Cadmium acts as a mutagen by inhibiting mismatch repair. (Genotoxicity caused by inhibition of a mutation avoidance system) Dmitry A. Gordenin National Institute of Environmental Health Sciences (NIEHS) COLLABORATORS (all from NIEHS): Yong Hwan (Yohan) Jin Hanan Al-Refai Michael Resnick Alan Clark Tom Kunkel Rob Slebos Jack Taylor Environmental factors can cause genome instability ErrorError DamageDamage NormalNormal DNADNA MutantMutant DNADNA RepairRepair Can hyper-mutability be caused by environmental factor inhibiting repair? ErrorError DamageDamage NormalNormal DNADNA MutantMutant DNADNA RepairRepair High Error Rate (R) in DNA Replication is Reduced by HighProofreading Risks of and Genome Mismatch Instability Repair. Combination of MMR and proofreading defects causeDNA catastrophic Pol elongation mutability(R=10-5) (Morrison & Sugino; Schaaper) DNA Pol proofreading (R=10-2) – lethality (error catastrophe) in haploids Mismatch repair, MMR (R=10-3) – synergy between mutators Resulting error rate 10-10 errors/nt At-Risk Motifs (ARMs) -- long homonucleotide runs 3' → 5' Exo DNA - Pol Consequences: •Synergy between a mutator and an ARM LongFrameshift homonucleotide intermediates (bulges) in long runs homonucleotide are runs and other microsatellites escape DNA polymerase proofreading in vivo hyper-mutablethus leaving all burden ofin preventi MMR-deficientng mutations to mismatch cells repair. Long homonucleotide runs are hyper-mutable in MMR-deficient yeast Homonucleotide run Mutation rate (x10-9) msh2 mutator (frameshift in run) effect Mutation reporter - frameshiftsWild type in msh2lys2-A14 A4At-risk (-1 nt) motif hyper-mutable0.4 in MMR-mutants31 x 78 A7 (-1 nt) 4 1,550 x 408 A10Allows (-1 nt) to detect as little 47as 0.1% 314,000unrepaired mismatchesx 6,681 A14 (-1 nt) 164 1,600,000 x 9,756 A5 (+1 nt) 1 37 x 34 A8 (+1 nt) 10 3,440 x 344 A12 (+1 nt) 190 84,000 x 444 Cadmium is hyper-mutagenic in a yeast long homonucleotide run (lys2-A14) 1x >>100x Cadmium (Cd++) in nature (IARC, Vol. 58, 1993) 112 µg=1µmole •Natural occurrence: 100-500 µg/kg of the Earth's crust (mainly associated with zinc) •Air: 0.05-0.5 µg/m3; occupational - mining, battery, paint, metal industries. •Water: <0.005 µg/L - 405 µg/L •Soil and plants: <1,000 µg/kg - 800,000 µg/kg •Cigarette smoke: A smoker can accumulate 500 µg/year. •Food: 10µg/day to 500 µg/day •Animal and human tissues: Liver, kidney, prostate (0.1-500 mg/kg). (The half-life of cadmium in human kidneys is around 10-20 years.) Cadmium carcinogenicity and genotoxicity (IARC, Vol. 58, 1993; NTP 10th Report on Carcinogens, 2002) Carcinogenicity: • lung cancer and prostate cancer (limited evidence) in humans • lung, testicular, adrenal, liver, prostate tumors as well as lymphomas in experimental animals Genotoxicity (mostly from acute short-term treatment): • chromosomal aberrations in lymphocytes of exposed workers • chromosomal aberrations and strand breaks in cultured mammalian cells • gene mutations in cultured mammalian cells (hprt, gpt) • intra-chromosomal recombination in yeast Multiple Potential Mechanisms of Metal Genotoxicity inhibiting repair suppressing fidelity oxidative damage to DNA oxidative damage to proteins complexes with DNA competing with "physiological“ metals for metalothioneins Cadmium caused hyper-mutability in yeast, unlike other ions tested H2OCoClCdCl2 2 CuSO4 MnCl2 NiSO4 ZnSO4 Mutation reporter - frameshifts in lys2-A14 At-risk motif hyper-mutable in MMR-mutants Multiple Potential Mechanisms of Cadmium Genotoxicity inhibiting repair suppressing fidelity oxidative damage to DNA oxidative damage to proteins complexes with DNA competing with "physiological“ metals for metalothioneins Hyper-mutability is not due to general oxidative damage H2O CdCl2 H2O2 t-BOOH Paraquat FeCl3 1x>>100x 1x 1x 1x 1x In vitro MMR assay system (Umar et al., Cell 1996) Cadmium inhibits DNA mismatch repair in extract from human cells (0%) (2.7%) (28%) (61%) (82%) % repair (95%) CdCl2 (µM) Cadmium is hyper-mutagenic (as much as x2,000) to wild type yeast cells Cadmium is not mutagenic to yeast cells that are deficient in MMR (msh2) lys2-14A -2 msh2 -3 -4 -5 WT -6 log (mutation rate) -7 -8 0 1 2 3 4 5 µM CdCl2 Interaction between mutator effects of cadmium and MMR-null resembles epistatic interactions between two mutator defects in the same pathway. How to prove that yeast MMR is in vivo target for cadmium ? Compare wild type yeast grown on cadmium with MMR-deficient yeast mutants High Error Rate (R) in DNA Replication is Reduced by HighProofreading Risks of and Genome Mismatch Instability Repair. Combination of MMR and proofreading -5 defects causeDNA catastrophic Pol elongation mutability(R=10 ) (Morrison & Sugino; Schaaper) DNA Pol proofreading (R=10-2) – lethality (error catastrophe) in haploids Mismatch repair, MMR (R=10-3) – synergy between mutators Resulting error rate 10-10 errors/nt Cadmium Causes Synergistic Hyper-mutability Combined with Defects in Proofreading -2 his7-2-2 CAN1 -3 -3 Wild Types -4 -4 (1n) (2n) -5 -5 -6 -6 Proofreading Defects log (mutation rate) -7 -7 (ε) pol2-4 (1n) (δ) pol3-01 (2n) -8 -8 0 1 2 3 4 5 0 1 2 3 4 5 µM CdCl2 Cadmium reduces viability of Pol δ Exo-deficient haploid (Note: viability of isogenic diploids is not reduced) 100 1n pol2-4 80 2n pol3-01 (Pol δ-Exo- diploid) 2n wild type 1n wild type 60 40 % viability % - 20 1n pol3-01 (Pol δ-Exo haploid) 0 0 1 2 3 4 5 CdCl2 (µM) “Synthetic lethality” of cadmium with Pol δ Exo-deficiency can be due to catastrophic rate of recessive lethals. Compare MMR-Deficient Yeast with Yeast Grown on Cadmium MMR deficiency CadmiumCadmium Synergy with Proofreading Defects yes -hyper-mutability -synthetic lethality Cadmium Can Induce Base Substitutions and Frameshifts Frameshifts Base substitutions 1000 1000 800 800 -7 -7 600 600 400 Rate x 10 400 Rate x 10 200 200 0 0 WT pol2-4 WT pol2-4 0.4 2.2 0 uM 0 uM 310 182 960 5 uM 5 uM 48 840 Mutations in the yeast CAN1 gene Compare MMR-Deficient Yeast with Yeast Grown on Cadmium MMR deficiency CadmiumCadmium Synergy with Proofreading Defects yes -hyper-mutability -synthetic lethality Base substitutions and frameshifts yes At-Risk Motifs (ARMs) -- long homonucleotide runs and other microsatellites are poor substrates for the 3' →5' Exo of DNA Pol Consequences: •homonucleotide runs are hyper-mutable in MMR-deficient cells •mutation rate depends on the size of run and type of frameshift - homonucleotide runs are hyper-mutable in MMR-deficient cells - mutation rate depends on the size of run and type of frameshift Homonucleotide run Mutation rate (x10-9) mutator effect (frameshift in run) of the msh2 Wild type msh2 A4 (-1 nt) 0.4 31 x 78 A7 (-1 nt) 4 1,550 x 408 A10 (-1 nt) 47 314,000 x 6,681 A14 (-1 nt) 164 1,600,000 x 9,756 A5 (+1 nt) 1 37 x 34 A8 (+1 nt) 10 3,440 x 344 A12 (+1 nt) 190 84,000 x 444 Size of a run and a type of frameshift: Relative mutability of runs mimics MMR-deficiency -2 -3 14A, wt A14, msh2 A10, msh2 10A, wt -4 A12, msh2 12A, wt -5 -6 log (mutation rate) -7 1 2 3 4 5 -8 µ 0 M CdCl 2 A14, -1 nt mutation A10, -1 nt mutation A12, +1 nt mutation Relative mutability of G-, C-, T-, and A- runs mimics MMR-deficiency -2 -3 10G-; 10C- mutation rates in 10T- MMR-null (msh2) mutants -4 }10A- (from Harfe and Jinks-Robertson, 2000) -5 SJR, lys2-10G -6 log (mutation rate) SJR, lys2-10C SJR, lys2-10T -7 SJR, lys2-10A -8 0 5 10 15 20 µM CdCl2 Compare MMR-Deficient Yeast with Yeast Grown on Cadmium MMR deficiency CadmiumCadmium Synergy with Proofreading Defects yes -hyper-mutability -synthetic lethality Base substitutions and frameshifts yes Mutator signature in homonuleotide runs: yes -type of frameshift -size of a run -sequence of a run Compare MMR-Deficient Yeast with Yeast Grown on Cadmium MMR deficiency = CadmiumCadmium Synergy with Proofreading Defects -hyper-mutability yes -synthetic lethality WildBaseWild substitutions typetype and frameshiftsyeastyeast growngrownyes onon cadmiumcadmiumMutator signature inbehavebehave asas ifif theythey homonuleotide runs: yes areare deficientdeficient-type of frameshift inin mismatchmismatch -size of a run -sequencerepairrepair of a run Hyper-mutability is caused by cadmium via inhibiting post-replication mismatch repair (MMR) Very low (micromolar) concentrations of Cd2+ ions, similar to those present in the environment and accumulated in organisms, inhibit MMR in yeast and human cell extract leaving about 20- 50% mismatches unrepaired. Cadmium is a new kind of mutagen that causes hyper-mutability by inhibiting mutation avoidance DNA repair system, rather than by damaging DNA. Specific notes of relevance: Cadmium is common in the environment. MMR system prevents mutations and cancer. What is the target for cadmium inside eukaryotic MMR? Mismatch recognition MutL Mlh1 Pms1 Mlh1 Mlh3 homologs MutS Msh2 Msh6 PCNA; Msh2 Msh3 homologs + RPA; RFC 1 nt ≥2nt G T Mismatch removal and gap filling Exo1; Pol δ; Pol ε OTHER QUESTIONS: Effect of cadmium on MMR in other species (pathogenic fungi, adaptive changes, evolution) Effect of cadmium on other functions of MMR proteins (meiosis, recombination, apoptosis, damage recognition, tolerance to alkylation damage) Effect of cadmium on mammalian MMR (species, cell types, tissues, genotypes, relation to carcinogenesis). Other environmental factors and drugs that could inhibit MMR Mutagenesis caused by inhibiting other repair and fidelity systems with environmental factors and drugs Mutagenesis caused by inhibiting repair and fidelity systems by environmental factors and drugs Where to look for targets? Identify sensitized motifs in protein structures Identify repair components present at the "threshold" level Identify repair components present at the "threshold" level 100% repair Repaired fraction of mismatches at different concentrations of cadmium.