DNA polymerase f (Polf) participates in translesion DNA synthesis and is involved in the generation of the majority of mutations induced by DNA damage. The mechanisms that license access of Polf to the primer terminus and regulate the extent of its participation in genome replication are poorly understood. The Polf-dependent damageinduced mutagenesis requires monoubiquitination of proliferating cell nuclear antigen (PCNA) that is triggered by exposure to mutagens. We show that Polf contributes to DNA replication and causes mutagenesis not only in response to DNA damage but also in response to malfunction of normal replicative machinery due to mutations in replication genes. These replication defects lead to ubiquitination of PCNA even in the absence of DNA damage. Unlike damage-induced mutagenesis, the Polf-dependent spontaneous mutagenesis in replication mutants is reduced in strains defective in both ubiquitination and sumoylation of Lys164 of PCNA. Additionally, studies of a PCNA mutant defective for functional interactions with Polf, but not for monoubiquitination by the Rad6/Rad18 complex demonstrate a role for PCNA in regulating the mutagenic activity of Polf separate from its modification at Lys164.

Loss of DNA mismatch repair due to mutation or diminished expression of the MLH1 gene is associated with genome instability and cancer. In this study, we used a yeast model system to examine three circumstances relevant to modulation of MLH1 function. First, overexpression of wild-type MLH1 was found to cause a strong elevation of mutation rates at three different loci, similar to the mutator effect of MLH1 gene inactivation. Second, haploid yeast strains with any of six mlh1 missense mutations that mimic germ line mutations found in human cancer patients displayed a strong mutator phenotype consistent with loss of mismatch repair function. Five of these mutations affect amino acids that are homologous to residues suggested by recent crystal structure and biochemical analysis of Escherichia coli MutL to participate in ATP binding and hydrolysis. Finally, using a highly sensitive reporter gene, we detected a mutator phenotype of diploid yeast strains that are heterozygous for mlh1 mutations. Evidence suggesting that this mutator effect results not from reduced mismatch repair in the MLH1/mlh1 cells but rather from loss of the wild-type MLH1 allele in a fraction of cells is presented. Exposure to bleomycin or to UV irradiation strongly enhanced mutagenesis in the heterozygous strain but had little effect on the mutation rate in the wild-type strain. This damage-induced hypermutability may be relevant to cancer in humans with germ line mutations in only one MLH1 allele.The stability of eukaryotic genomes depends heavily on several DNA repair processes, including correction of DNA replication errors by the DNA mismatch repair (MMR) system (reviewed in references 19, 26, and 46). Mutations in genes that inactivate mismatch repair strongly elevate spontaneous mutation rates and predispose humans to cancer. Current evidence suggests that germ line human MSH2 and MLH1 mutations account for a majority of hereditary nonpolyposis colorectal cancer (HNPCC) cases (31). Many of these result in loss of intact protein and are thus predicted to completely inactivate MMR. Others, such as the MLH1 missense mutations found in more than 30 HNPCC families (15,25,31), are often inferred to be pathogenic if they are nonconservative changes in evolutionary conserved amino acids, if they cosegregate with the disease, and/or if they are not observed in the normal population. However, unlike mutations that lead to protein truncation, single amino acid changes may not impair protein function or may only be partially inactivating.To assess the functional consequences of missense mutations in human MLH1, Shimodaira et al. (40) developed an assay in yeast, based on elimination of a dominant mutator phenotype conferred by expression of human MLH1 cDNA. They demonstrated that several human MLH1 missense mutations identified in HNPCC patients impair the function required for this dominant mutator effect. A more direct approach to address the effect of MLH1 missense mutations on MMR efficiency is based on the fact that the amino acid sequences of...

Translesion synthesis DNA polymerases contribute to DNA damage tolerance by mediating replication of damaged templates. Due to the low fidelity of these enzymes, lesion bypass is often mutagenic. We have previously shown that, in Saccharomyces cerevisiae, the contribution of the error-prone DNA polymerase z (Polz) to replication and mutagenesis is greatly enhanced if the normal replisome is defective due to mutations in replication genes. Here we present evidence that this defective-replisome-induced mutagenesis (DRIM) results from the participation of Polz in the copying of undamaged DNA rather than from mutagenic lesion bypass. First, DRIM is not elevated in strains that have a high level of endogenous DNA lesions due to defects in nucleotide excision repair or base excision repair pathways. Second, DRIM remains unchanged when the level of endogenous oxidative DNA damage is decreased by using anaerobic growth conditions. Third, analysis of the spectrum of mutations occurring during DRIM reveals the characteristic error signature seen during replication of undamaged DNA by Polz in vitro. These results extend earlier findings in Escherichia coli indicating that Y-family DNA polymerases can contribute to the copying of undamaged DNA. We also show that exposure of wild-type yeast cells to the replication inhibitor hydroxyurea causes a Polz-dependent increase in mutagenesis. This suggests that DRIM represents a response to replication impediment per se rather than to specific defects in the replisome components.

We have purified wild type and exonuclease-deficient four-subunit DNA polymerase ⑀ (Pol ⑀) complex from Saccharomyces cerevisiae and analyzed the fidelity of DNA synthesis by the two enzymes. Wild type Pol ⑀ synthesizes DNA accurately, generating single-base substitutions and deletions at average error rates of <2 ؋ 10 ؊5 and <5 ؋ 10 ؊7 , respectively. Pol ⑀ lacking 3 3 5 exonuclease activity is less accurate to a degree suggesting that wild type Pol ⑀ proofreads at least 92% of base substitution errors and at least 99% of frameshift errors made by the polymerase. Surprisingly the base substitution fidelity of exonuclease-deficient Pol ⑀ is severalfold lower than that of proofreading-deficient forms of other replicative polymerases. Moreover the spectrum of errors shows a feature not seen with other A, B, C, or X family polymerases: a high proportion of transversions resulting from T⅐dTTP, T⅐dCTP, and C⅐dTTP mispairs. This unique error specificity and amino acid sequence alignments suggest that the structure of the polymerase active site of Pol ⑀ differs from those of other B family members. We observed both similarities and differences between the spectrum of substitutions generated by proofreading-deficient Pol ⑀ in vitro and substitutions occurring in vivo in a yeast strain defective in Pol ⑀ proofreading and DNA mismatch repair. We discuss the implications of these findings for the role of Pol ⑀ polymerase activity in DNA replication.Replication of chromosomes in eukaryotes is believed to require three DNA polymerases, Pol ␣, 1 Pol ␦, and Pol ⑀ (for reviews, see Refs. 1-3). Pol ␣ has an associated DNA primase activity and synthesizes short RNA-DNA primers to initiate replication at origins and start Okazaki fragments on the lagging DNA strand. The roles of Pol ␦ and Pol ⑀ in chromosomal replication are not clearly defined. Evidence for essential roles of Pol ␦ and Pol ⑀ in replication comes from genetic studies in Saccharomyces cerevisiae. Mutational inactivation of catalytic subunits and some of the additional subunits of both polymerases is lethal. Strains carrying temperature-sensitive mutations in POL3 gene (encoding the catalytic subunit of Pol ␦), POL2, or DPB2 genes (encoding subunits of Pol ⑀) arrest in S phase of the cell cycle with a terminal morphology characteristic of a DNA replication defect upon shift to non-permissive temperature (4 -6). Incorporation of labeled precursors into DNA stops in these mutants at non-permissive temperature (4, 5, 7). The POL3, POL2, and DPB2 genes, as well as the DPB3 gene encoding the third subunit of Pol ⑀, are expressed periodically in the cell cycle with a peak in G 1 to S phase transition (4, 5, 8), which is characteristic of genes encoding DNA replication proteins. Both Pol ␦ and Pol ⑀ have intrinsic 3Ј 3 5Ј exonuclease activities that correct polymerase errors during replication (9, 10). Mutations in POL3 or POL2 inactivating the exonucleases result in a mutator phenotype (11,12).To accommodate roles for Pol ␦ and Pol ⑀ at a replication fork, it was proposed...