EXO1 interacts with MSH2 and MLH1 and has been proposed to be a redundant exonuclease that functions in mismatch repair (MMR). To better understand the role of EXO1 in mismatch repair, a genetic screen was performed to identify mutations that increase the mutation rates caused by weak mutator mutations such as exo1⌬ and pms1-A130V mutations. In a screen starting with an exo1 mutation, exo1-dependent mutator mutations were obtained in MLH1, PMS1, MSH2, MSH3, POL30 (PCNA), POL32, and RNR1, whereas starting with the weak pms1 allele pms1-A130V, pms1-dependent mutator mutations were identified in MLH1, MSH2, MSH3, MSH6, and EXO1. These mutations only cause weak MMR defects as single mutants but cause strong MMR defects when combined with each other. Most of the mutations obtained caused amino acid substitutions in MLH1 or PMS1, and these clustered in either the ATP-binding region or the MLH1-PMS1 interaction regions of these proteins. The mutations showed two other types of interactions: specific pairs of mutations showed unlinked noncomplementation in diploid strains, and the defect caused by pairs of mutations could be suppressed by high-copy-number expression of a third gene, an effect that showed allele and overexpressed gene specificity. These results support a model in which EXO1 plays a structural role in MMR and stabilizes multiprotein complexes containing a number of MMR proteins. A similar role is proposed for PCNA based on the data presented.Postreplicative DNA mismatch repair (MMR) enhances the fidelity of DNA replication by repairing errors made by the replicative DNA polymerases. Studies of the Escherichia coli MutHLS MMR system have been instrumental in providing insights into the general mechanism of MMR (for reviews, see references 40 and 50). A central player in E. coli MMR is MutS, which is the mismatch recognition factor. After MutS binds a mismatch, the MutL protein binds to MutS and activates the MutH endonuclease, which nicks hemimethylated DNA at unmethylated GATC sites. Subsequently, the action of one of a number of redundant single-stranded DNA-specific exonucleases and a DNA helicase, UvrD, degrades the mismatch-containing DNA from the nick (27,50,78). The resulting gap is filled in by the replicative machinery, including DNA polymerase III. Eukaryotic MMR is related to bacterial MMR in that it utilizes MutS-and MutL-related proteins, but it appears to be more complex (for reviews, see references 40, 41, and 50). Instead of a single MutS protein, eukaryotic MMR utilizes three MutS-related proteins, MSH2, MSH3, and MSH6, that form two different heterodimeric complexes (1,15,21,48,53). The MSH2 and MSH6 proteins form a complex that is important for the recognition of single-base mispairs and small insertion-deletion loops, whereas the MSH2 and MSH3 proteins form a complex that recognizes insertion-deletion loops (21,48,62). Similarly, eukaryotic MMR uses three MutL-related proteins, MLH1, PMS1, and MLH3, that also form heterodimeric complexes (20,45,51,57,58). Although the exact function of ...
DNA mismatch repair plays a key role in the maintenance of genetic fidelity. Mutations in the human mismatch repair genes hMSH2, hMLH1, hPMS1, and hPMS2 are associated with hereditary nonpolyposis colorectal cancer. The proliferating cell nuclear antigen (PCNA) is essential for DNA replication, where it acts as a processivity factor. Here, we identify a point mutation, pol30 -104, in the Saccharomyces cerevisiae POL30 gene encoding PCNA that increases the rate of instability of simple repetitive DNA sequences and raises the rate of spontaneous forward mutation. Epistasis analyses with mutations in mismatch repair genes MSH2, MLH1, and PMS1 suggest that the pol30 -104 mutation impairs MSH2/ MLH1/PMS1-dependent mismatch repair, consistent with the hypothesis that PCNA functions in mismatch repair. MSH2 functions in mismatch repair with either MSH3 or MSH6, and the MSH2-MSH3 and MSH2-MSH6 heterodimers have a role in the recognition of DNA mismatches. Consistent with the genetic data, we find specific interaction of PCNA with the MSH2-MSH3 heterodimer.In both prokaryotes and eukaryotes, defects in DNA mismatch repair cause elevated spontaneous mutation rates and increased instability of simple repeat DNA sequences. Mutations in any of the human mismatch repair genes hMSH2, hMLH1, hPMS1, and hPMS2 are associated with hereditary nonpolyposis colorectal cancer. Cell lines from these cancers are defective in DNA mismatch repair and display increased levels of spontaneous mutations and frequent alterations of microsatellite repeat sequences (1, 2).Epistasis analyses in yeast have suggested that MSH2 protein functions in conjunction with MSH3 or MSH6 protein in mismatch recognition. Genetic and biochemical studies in both yeast and humans have further indicated that the MSH2-MSH3 and MSH2-MSH6 complexes differ in substrate specificities. In yeast, mutations in MSH3 cause an increase in instability of microsatellite tracts but have little effect on single-base mispairs, whereas mutations in MSH6 have a more prominent effect on the incidence of single-base mispairs than on microsatellite tract instability (3-5). From these and other genetic observations, it has been inferred that MSH2-MSH3 complex is more proficient in the removal of insertion-deletion mismatches of two or more nucleotides (4), whereas MSH2-MSH6 is better at removing single nucleotide mismatches (4, 5). Human cell lines defective in the MSH6 component of the MSH2-MSH6 heterodimer hMutS␣ exhibit a selective loss in the repair of base-base and single-nucleotide insertion-deletion mismatches; the repair of two-, three-, and four-nucleotide insertion-deletion mismatches is reduced 2-4-fold in these cell lines (6, 7). Consistent with genetic observations, hMutS␣ binds a G/T mismatch or a one nucleotide insertion-deletion mismatch with high efficiency (6). By contrast, the yeast MSH2-MSH3 heterodimer exhibits little affinity for a G/T mismatch but binds insertion-deletion mismatches with high specificity (8). The manner by which PMS1 and MLH1 function in mismatch...
The Saccharomyces cerevisiae DNA polymerase delta proofreading exonuclease-defective mutation pol3-01 is known to cause high rates of accumulating mutations. The pol3-01 mutant was found to have abnormal cell cycle progression due to activation of the S phase checkpoint. Inactivation of the S phase checkpoint suppressed both the pol3-01 cell cycle progression defect and mutator phenotype, indicating that the pol3-01 mutator phenotype was dependent on the S phase damage checkpoint pathway. Epistasis analysis suggested that a portion of the pol3-01 mutator phenotype involves members of the RAD6 epistasis group that function in both error-free and error-prone repair. These results indicate that activation of a checkpoint in response to certain types of replicative defects can result in the accumulation of mutations.
To identify the regions of the proliferating cell nuclear antigen (PCNA) that are important for function in vivo, we used random mutagenesis to isolate 10 cold-sensitive (Cs−) and 31 methyl methanesulfonate-sensitive (Mmss) mutations of the PCNA gene (POL30) in Saccharomyces cerevisiae. Unlike the Mmss mutations, the CsC mutations are strikingly clustered in the interdomain region of the three-dimensional PCNA monomer structure. At the restrictive temperature, the Cs− pol30 mutants undergo a RAD9 dependent arrest as large-budded cells with a 2c DNA content. Defects in DNA synthesis are suggested by a significant delay in the progression of synchronized pol30 cells through S phase at the restrictive temperature. DNA repair defects are revealed by the observation that Cs− pol30 mutants are very sensitive to the alkylating agent MMS and mildly sensitive to ultraviolet radiation, although they are not sensitive to gamma radiation. Finally, analysis of the chromosomal DNA in pol30 cells by velocity sedimentation gradients shows that pol30 cells accumulate single-stranded DNA breaks at the restrictive temperature. Thus, our results show that PCNA plays an essential role in both DNA replication and DNA repair in vivo.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.