Genome Stability, Genetic Diseases and Cancer Lecture 1 DNA damage. Damage Reversal. Base excision repair. Mismatch repair Lecture 2 Nucleotide excision repair: cellular and clinical aspects Nucleotide excision repair: genes and proteins Lecture 3 Replication of damaged DNA. Mutagenesis and carcinogenesis Learning outcomes (Lecture 1a) Understanding: • Different types of DNA damage • Three examples of ways in which cells can reverse DNA damage in situ • Basic mechanism of Base Excision Repair 1.1 Procarcinogen Inactive Products Ultimate Carcinogen DNA DNA Damage Mutations Chromosome Rearrangements Cell Death Activation of Oncogenes Inactivation of Tumour Suppressor Genes Cancer Pre-cancerous cells Defence Mechanisms DNA Repair Detoxification 1.2 Cell cycle arrest - + + + + + + + + - + + - - - + - + + + Non distorting chemical damage Minor distorting chemical damage Major distorting chemical damage Interstrand cross links Strand breaks UV Ionizing Monofuctional Bifunctional Radiation Chemicals Chemicals DNA Damage H3C N N H O OH3C N N H O O 1 23 4 5 6 Cyclobutane pyrimidine dimer (CPD) H3C N NH2 O O 1 6 5 4 32 N N O 1 6 5 4 3 2 N TC (6-4) photoproduct (6-4PP) CH3 7-methylguanine O CH3 O-6-methylguanine CH3 3-methyladenine Major UV photoproducts Methylated purines 1.3 CH3 1-methyladenine 1 2 3 4 6 5 O CH3 3-methylcytosine Aspects of DNA repair 1. Initial damage 2. Repair of damage 3. Genes involved 4. Mechanism of action of gene products 5. Replication of unremoved damage. Cell cycle progression. 6. Biological consequences of damage, repair and failure to repair. 1.4 Damage reversal PR light No PR light UV Dose Survival 1. Photoreactivation 1.5 Cyclobutanepyrimidinedimers Time Friedberg et al, 2005 DNA Repair and Mutagenesis Photolyase mechanism 1.6 Friedberg et al, 2005 DNA Repair and Mutagenesis O CH3 CH3 CH3 Thymine O-6-methylguanine Mispairing of O-6-methylguanine with thymine 1.7 2. Repair of O6-methylguanine Damage reversal 6 6 Dual activities of Ada methyltransferase 1.9 Friedberg et al, 2005 DNA Repair and Mutagenesis Induction of ada gene 1.10 Ogt gene is not inducible Friedberg et al, 2005 DNA Repair and Mutagenesis • Similar mechanism to E. coli, but for O-6-meG alone, like Ogt, not inducible. • K/o mouse constructed, very sensitive to carcinogenesis by methylating agents. • Conversely transgenic mice bearing MGMT gene are more resistant. • Many cancer cell lines are Mex-. MGMT silenced by methylation in about 50% of tumours. • Mex- cells are sensitive to killing and mutagenesis by alkylating agents. • Many cancer therapy drugs are alkylating agents, eg temozolomide. • Patrin2 binds MGMT and depletes it. Currently in clinical trials together with temozolomide. Alkyltransferases in mammalian cells 1.11 Damage reversal 3. Oxidative demethylation (A4) wt alkB alkB survival Mechanism α-Ketoglutarate 1.12 Friedberg et al, 2005 DNA Repair and Mutagenesis Base Excision Repair 8-hydroxyguanine DNA glycosylases Enzyme E. coli Human Size (aa) Chromosome location of gene Altered base removed from DNA ung UNG2 313 12q23-q24 U and 5-hydroxyuracil (rep fork) MUG 410 12q24.1 U or T opposite G, ethenocytosine hSMUG1 270 12q13.1-q14 U (from G:U mismatches) MBD4 580 3q21 U or T opposite G at CpG sequences Fpg (MutM) hOGG1 345 3p25 8-oxo G opposite C, formamidopyrimidine MutY MYH 521 1p32.1-p34.3 A opposite 8-oxo G Nth hNTH1 312 16p13.2- Thymine glycol, cytosine glycol, dihydrouracil, formamidopyrimidine AlkA and Tag AAG 293 16p (near telomere) 3-MeA, ethenoadenine, hypoxanthine Nei Neil 1 Oxidised pyrimidines (rep fork) Neil2 Oxidised pyrimidines Neil3 Deamination of bases 5 3-d structures of glycosylases Ung Ogg1 AAG MutM 6 Protection from 8-oxoguanine Base Excision-Repair 7 XRCC1 P polβ or 3-d structures of APendonucleases APE1 ENDO IV Summary (Lecture 1a) • DNA damage can cause distortions of different severity • With visible light, UV damage is repaired by photoreversal (not in placental mammals) • O6-methylguanine is repaired by a specific methyltransferase • 1-methyladenine and 3methylcytosine are repaired by oxidative demethylation • Spontaneous lesions are removed by Base Excision Repair 1.13 Learning outcomes (Lecture 1b) Understanding: • Detailed mechanism of mismatch repair in E. coli and eukaryotes • How mismatch repair is important both for cancer protection and cancer therapy 1.14 Mismatch Repair (A5, A6) • DNA polymerases replicate DNA very faithfully. Accurate insertion Associated 3’-5’ exonuclease for proof-reading Error rates c. 10-6 or less • But genomes are big: E. coli 3x106 bp, mammals 3x109 • Errors can be single base mismatches or small insertions or deletions caused by base slippage • Mismatches are repaired by the MMR system which recognises the mismatched bases • But there’s a problem 1.15 Methylation-directed mismatch repair Dam- strains (methylation deficient) are mutators Dam methylase 1.16 Friedberg et al, 2005 DNA Repair and Mutagenesis Mismatch recognition and strand discrimination in E. coli 1.17 MutH, MutL and MutS- strains are mutators CH3 CH3 S2 L2 H 3-D structure of MutS 1.18 Structural homodimer Functional heterodimer Jiricny, Current Biology, 2000 Jiricny, MRMCB, 2006 Activities of MutS Sliding clamp Crucial Phe in Phe-X-Glu contacts mismatch Late steps in MMR in E. coli 1.19 Friedberg et al, 2005 DNA Repair and Mutagenesis UvrD helicase II unwinds SSB covers exposed ss DNA DNA Ligase Human homologues of MutH,L,S MutS: Msh2 MMR Msh3 MMR Msh4 Meiosis Msh5 Meiosis Msh6 MMR MutL: Mlh1 MMR Mlh2 ? Mlh3 MMR Pms1 ? Pms2 MMR (= Pms1 in yeast) MutH: No homologues Neither yeast nor Drosophila has methylated DNA Strand discrimination based on nicks/ends in daughter DNA MMR proteins interact with PCNA at replication fork 1.20 G T T GT GT GTC GTAC GTC Msh2 Msh3 MutSα Preference for single base mismatches and small IDL MutSβ Preference for large IDL G T Msh2 Msh6 Pms2 Mlh1 G T Msh2 Msh6 GTC Msh2 Msh3 Pms2 Mlh1 PCNA PCNA G C T A GT CA GT CA GTC CAG GTAC CATG Mismatch Repair in eukaryotes Primary recognition Secondary recognition Removal and restoration MutLα 1.21 IDL (Insertion-deletion loop) MutSα or β binds mismatch MutSα/MutLα translocates to end PCNA Lagging strand: Exo1 degrades 5’ to 3’ 1.22 Mismatch Repair in eukaryotes Leading strand: MutLα (Pms2 subunit) cleaves on 5’ side of mismatch. Exo1 degrades 5’ to 3’ 5’ 5’ 3’ 3’ Modified from Jiricny, Nature Rev Mol Cell Biol 2006 (ExoI) Microsatellite instability in tumour tissue from HNPCC (Hereditary non-polyposis colon carcinoma) Aaltonen, et al. Science 260, 812 (1993) CA CA CA CA CA CA CA GT GT GT GT GT GT GT Primer 1 Primer 2 CA CA CA CA CA CA GT GT GT GT GT GT CA Replication Slippage 7 microsatellite repeats 6 repeats1.23 2-hit tumour suppressor model 1 germ-line mutation Somatic mutation Sporadic colon cancer 1st somatic mutation 2nd somatic mutation Normal allele Mutant allele Most HNPCC result from mutations in hMsh2 or hMlh1 1.24 Extracts of tumour cells are deficient in MMR of dinucleotide loops and single base mismatches HNPCC HNPCC is autosomal dominant MMR proficient MMR deficient MMR proficient MMR proficient MMR deficient Microsatellite instability results from loss of Mismatch Repair 5' T-G-T-G-T-G-T-G-T-G 3' A-C-A-C-A-C-A-C-A-C-A-C-5' misalignmentT-G | | 5' T-G T-G-T-G-T-G 3' A-C-A-C-A-C-A-C-A-C-A-C T-G | | 5' T-G T-G-T-G-T-G-T-G-T-G 3' A-C-A-C-A-C-A-C-A-C-A-C 5' T-G-T-G-T-G-T-G-T-G 3' A-C-A-C-A-C-A-C-A-C-A-C 5' T-G-T-G-T-G-T-G-T-G-T-G 3' A-C-A-C-A-C-A-C-A-C-A-C extension reversal (proofreading) mismatch repair 1.25 • Microsatellite instability is a useful diagnostic tool. It’s not the cause of the cancers • Cancers arise from high rate of single-base mismatches during replication • These lead to high frequency of somatic mutations • Why only in colon? Not known O CH3 CH3 CH3 Thymine O-6-methylguanine Mispairing of O-6-methylguanine with thymine 1.7 2. Repair of O6-methylguanine Damage reversal 6 6 • In many tumour cells MGMT is silenced. So alkylating agents are good for therapy. • But often develop resistance. • Select for alkylation-resistance in cells. • MGMT not restored. O6-MeG remains in DNA. Instead cells have lost one of the MMR genes. • Implies MMR somehow sensitises cells to alkylation damage. • Result of futile cycles. O6-MeG:C and O6-MeG:T both recognised as mismatches. • C or T opposite O6-MeG removed by MMR and replaced with C or T. Futile cycles. • Results in cell cycle arrest or apoptosis MMR and resistant tumours 1.26 Loss of MMR protects against MNNG apoptosis 1.27 MMR and cancer MMR deficiency • Increases cancer susceptibility (HNPCC) • Results in resistance to cancer therapy Friedberg et al, 2005 DNA Repair and Mutagenesis Summary (Lecture 1b) • Mismatches are repaired by the Mut(H),L,S system • Mismatches are recognised by MutS and its homologues • Strand discrimination is brought about by methylation in E. coli and nicks/ends in daughter strands in eukaryotes • MMR deficiency leads to HNPCC and is detected by microsatellite instability • Loss of MMR results in resistance to alkylating agents 1.28