Most errors that arise during DNA replication can be corrected by DNA polymerase proofreading or by post-replication mismatch repair (MMR). Inactivation of both mutation-avoidance systems results in extremely high mutability that can lead to error catastrophe 1,2 . High mutability and the likelihood of cancer can be caused by mutations and epigenetic changes that reduce MMR 3,4 . Hypermutability can also be caused by external factors that directly inhibit MMR. Identifying such factors has important implications for understanding the role of the environment in genome stability. We found that chronic exposure of yeast to environmentally relevant concentrations of cadmium, a known human carcinogen 5 , can result in extreme hypermutability. The mutation specificity along with responses in proofreading-deficient and MMR-deficient mutants indicate that cadmium reduces the capacity for MMR of small misalignments and base-base mismatches. In extracts of human cells, cadmium inhibited at least one step leading to mismatch removal. Together, our data show that a high level of genetic instability can result from environmental impediment of a mutation-avoidance system.To identify factors that might influence MMR, we used long homonucleotide runs in the yeast Saccharomyces cerevisiae as reporters of altered MMR capacity. Long homonucleotide runs and other microsatellites are at-risk motifs in MMR-deficient cells 6 . These motifs are sensitive reporters for low levels of MMR 7-10 and for MMR-deficient cancers 3,4,11 because the correction of frequently occurring spontaneous frameshift errors in long homonucleotide runs and microsatellites is accomplished primarily by MMR. Frameshifts in shorter runs and base substitutions are corrected by proofreading as well as by MMR 8,12 .We chose to test divalent cations as potential inhibitors of MMR because they can affect many enzymatic reactions. Several divalent cations that are also carcinogens can decrease the fidelity of DNA synthesis in vitro 13 . Thus, it seemed possible that these cations could also target mutation-avoidance systems in vivo. We found that chronic exposure to low, non-lethal doses of cadmium (CdCl 2 ) caused a substantial increase in mutability (as much as 2,000-fold) of long homonucleotide runs in the yeast gene LYS2 (Fig. 1a and Because the stability of long homonucleotide runs is extremely sensitive to reduction in MMR capacity, we proposed that MMR itself is a target of cadmium. Mutations that completely eliminate DNA-damage repair pathways have never been reported to cause such strong mutator effects as are caused by MMR defects in long homonucleotide runs and other microsatellites 3 . Our experiments with isogenic strains that lack base-excision repair (rad27-Δ16), nucleotide excision repair (rad1-Δ, data not shown) and double-strand break repair (rad52-Δ, data not shown) identified only moderate mutator effects as compared with those caused by defects in MMR. The hypothesis about MMR inhibition was supported by a comparison of the effects of cadm...

A variety of pathologies are associated with exposure to supraphysiological concentrations of essential metals and to non-essential metals and metalloids. The molecular mechanisms linking metal exposure to human pathologies have not been clearly defined. To address these gaps in our understanding of the molecular biology of transition metals, the genomic effects of exposure to Group IB (copper, silver), IIB (zinc, cadmium, mercury), VIA (chromium), and VB (arsenic) elements on the yeast Saccharomyces cerevisiae were examined. Two comprehensive sets of metal-responsive genomic profiles were generated following exposure to equi-toxic concentrations of metal: one that provides information on the transcriptional changes associated with metal exposure (transcriptome), and a second that provides information on the relationship between the expression of ∼4,700 non-essential genes and sensitivity to metal exposure (deletome). Approximately 22% of the genome was affected by exposure to at least one metal. Principal component and cluster analyses suggest that the chemical properties of the metal are major determinants in defining the expression profile. Furthermore, cells may have developed common or convergent regulatory mechanisms to accommodate metal exposure. The transcriptome and deletome had 22 genes in common, however, comparison between Gene Ontology biological processes for the two gene sets revealed that metal stress adaptation and detoxification categories were commonly enriched. Analysis of the transcriptome and deletome identified several evolutionarily conserved, signal transduction pathways that may be involved in regulating the responses to metal exposure. In this study, we identified genes and cognate signaling pathways that respond to exposure to essential and non-essential metals. In addition, genes that are essential for survival in the presence of these metals were identified. This information will contribute to our understanding of the molecular mechanism by which organisms respond to metal stress, and could lead to an understanding of the connection between environmental stress and signal transduction pathways.

Many DNA polymerases (Pol) have an intrinsic 335 exonuclease (Exo) activity which corrects polymerase errors and prevents mutations. We describe a role of the 335 Exo of Pol ␦ as a supplement or backup for the Rad27͞Fen1 5 flap endonuclease. A yeast rad27 null allele was lethal in combination with Pol ␦ mutations in Exo I, Exo II, and Exo III motifs that inactivate its exonuclease, but it was viable with mutations in other parts of Pol ␦. The rad27-p allele, which has little phenotypic effect by itself, was also lethal in combination with mutations in the Pol ␦ Exo I and Exo II motifs. However, rad27-p Pol ␦ Exo III double mutants were viable. They exhibited strong synergistic increases in CAN1 duplication mutations, intrachromosomal and interchromosomal recombination, and required the wild-type double-strand break repair genes RAD50, RAD51, and RAD52 for viability. Observed effects were similar to those of the rad27-null mutant deficient in the removal of 5 flaps in the lagging strand. These results suggest that the 335 Exo activity of Pol ␦ is redundant with Rad27͞Fen1 for creating ligatable nicks between adjacent Okazaki fragments, possibly by reducing the amount of strand-displacement in the lagging strand.

To address the different functions of Pol ␦ and FEN1 (Rad27) in Okazaki fragment maturation, exonucleasedeficient polymerase Pol ␦-01 and Pol ␦-5DV (corresponding to alleles pol3-01-(D321A, E323A) and pol3-5DV-(D520V), respectively) were purified and characterized in this process. In the presence of the replication clamp PCNA, both wild-type and exo ؊ Pol ␦ carried out strand displacement synthesis with similar rates; however, initiation of strand displacement synthesis was much more efficient with Pol ␦-exo ؊ . When Pol ␦-exo ؊ encountered a downstream primer, it paused with 3-5 nucleotides of the primer displaced, whereas the wild type carried out precise gap filling. Consequently, in the absence of FEN1, Pol ␦ exonuclease activity was essential for closure of simple gaps by DNA ligase. Compared with wild type, Okazaki fragment maturation with Pol ␦-exo ؊ proceeded with an increased duration of nick translation prior to ligation. Maturation was efficient in the absence of Dna2 and required Dna2 only when FEN1 activity was compromised. In agreement with these results, the proposed generation of double strand breaks in pol3-exo ؊ rad27 mutants was suppressed by the overexpression of DNA2. Further genetic studies showed that pol3-exo ؊ rad27 double mutants were sensitive to alkylation damage consistent with an in vivo defect in gap filling by exonucleasedeficient Pol ␦.Efficient and faithful maturation of Okazaki fragments during DNA replication in eukaryotes depends on a coordinated degradation of the RNA primer strand by one or more nucleases along with gap-filling DNA synthesis by a replicative DNA polymerase followed by ligation of the remaining nick. Previous models based on a combination of biochemical and genetic studies have indicated a role for the flap 5Ј-endonuclease FEN1 and the nuclease/helicase Dna2 in carrying out degradation including the removal of a displaced flap and a role for DNA polymerase ␦ (Pol ␦) 1 to carry out DNA synthesis (1). However, biochemical experiments in the accompanying paper (3) indicate that the main degradative force is provided by FEN1. The activity of Dna2 becomes crucial only in cases where strand displacement proceeds to the extent that proteins inhibitory to FEN1 can bind to the displaced 5Ј-strand (2, 3). Many DNA polymerases have an intrinsic 3Ј-5Ј exonuclease activity, which corrects polymerase errors and prevents mutations. Recently, we provided genetic evidence for the action of the 3Ј-5Ј-exonuclease of Pol ␦ in the process of Okazaki fragment maturation in vivo (4, 5). This was indicated by synthetic lethality of rad27 (FEN1) mutants with several exodeficient mutants in Pol ␦ and by a dramatic increase in duplication mutations in viable pol3-exo Ϫ rad27 double mutants. We have suggested that the 3Ј-5Ј-exonuclease could be specifically involved in preventing the excessive formation of 5Ј-flaps by strand displacement synthesis.Okazaki fragment maturation is mediated by the concerted strand displacement of Pol ␦ and degradation of the displaced strand by the nucleas...