Learning outcomes (Lecture 3a) Replication of damaged DNA Understanding: • Basic mechanism of damage avoidance by recombination repair in E. coli • Concept of translesion synthesis • Y-family polymerases and XP variants • Polymerase switching H3C N N H O H3C N N H O O 1 23 4 5 6 Cyclobutane thymine dimer H3C N NH2 O1 6 5 4 32 N TC (6-4) photoproduct Major UV photoproducts CH3 CH3 O CH3 7-methylguanine 3-methyladenine O-6-methylguanine Methylated purines 1. No effect, eg 7Me-G. 2. Misreplication, eg O6-MeG 3. Lesion obstructs fork progression 4. Lesion stops initiation 5. Lesion arrests cell cycle. Effects of DNA damage on replication 3.1 Model for recombination repair of daughter-strand gaps uvrA- strains tolerate 50 CPD per genome New DNA is small, gets bigger. Repair synthesis and Major mechanism in E. coli Friedberg et al, 2005 DNA Repair and Mutagenesis 3.2 Mutations UV dose uvrA- umuDC- recA- wt Survival UV dose uvrA- umuDC- recA- wt Genetics of UV mutagenesis A. E. coli uvrA- recA- 3.3 SOS Response In E.coli, recA, umuCD mutants are not mutable by UV light. LexA is a repressor of about 30 genes including recA, umuCD (as well as NER genes). LexA recA umuD umuC LexA LexA UmuD’ UmuD’ UmuC RecA RecA* RecA is activated by ssDNA, exposed at replication fork when it encounters DNA damage (RecA*). RecA* catalyses cleavage and inactivation of lexA repressor. Results in increased levels of RecA* and UmuDC. RecA* also catalyses cleavage of N-terminal 24aa from UmuD UmuD’ UmuD’2C is DNA Pol V, which, unlike Pol III, can synthesise past DNA damage – but it makes errors Pol III Pol III ^ Pol V vvvvvvvvv vvvvvvvvv ^ vvvvvvvvvvv ^ b-clamp Translesion synthesis (TLS) (A8, A10) Quantitatively minor, but v important 3.4 Translesion Synthesis (TLS) Replication restart Replacement of by TLS DNA polymerase TLS polymerase can incorporate either correct or incorrect nucleotide Replicative DNA synthesis is blocked by DNA lesion 3.5 DNA Polymerases Name Function 3’-5’ Exonuclease Proofreading Processivity Fidelity TLS Pol I Removal of RNA primers; Repair synthesis: BER and NER Yes High High No Pol II TLS Yes (Weak) High Yes Pol III Replication, MMR Yes V. high V. high No *Pol IV TLS No Low Low Yes E. coli *Pol V TLS No Low Low Yes Pol α RNA-DNA priming during replication No Low High No Pol β BER No Moderate Moderate Poor Pol δ Replication, NER Yes V. high V. high No Pol ε Replication, NER Yes V. high V. high No Pol ζ TLS No Low Low Yes *Rev1 TLS No Low Yes *Pol η TLS (CPD) No Low Low Yes *Pol TLS No Low Low Yes Mammalian *Pol  TLS No Low Low Yes * Y-family of DNA polymerases 3.6 • Conserved catalytic domain at N-terminus • Finger, palm and thumb domains characteristic of DNA polymerases • Extra Little finger domain • C-terminal third involved in protein-protein interactions • Catalytic domains have more open structure • Can accommodate damaged bases in active sites • Error-prone on undamaged DNA • Poor processivity Properties of Y-family polymerases Structure of a Y-family polymerase Ling et al., Cell 20013.7 High-fidelity (closed) replicative DNA polymerase Low-fidelity (open) TLS DNA polymerase Xeroderma Pigmentosum Patients XP-C XP variant XP-D Robbins et al 1974 Properties of XP, CS and TTD complementation groups 1. Clinical features Repair characteristics Group Skin Cancer 2. Neurological abnormalities 3. Relative frequency of occurrence 4. UV- sensitivity 5. Residual UDS* 6. Remarks XP-A + ++ high +++ <5 XP-B +/- +++/+ very rare ++ <10 Combined XP/CS or TTD XP-C + - high + 15-30 Deficient in ‘global genome’ repair. Normal transcription-coupled repair XP-D + ++/- intermediate ++ 15-50 Includes patients with TTD and patients with XP/CS XP-E +/- - rare + >50 XP-F +/- - rare/ intermediate + 15-30 Repair slow but prolonged XP-G +/- +++/+ rare ++ <10 Includes patients with XP/CS XP-V + - high + 100 Defective in post-replication repair. Normal NER CS-A - ++ rare + 100 Defective in transcriptioncoupled repair ‘Global genome’ repair normal CS-B - ++ high + 100 Defective in transcriptioncoupled repair ‘Global genome’ repair normal TTD-A - + very rare + 15 TTD *Unscheduled DNA synthesis as a percentage of wild-type activity • XP-Variant patients are hypersensitive to sunlight-induced pigmentation changes and skin cancer • XP-V cells carry out normal nucleotide excision repair but are defective in their replication of UV-damaged DNA (postreplication repair) • The cells are only mildly sensitive to killing by UV • This sensitivity can be increased with caffeine (diagnostic test) • They are hypermutable with UV light XP variants 3.8 Diagnostic test for XP Variant Patients • UDS is normal • Cell survival after UV is close to normal • Cell survival after UV is reduced by caffeine Survival UV dose XP-V wt Wt +caffeine XP-V+caffeine 3.9 Mutations UV dose uvrA- umuDC- recA- wt Survival UV dose uvrA- umuDC- recA- wt Genetics of UV mutagenesis A. E. coli Survival UV dose XP-V XP-A wt B. Human cells Mutations UV dose XP-A XP-V wt uvrA- recA- 3.10 • XP-Variant patients are hypersensitive to sunlight • XP-V cells carry out normal nucleotide excision repair but are defective in their replication of UV-damaged DNA (postreplication repair) • The cells are only mildly sensitive to killing by UV • This sensitivity can be increased with caffeine (diagnostic test) • They are hypermutable with UV light • They are defective in Polh XP variants 3.11 DNA polymerase h • Member of Y-family • Can carry out TLS past CPDs • Puts correct bases opposite CPD! • Can carry out TLS past other lesions inefficiently • Inaccurate on undamaged template NB TLS is the major pathway in mammalian cells What do the other Y-family pols do? Different lesions Insertion and extension? DNA Polymerases Name Function 3’-5’ Exonuclease Proofreading Processivity Fidelity TLS Pol I Removal of RNA primers; Repair synthesis: BER and NER Yes High High No Pol II TLS? Yes (weak) High Yes Pol III Replication, MMR Yes V. high V. high No *Pol IV TLS No Low Low Yes E. coli *Pol V TLS No Low Low Yes Pol α RNA-DNA priming during replication No Low High No Pol β BER No Moderate Moderate Poor Pol δ Replication, NER Yes V. high V. high No Pol ε Replication, NER Yes V. high V. high No Pol ζ TLS No Low Low Yes *Rev1 TLS No Low Yes *Pol η TLS (CPD) No Low Low Yes *Pol TLS No Low Low Yes Mammalian *Pol  TLS No Low Low Yes Insertion and extension Replication restart Replacement of by first TLS DNA polymerase First TLS polymerase inserts opposite damage Replicative DNA synthesis is blocked by DNA lesion 3.12 Replacement with second TLS polymerase and extension of chain Polz is a good extender: needed for replication past most lesions (except CPD – polh can do it all) Proteins involved in replication of DNA damage in S. cerevisiae • Rad6 and Rad18 are required for all processes of postreplication repair • Mms2, Ubc13 and Rad5 are involved in an error-free branch • Rad6 and Ubc13-Mms2 are E2 Ubiquitin conjugating enzymes • Rad18 and Rad5 are E3 ubiquitin ligases • Multiple interactions (Ulrich and Jentsch) Rad18 Rad5 Rad6 Mms2 Ubc13 3.13 E2 (Ub-lys63) E2 E3E3 Polymerase Switch A9 Ub + E1 Ub-E1 Ub-E2 Ub-target E2 E1 E3 PCNA is the ubiquitination substrate B5 3.14 K164 K164 K164 DNA PIP-box proteins bind here Interdomain connecting loop Switching via ubiquitination of PCNA Ubiquitination-mediated switch TLS pold polh Switch back PIR pold polh PCNA PIRUBZ polh U PIR UBZ UBZ (B5, A9) Rad6-Rad18 Y-family pols have ub-binding motifs polh U PIR UBZ Poly-ubiquitination (Lys 63) UUU Mms2-Ubc13 Rad5 Template switch damage avoidance U U Replication of damage and errors • All Y-family polymerases have ubiquitin-binding domains • So they can all bind to Ubiquitinated PCNA • With UV-irradiated DNA, polh makes few errors • In its absence, others can substitute. They make more errors • May need two pols to get past some types of damage, for insertion and extension • TLS can be error-free, but is usually error-prone • The template – switch mechanism is error-free 3.16 Mutations UV dose uvrA- umuCD- recA- wt Survival UV dose uvrA- umuCD- recA- wt Genetics of UV mutagenesis A. E. coli Survival UV dose XP-V XP-A wt B. Human cells Mutations UV dose XP-A XP-V wt 3.10 (1) Replication A G C T T T A A G C T T T A T C G A A A T A G C T T TA A G C T T T A T C G A A A T (2) UV(3) Nucleotide Excision repair (4) a. Accurate TLS polymerase or b. Homologous recombination c. Template switching ^ ^ A G C T T T A T C G A G A T (5) Inaccurate TLS polymerase ^ In bacteria, UvrABC proteins needed for (3) Therefore in uvr ABC- cells, more mutations via step (5) RecA needed for (4) and (5). So no mutations in recA- cells UmuCD needed for (5). So no mutations in umuCD- cells In humans, no excision-repair in excision-defective XPs, so more mutations via step (5) In XP variants, step (4) a. is deficient, so more mutations via step (5) Ubiquitination of PCNA modulates channelling into different pathways UV mutagenesis 3.17 Cancer Summary (Lecture 3a) • In E. coli avoidance of damage by recombination is the major pathway • Mutations are generated by translesion synthesis (TLS) using PolV • TLS is carried out by the specialised Y-family of DNA polymerases • XP variants are defective in polh • Polymerase switching is mediated by the ubiquitination of PCNA Learning outcomes (Lecture 3b) Understanding: • Age-related incidence of cancer • Interpretation of mutation signatures in tumours • Links between DNA damage and ageing Procarcinogen Inactive Products Ultimate Carcinogen DNA DNA Damage Mutations Chromosome Rearrangements Cell Death Activation of Oncogenes Inactivation of Tumour Suppressor Genes CancerPre-cancerous cells Defence Mechanisms DNA Repair Detoxification Cell cycle arrest Other disorders 1.1 Age-related cancer incidence Cancer incidence proportional to (Age)6 Interpreted to indicate need for 6 events (mutations, chromosome rearrangements) Slope = 6 3.18 Mutations in skin cancer (A11) • Database of mutations in p53 gene • 60% skin cancers have p53 mutations. All at dipyrimidines, 65% CT • BCC 12% CC  TT, SCC 15%, very characteristic of UV mutations, very different from internal tumours. • More striking in XP tumours as well. 90% C T; 60%CC  TT. • Strong evidence that sunlight induced damage results in p53 mutations. P53 • Skin cancers Basal Cell Carcinoma (BCC) Squamous cell carcinoma (SCC) Malignant Melanoma (MM) • Cell culture: UV mutations are mainly C T; CCTT at dipyrimidine sites. • Gorlin’s syndrome – high frequency of BCC. • Gene cloned and found to be PTCH1, human homologue of Drosophila patched. • Protein is a transmembrane glycoprotein receptor for Hedgehog signalling. Involved in control of differentiation and proliferation. Not a DNA repair gene • Mutations in PTCH1 gene in BCCs in XPs. • Found in 73% XP BCCs, half are CC to TT. Implies important step in BCC development. PTCH1 3.19 Colon cancer Loss of a TSG Change in Gene expression Activation of oncogene Loss of a TSG Loss of a TSG Associated with familial polyposis coli (FPC) Associated with familial nonpolyposis coli (HNPCC) 3.20 • Mismatch repair deficiency results in general increase in mutation frequency • 65% of HNPCC tumours have p53 mutations • Mutations mainly C T, but not at dipyrimidine sites, at CpG sites • Cytosine spontaneously hydrolyses to uracil, which is removed by BER • Cytosines are methylated at 5 position at many CpG sites • 5MeC hydrolyses to thymine, resulting in a G:T mismatch, repaired by MMR not BER • In HNPCC, G:T mismatches repaired poorly. • This is the major source of p53 mutations in HNPCC p53 mutations in HNPCC 3.21 G: G: G: G: Cancer in XP, TTD, CS • Why no cancer in TTD and CS despite NER defects? • TTD? Transcription defect interferes with cancer progression? • What about CS, not essential genes? Most mutations nulls. • How can we explain the complex combined features of XP and CS, in some XP-B, XP-D, XP-G patients? Unanswered questions in XP, CS and TTD XP TTD CS Neurological abnormalities XP-A, D, G progressive neurological degeneration CS, TTD dysmyelination, mental retardation ?oxidative damage in brain? 3.22 TTD mouse Ageing (A12, B6) • Long-standing hypothesis that decreased repair is a cause of ageing. • Aspects of premature ageing in CS. • TTD mouse: after 1 year looks very old. • XP-A/TTD double even more extreme, implies DNA damage and transcriptional defect result in premature ageing. What is damage? DNA damage Cell death Ageing Mutations Cancer TCRGGR Hoeijmakers hypothesis of ageing and cancer XP CS 3.23 Summary (Lecture 3b) • Cancer results from about 6 genetic changes • Mutation signatures in skin cancers show importance of UV damage in p53 and PTCH1 genes • p53 mutations at CpG sites are important in HNPCC • Unrepaired DNA damage plays a role in ageing