•PhotolyasesPhotolyases•AlkyltransferasesAlkyltransferases

•Nucleotide Excision Repair (NER)Nucleotide Excision Repair (NER)

Double-Strand BreakRepair:

•Non-homologous end-joining (NHEJ)Non-homologous end-joining (NHEJ)•Homologous recombination (HEJ)Homologous recombination (HEJ)

•Mismatch repair (MMR)Mismatch repair (MMR)

Excision-Repair: •Base Excision RepairBase Excision Repair

Error-prone Repair:•Trans-lesion replication (TLS) (SOS)Trans-lesion replication (TLS) (SOS)

•Transcription-coupled Repair (NER)Transcription-coupled Repair (NER)

DNA photolyase

DNA methyltransferates

Glycosylases use base flipping to identify modified bases

Glycosylases use base flipping to identify modified

bases

Nucleotide deamination

The cell can use multiple mechanisms to remove damaged bases

DNA mismatch repair

DAM methylation

Removal of mismatched DNA

Bacterial Nucleotide Excision Repair

Transcription-Coupled NER

Recognition of Stalled RNA Pol.Recognition of Stalled RNA Pol.

Recruitment of NER factorsRecruitment of NER factors

Removal of 24 to 32 bp Removal of 24 to 32 bp oligonucleotideoligonucleotide

Fill-in of GapFill-in of Gap

DNA lesionDNA lesionRNAPRNAP

Methods for repairing double stranded breaks

Rad51

DSB -> homologous recombination in mitosis

Formulated to account for several experimental observationsin budding yeast… DNA transformation experiments

•Linearized plasmids, cut in a region of Linearized plasmids, cut in a region of similarity (“homology”) with a chromosome, similarity (“homology”) with a chromosome, transformed into yeast are repaired and transformed into yeast are repaired and integrated into the chromosome efficientlyintegrated into the chromosome efficiently•Plasmids containing a gap in the homologous Plasmids containing a gap in the homologous region are repaired by filling-in of all of region are repaired by filling-in of all of the information present on the template but the information present on the template but missing on the plasmidmissing on the plasmid

chromosome

gapped plasmid

chromosome

repaired plasmid

Damaged molecule gains information from the donor

Non-homologousEnd 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

Non-homologousEnd Joining

(NHEJ)One function of the One function of the MRE11MRE11//RAD50RAD50//NBS1NBS1 (MRN) complex may be to bring the ends (MRN) complex may be to bring the ends togethertogether

Rad50 has structure similar Rad50 has structure similar to SMC proteinsto SMC proteins

(Xsr2)(Xsr2)

Human syndromes associated with defects in DSB repair

Ataxia telangiectasia

Nijmegen breakage syndrome

Breast and Ovarian Cancer (BRCA1-2)

General strategies for DNA repair

Direct Reversal of DNA damage:•PhotolyasesPhotolyases•AlkyltransferasesAlkyltransferases

•Nucleotide Excision Repair (NER)Nucleotide Excision Repair (NER)

Double-Strand BreakRepair:

•Non-homologous end-joining (NHEJ)Non-homologous end-joining (NHEJ)•Homologous recombination (HEJ)Homologous recombination (HEJ)

•Mismatch repair (MMR)Mismatch repair (MMR)

Excision-Repair: •Base Excision RepairBase Excision Repair

Error-prone Repair:•Trans-lesion replication (TLS) (SOS)Trans-lesion replication (TLS) (SOS)

•Transcription-coupled Repair (NER)Transcription-coupled Repair (NER)

Enzyme Gene Function

I polA repair

II polB replication start

III polC replicase

IV dinB translesion replication

V umuD’2C translesion replication

E. coli DNA polymerases

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

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

Translesion synthesisDNA 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 296 31 MAY 2002

Translesion synthesis

TLSN M

Primer ExtensionN M

Resumption of ReplicationN M

Repair of LesionN MN M

Errol C. Friedberg, Robert Wagner, Miroslav Radman Specialized DNA Polymerases, Cellular Survival, and the Genesis of Mutations SCIENCE VOL 296 31 MAY 2002

TABLE 1 Lesion bypass by one or two DNA polymerasesLesion bypass by

Two Pols Error-free orDNA lesion One Pol Inserter Extender

mutagenic8-oxoG Polη Error-freeCPDs at TT, TC, CC sites Polη Error-free(6-4) photoproducts:at TT Polη Polζ Mutagenicat TC and CC Polη Polζ Error-freeat TT, TC, and CC Polι Polζ MutagenicAbasic sites Polδ Polζ Mutagenic

PolηRev1Polι

Tg Polδ Polζ Error-freeAAF- or AF-adducted G Polη Polζ Error-freeγ -HOPdG Rev1 Polζ Error-free

Polι Polκ Error-free

CPDs = cyclobutane pyrimidine dimers6-4 = another UV photo damage Tg = Thymine glycolN-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. 2005. 74:317–53

Eukaryotic DNA polymerases

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.

MicroReviewEvolution of mutation rates in bacteria. E. Denamur and I. MaticMolecular Microbiology (2006) 60(4), 820–827