scholarly journals Mutations in the Bacillus subtilis β Clamp That Separate Its Roles in DNA Replication from Mismatch Repair

2010 ◽  
Vol 192 (13) ◽  
pp. 3452-3463 ◽  
Author(s):  
Nicole M. Dupes ◽  
Brian W. Walsh ◽  
Andrew D. Klocko ◽  
Justin S. Lenhart ◽  
Heather L. Peterson ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Dna Replication ◽  
Mismatch Repair ◽  
Mutation Frequency ◽  
Elevated Temperatures ◽  
Temperature Sensitive ◽  
Mutant Form ◽  
Appreciable Effect ◽  
Missense Mutations ◽  
Dna Mismatch

ABSTRACT The β clamp is an essential replication sliding clamp required for processive DNA synthesis. The β clamp is also critical for several additional aspects of DNA metabolism, including DNA mismatch repair (MMR). The dnaN5 allele of Bacillus subtilis encodes a mutant form of β clamp containing the G73R substitution. Cells with the dnaN5 allele are temperature sensitive for growth due to a defect in DNA replication at 49°C, and they show an increase in mutation frequency caused by a partial defect in MMR at permissive temperatures. We selected for intragenic suppressors of dnaN5 that rescued viability at 49°C to determine if the DNA replication defect could be separated from the MMR defect. We isolated three intragenic suppressors of dnaN5 that restored growth at the nonpermissive temperature while maintaining an increase in mutation frequency. All three dnaN alleles encoded the G73R substitution along with one of three novel missense mutations. The missense mutations isolated were S22P, S181G, and E346K. Of these, S181G and E346K are located near the hydrophobic cleft of the β clamp, a common site occupied by proteins that bind the β clamp. Using several methods, we show that the increase in mutation frequency resulting from each dnaN allele is linked to a defect in MMR. Moreover, we found that S181G and E346K allowed growth at elevated temperatures and did not have an appreciable effect on mutation frequency when separated from G73R. Thus, we found that specific residue changes in the B. subtilis β clamp separate the role of the β clamp in DNA replication from its role in MMR.

scholarly journals Phosphorylation of PCNA by EGFR inhibits mismatch repair and promotes misincorporation during DNA synthesis

2015 ◽  
Vol 112 (18) ◽  
pp. 5667-5672 ◽  
Author(s):  
Janice Ortega ◽  
Jessie Y. Li ◽  
Sanghee Lee ◽  
Dan Tong ◽  
Liya Gu ◽  
...  
Keyword(s):  
Dna Replication ◽  
Dna Synthesis ◽  
Mismatch Repair ◽  
Posttranslational Modifications ◽  
Growth Factor Receptor ◽  
Nuclear Antigen ◽  
Dna Mismatch ◽  
Epidermal Growth ◽  
Mismatch Recognition ◽  
Proliferating Cell

Proliferating cell nuclear antigen (PCNA) plays essential roles in eukaryotic cells during DNA replication, DNA mismatch repair (MMR), and other events at the replication fork. Earlier studies show that PCNA is regulated by posttranslational modifications, including phosphorylation of tyrosine 211 (Y211) by the epidermal growth factor receptor (EGFR). However, the functional significance of Y211-phosphorylated PCNA remains unknown. Here, we show that PCNA phosphorylation by EGFR alters its interaction with mismatch-recognition proteins MutSα and MutSβ and interferes with PCNA-dependent activation of MutLα endonuclease, thereby inhibiting MMR at the initiation step. Evidence is also provided that Y211-phosphorylated PCNA induces nucleotide misincorporation during DNA synthesis. These findings reveal a novel mechanism by which Y211-phosphorylated PCNA promotes cancer development and progression via facilitating error-prone DNA replication and suppressing the MMR function.

scholarly journals Tumors arising in DNA mismatch repair-deficient mice show a wide variation in mutation frequency as assessed by a transgenic reporter gene

2000 ◽  
Vol 21 (6) ◽  
pp. 1259-1262
Author(s):  
Agnes Baross-Francis ◽  
M.Kate Milhausen ◽  
Susan E. Andrew ◽  
Gareth Jevon ◽  
Frank R. Jirik
Keyword(s):  
Mismatch Repair ◽  
Reporter Gene ◽  
Mutation Frequency ◽  
Dna Mismatch Repair ◽  
Dna Mismatch ◽  
Deficient Mice

scholarly journals Characterization of Pathogenic Human MSH2 Missense Mutations Using Yeast as a Model System: A Laboratory Course in Molecular Biology

2004 ◽  
Vol 3 (1) ◽  
pp. 31-48 ◽  
Author(s):  
Alison E. Gammie ◽  
Naz Erdeniz
Keyword(s):  
Molecular Biology ◽  
Mismatch Repair ◽  
Recombinant Dna ◽  
Dna Mismatch Repair ◽  
Genetic Material ◽  
Molecular Techniques ◽  
Original Research ◽  
Missense Mutations ◽  
Laboratory Course ◽  
Dna Mismatch

This work describes the project for an advanced undergraduate laboratory course in cell and molecular biology. One objective of the course is to teach students a variety of cellular and molecular techniques while conducting original research. A second objective is to provide instruction in science writing and data presentation by requiring comprehensive laboratory reports modeled on the primary literature. The project for the course focuses on a gene, MSH2, implicated in the most common form of inherited colorectal cancer. Msh2 is important for maintaining the fidelity of genetic material where it functions as an important component of the DNA mismatch repair machinery. The goal of the project has two parts. The first part is to create mapped missense mutation listed in the human databases in the cognate yeast MSH2 gene and to assay for defects in DNA mismatch repair. The second part of the course is directed towards understanding in what way are the variant proteins defective for mismatch repair. Protein levels are analyzed to determine if the missense alleles display decreased expression. Furthermore, the students establish whether the Msh2p variants are properly localized to the nucleus using indirect immunofluorescence and whether the altered proteins have lost their ability to interact with other subunits of the MMR complex by creating recombinant DNA molecules and employing the yeast 2-hybrid assay.

scholarly journals Control of Initiation of Sporulation by Replication Initiation Genes in Bacillus subtilis

2000 ◽  
Vol 182 (10) ◽  
pp. 2989-2991 ◽  
Author(s):  
Katherine P. Lemon ◽  
Iren Kurtser ◽  
Judy Wu ◽  
Alan D. Grossman
Keyword(s):  
Bacillus Subtilis ◽  
Dna Replication ◽  
Replication Initiation ◽  
Spore Formation ◽  
Temperature Sensitive ◽  
Temperature Sensitive Mutation ◽  
Initiation Of Dna Replication ◽  
Replication Initiation Proteins

ABSTRACT Initiation of spore formation in Bacillus subtilisappears to depend on initiation of DNA replication. This regulation was first identified using a temperature-sensitive mutation indnaB. We found that mutations in the replication initiation genes dnaA and dnaD also inhibit sporulation, indicating that inhibition of sporulation is triggered by general defects in the function of replication initiation proteins.

scholarly journals Somatic Hypermutation in Muts Homologue (Msh)3-, Msh6-, and Msh3/Msh6-Deficient Mice Reveals a Role for the Msh2–Msh6 Heterodimer in Modulating the Base Substitution Pattern

2000 ◽  
Vol 191 (3) ◽  
pp. 579-584 ◽  
Author(s):  
Margrit Wiesendanger ◽  
Burkhard Kneitz ◽  
Winfried Edelmann ◽  
Matthew D. Scharff
Keyword(s):  
Immune Response ◽  
Dna Replication ◽  
Mismatch Repair ◽  
Dna Mismatch Repair ◽  
Somatic Hypermutation ◽  
Hot Spot ◽  
Base Substitution ◽  
Substitution Pattern ◽  
Dna Mismatch ◽  
Deficient Mice

Although the primary function of the DNA mismatch repair (MMR) system is to identify and correct base mismatches that have been erroneously introduced during DNA replication, recent studies have further implicated several MMR components in somatic hypermutation of immunoglobulin (Ig) genes. We studied the immune response in mice deficient in MutS homologue (MSH)3 and MSH6, two mutually exclusive partners of MSH2 that have not been examined previously for their role in Ig hypermutation. In Msh6−/− and Msh3−/−/Msh6−/− mice, base substitutions are preferentially targeted to G and C nucleotides and to an RGYW hot spot, as has been shown previously in Msh2−/− mice. In contrast, Msh3−/− mice show no differences from their littermate controls. These findings indicate that the MSH2–MSH6 heterodimer, but not the MSH2–MSH3 complex, is responsible for modulating Ig hypermutation.

scholarly journals mlh3 separation of function and endonuclease defective mutants display an unexpected effect on meiotic recombination outcomes

2017 ◽  
Author(s):  
Najla Al-Sweel ◽  
Vandana Raghavan ◽  
Abhishek Dutta ◽  
V. P. Ajith ◽  
Luigi Di Vietro ◽  
...  
Keyword(s):  
Dna Replication ◽  
Mismatch Repair ◽  
Protein Interactions ◽  
Meiotic Recombination ◽  
Meiotic Division ◽  
Dna Mismatch Repair ◽  
Crossing Over ◽  
Baker's Yeast ◽  
Protein Protein Interactions ◽  
Dna Mismatch

AbstractMlh1-Mlh3 is an endonuclease hypothesized to act in meiosis to resolve double Holliday junctions into crossovers. It also plays a minor role in eukaryotic DNA mismatch repair (MMR). To understand how Mlh1-Mlh3 functions in both meiosis and MMR, we analyzed in baker’s yeast 60 new mlh3 alleles. Five alleles specifically disrupted MMR, whereas one (mlh3-32) specifically disrupted meiotic crossing over. Mlh1-mlh3 representatives for each separation of function class were purified and characterized. Both Mlh1-mlh3-32 (MMR+, crossover-) and Mlh1-mlh3-45 (MMR-, crossover+) displayed wild-type endonuclease activities in vitro. Msh2-Msh3, an MSH complex that acts with Mlh1-Mlh3 in MMR, stimulated the endonuclease activity of Mlh1-mlh3-32 but not Mlh1-mlh3-45, suggesting that Mlh1-mlh3-45 is defective in MSH interactions. Whole genome recombination maps were constructed for two mlh3 mutants with opposite separation of function phenotypes, and an endonuclease defective mutant. Unexpectedly, all three showed increases in the number of non-crossover events that were not observed in mlh3Δ. Our observations provide a structure-function map for Mlh3 that reveals the importance of protein-protein interactions in regulating Mlh1-Mlh3’s enzymatic activity. They also illustrate how defective meiotic components can alter the fate of meiotic recombination intermediates, providing new insights for how meiotic recombination pathways are regulated.Author SummaryDuring meiosis, diploid germ cells that become eggs or sperm undergo a single round of DNA replication followed by two consecutive chromosomal divisions. The segregation of chromosomes at the first meiotic division is dependent in most organisms on at least one genetic exchange, or crossover event, between chromosome homologs. Homologs that do not receive a crossover frequently undergo non-disjunction at the first meiotic division, yielding gametes that lack chromosomes or contain additional copies. Such events have been linked to human disease and infertility. Recent studies suggest that the Mlh1-Mlh3 complex is an endonuclease that resolves recombination intermediates into crossovers. Interestingly, this complex also acts as a matchmaker in DNA mismatch repair (MMR) to remove DNA replication errors. How does one complex act in two different processes? We investigated this question by performing a mutational analysis of the baker’s yeast Mlh3 protein. Five mutations were identified that disrupted MMR but not crossing over, and one mutation disrupted crossing over while maintaining MMR. Using a combination of biochemical and genetic analyses to further characterize these mutants we illustrate the importance of protein-protein interactions for Mlh1-Mlh3’s activity. Importantly, we illustrate how defective meiotic components can alter the outcome of meiotic recombination events. They also provide new insights in our understanding of the basis of infertility syndromes.

scholarly journals Characterization of the Repeat-Tract Instability and Mutator Phenotypes Conferred by a Tn3 Insertion in RFC1, the Large Subunit of the Yeast Clamp Loader

Genetics ◽  
1999 ◽  
Vol 151 (2) ◽  
pp. 499-509 ◽  
Author(s):  
Yali Xie ◽  
Chris Counter ◽  
Eric Alani
Keyword(s):  
Dna Replication ◽  
Mismatch Repair ◽  
Mutation Frequency ◽  
Synthetic Lethality ◽  
Large Subunit ◽  
Repair System ◽  
Clamp Loader ◽  
Repeat Tract ◽  
Forward Mutation ◽  
Instability Phenotype

Abstract The RFC1 gene encodes the large subunit of the yeast clamp loader (RFC) that is a component of eukaryotic DNA polymerase holoenzymes. We identified a mutant allele of RFC1 (rfc1::Tn3) from a large collection of Saccharomyces cerevisiae mutants that were inviable when present in a rad52 null mutation background. Analysis of rfc1::Tn3 strains indicated that they displayed both a mutator and repeat-tract instability phenotype. Strains bearing this allele were characterized in combination with mismatch repair (msh2Δ, pms1Δ), double-strand break repair (rad52), and DNA replication (pol3-01, pol30-52, rth1Δ/rad27Δ) mutations in both forward mutation and repeat-tract instability assays. This analysis indicated that the rfc1::Tn3 allele displays synthetic lethality with pol30, pol3, and rad27 mutations. Measurement of forward mutation frequencies in msh2Δ rfc1:Tn3 and pms1Δ rfc1:Tn3 strains indicated that the rfc1::Tn3 mutant displayed a mutation frequency that appeared nearly multiplicative with the mutation frequency exhibited by mismatch-repair mutants. In repeat-tract instability assays, however, the rfc1::Tn3 mutant displayed a tract instability phenotype that appeared epistatic to the phenotype displayed by mismatch-repair mutants. From these data we propose that defects in clamp loader function result in DNA replication errors, a subset of which are acted upon by the mismatch-repair system.

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