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Richard D. Kolodner - One of the best experts on this subject based on the ideXlab platform.

  • reconstitution of saccharomyces cerevisiae dna polymerase e dependent mismatch repair with purified proteins
    Proceedings of the National Academy of Sciences of the United States of America, 2017
    Co-Authors: Nikki Bowen, Richard D. Kolodner
    Abstract:

    Mammalian and Saccharomyces cerevisiae mismatch repair (MMR) proteins catalyze two MMR reactions in vitro. In one, mispair binding by either the MutS homolog 2 (Msh2)–MutS homolog 6 (Msh6) or the Msh2–MutS homolog 3 (MSH3) stimulates 5′ to 3′ excision by exonuclease 1 (Exo1) from a single-strand break 5′ to the mispair, excising the mispair. In the other, Msh2–Msh6 or Msh2–MSH3 activate the MutL homolog 1 (Mlh1)–postmeiotic segregation 1 (Pms1) endonuclease in the presence of a mispair and a nick 3′ to the mispair, to make nicks 5′ to the mispair, allowing Exo1 to excise the mispair. DNA polymerase δ (Pol δ) is thought to catalyze DNA synthesis to fill in the gaps resulting from mispair excision. However, colocalization of the S. cerevisiae mispair recognition proteins with the replicative DNA polymerases during DNA replication has suggested that DNA polymerase e (Pol e) may also play a role in MMR. Here we describe the reconstitution of Pol e-dependent MMR using S. cerevisiae proteins. A mixture of Msh2–Msh6 (or Msh2–MSH3), Exo1, RPA, RFC-Δ1N, PCNA, and Pol e was found to catalyze both short-patch and long-patch 5′ nick-directed MMR of a substrate containing a +1 (+T) mispair. When the substrate contained a nick 3′ to the mispair, a mixture of Msh2–Msh6 (or Msh2–MSH3), Exo1, RPA, RFC-Δ1N, PCNA, and Pol e was found to catalyze an MMR reaction that required Mlh1–Pms1. These results demonstrate that Pol e can act in eukaryotic MMR in vitro.

  • Mlh2 Is an Accessory Factor for DNA Mismatch Repair in Saccharomyces cerevisiae
    PLoS genetics, 2014
    Co-Authors: Christopher S Campbell, Christopher D Putnam, Hans Hombauer, Arshad Desai, Anjana Srivatsan, Nikki Bowen, Kerstin Gries, Richard D. Kolodner
    Abstract:

    In Saccharomyces cerevisiae, the essential mismatch repair (MMR) endonuclease Mlh1-Pms1 forms foci promoted by Msh2-Msh6 or Msh2-MSH3 in response to mispaired bases. Here we analyzed the Mlh1-Mlh2 complex, whose role in MMR has been unclear. Mlh1-Mlh2 formed foci that often colocalized with and had a longer lifetime than Mlh1-Pms1 foci. Mlh1-Mlh2 foci were similar to Mlh1-Pms1 foci: they required mispair recognition by Msh2-Msh6, increased in response to increased mispairs or downstream defects in MMR, and formed after induction of DNA damage by phleomycin but not double-stranded breaks by I-SceI. Mlh1-Mlh2 could be recruited to mispair-containing DNA in vitro by either Msh2-Msh6 or Msh2-MSH3. Deletion of MLH2 caused a synergistic increase in mutation rate in combination with deletion of MSH6 or reduced expression of Pms1. Phylogenetic analysis demonstrated that the S. cerevisiae Mlh2 protein and the mammalian PMS1 protein are homologs. These results support a hypothesis that Mlh1-Mlh2 is a non-essential accessory factor that acts to enhance the activity of Mlh1-Pms1.

  • Mispair-specific recruitment of the Mlh1-Pms1 complex identifies repair substrates of the Saccharomyces cerevisiae Msh2-MSH3 complex.
    The Journal of biological chemistry, 2014
    Co-Authors: Anjana Srivatsan, Nikki Bowen, Richard D. Kolodner
    Abstract:

    DNA mismatch repair is initiated by either the Msh2-Msh6 or the Msh2-MSH3 mispair recognition heterodimer. Here we optimized the expression and purification of Saccharomyces cerevisiae Msh2-MSH3 and performed a comparative study of Msh2-MSH3 and Msh2-Msh6 for mispair binding, sliding clamp formation, and Mlh1-Pms1 recruitment. Msh2-MSH3 formed sliding clamps and recruited Mlh1-Pms1 on +1, +2, +3, and +4 insertion/deletions and CC, AA, and possibly GG mispairs, whereas Msh2-Msh6 formed mispair-dependent sliding clamps and recruited Mlh1-Pms1 on 7 of the 8 possible base:base mispairs, the +1 insertion/deletion mispair, and to a low level on the +2 but not the +3 or +4 insertion/deletion mispairs and not on the CC mispair. The mispair specificity of sliding clamp formation and Mlh1-Pms1 recruitment but not mispair binding alone correlated best with genetic data on the mispair specificity of Msh2-MSH3- and Msh2-Msh6-dependent mismatch repair in vivo. Analysis of an Msh2-Msh6/MSH3 chimeric protein and mutant Msh2-MSH3 complexes showed that the nucleotide binding domain and communicating regions but not the mispair binding domain of Msh2-MSH3 are responsible for the extremely rapid dissociation of Msh2-MSH3 sliding clamps from DNA relative to that seen for Msh2-Msh6, and that amino acid residues predicted to stabilize Msh2-MSH3 interactions with bent, strand-separated mispair-containing DNA are more critical for the recognition of small +1 insertion/deletions than larger +4 insertion/deletions.

  • functional studies and homology modeling of msh2 MSH3 predict that mispair recognition involves dna bending and strand separation
    Molecular and Cellular Biology, 2010
    Co-Authors: Jill M Dowen, Christopher D Putnam, Richard D. Kolodner
    Abstract:

    The Msh2-MSH3 heterodimer recognizes various DNA mispairs, including loops of DNA ranging from 1 to 14 nucleotides and some base-base mispairs. Homology modeling of the mispair-binding domain (MBD) of MSH3 using the related Msh6 MBD revealed that mismatch recognition must be different, even though the MBD folds must be similar. Model-based point mutation alleles of Saccharomyces cerevisiae MSH3 designed to disrupt mispair recognition fell into two classes. One class caused defects in repair of both small and large insertion/deletion mispairs, whereas the second class caused defects only in the repair of small insertion/deletion mispairs; mutations of the first class also caused defects in the removal of nonhomologous tails present at the ends of double-strand breaks (DSBs) during DSB repair, whereas mutations of the second class did not cause defects in the removal of nonhomologous tails during DSB repair. Thus, recognition of small insertion/deletion mispairs by MSH3 appears to require a greater degree of interactions with the DNA conformations induced by small insertion/deletion mispairs than with those induced by large insertion/deletions that are intrinsically bent and strand separated. Mapping of the two classes of mutations onto the MSH3 MBD model appears to distinguish mispair recognition regions from DNA stabilization regions.

  • Saccharomyces cerevisiae Msh2-MSH3 acts in repair of base-base mispairs.
    Molecular and cellular biology, 2007
    Co-Authors: Jill M. Harrington, Richard D. Kolodner
    Abstract:

    DNA mismatch repair is thought to act through two subpathways involving the recognition of base-base and insertion/deletion mispairs by the Msh2-Msh6 heterodimer and the recognition of insertion/deletion mispairs by the Msh2-MSH3 heterodimer. Here, through genetic and biochemical approaches, we describe a previously unidentified role of the Msh2-MSH3 heterodimer in the recognition of base-base mispairs and the suppression of homology-mediated duplication and deletion mutations. Saccharomyces cerevisiae MSH3 mutants did not show an increase in the rate of base substitution mutations by the CAN1 forward mutation assay compared to the rate for the wild type but did show an altered spectrum of base substitution mutations, including an increased accumulation of base pair changes from GC to CG and from AT to TA; MSH3 mutants also accumulated homology-mediated duplication and deletion mutations. The mutation spectrum of mlh3 mutants paralleled that of MSH3 mutants, suggesting that the Mlh1-Mlh3 heterodimer may also play a role in the repair of base-base mispairs and in the suppression of homology-mediated duplication and deletion mutations. Mispair binding analysis with purified Msh2-MSH3 and DNA substrates derived from CAN1 sequences found to be mutated in vivo demonstrated that Msh2-MSH3 exhibited robust binding to specific base-base mispairs that was consistent with functional mispair binding.

Eric Alani - One of the best experts on this subject based on the ideXlab platform.

  • mlh3 mutations in baker s yeast alter meiotic recombination outcomes by increasing noncrossover events genome wide
    PLOS Genetics, 2017
    Co-Authors: Najla Alsweel, Carol M Manhart, Jennifer A Surtees, Vandana Raghavan, Abhishek Dutta, V P Ajith, Luigi Di Vietro, Nabila Khondakar, K T Nishant, Eric Alani
    Abstract:

    Mlh1-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 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 wild-type and MMR+ crossover-, MMR- crossover+, endonuclease defective and null mlh3 mutants in an S288c/YJM789 hybrid background. Compared to wild-type, all of the mlh3 mutants showed increases in the number of noncrossover events, consistent with recombination intermediates being resolved through alternative recombination pathways. 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.

  • mlh3 separation of function and endonuclease defective mutants display an unexpected effect on meiotic recombination outcomes
    bioRxiv, 2017
    Co-Authors: Najla Alsweel, Carol M Manhart, Jennifer A Surtees, Abhishek Dutta, V P Ajith, Luigi Di Vietro, Nabila Khondakar, K T Nishant, Eric Alani
    Abstract:

    Mlh1-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 bakers 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 mlh3null. Our observations provide a structure-function map for Mlh3 that reveals the importance of protein-protein interactions in regulating Mlh1-Mlh3 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.

  • mlh1 mlh3 a meiotic crossover and dna mismatch repair factor is a msh2 MSH3 stimulated endonuclease
    Journal of Biological Chemistry, 2014
    Co-Authors: Maria V Rogacheva, Carol M Manhart, Jennifer A Surtees, Cheng Chen, Alba Guarne, Eric Alani
    Abstract:

    Crossing over between homologous chromosomes is initiated in meiotic prophase in most sexually reproducing organisms by the appearance of programmed double strand breaks throughout the genome. In Saccharomyces cerevisiae the double-strand breaks are resected to form three prime single-strand tails that primarily invade complementary sequences in unbroken homologs. These invasion intermediates are converted into double Holliday junctions and then resolved into crossovers that facilitate homolog segregation during Meiosis I. Work in yeast suggests that Msh4-Msh5 stabilizes invasion intermediates and double Holliday junctions, which are resolved into crossovers in steps requiring Sgs1 helicase, Exo1, and a putative endonuclease activity encoded by the DNA mismatch repair factor Mlh1-Mlh3. We purified Mlh1-Mlh3 and showed that it is a metal-dependent and Msh2-MSH3-stimulated endonuclease that makes single-strand breaks in supercoiled DNA. These observations support a direct role for an Mlh1-Mlh3 endonuclease activity in resolving recombination intermediates and in DNA mismatch repair.

  • genetic analysis of mlh3 mutations reveals interactions between crossover promoting factors during meiosis in baker s yeast
    G3: Genes Genomes Genetics, 2013
    Co-Authors: Megan Sonntag Brown, Cheng Chen, K T Nishant, Elisha Lim, Eric Alani
    Abstract:

    Crossing over between homologous chromosomes occurs during the prophase of meiosis I and is critical for chromosome segregation. In baker’s yeast, two heterodimeric complexes, Msh4-Msh5 and Mlh1-Mlh3, act in meiosis to promote interference-dependent crossing over. Mlh1-Mlh3 also plays a role in DNA mismatch repair (MMR) by interacting with Msh2-MSH3 to repair insertion and deletion mutations. Mlh3 contains an ATP-binding domain that is highly conserved among MLH proteins. To explore roles for Mlh3 in meiosis and MMR, we performed a structure−function analysis of eight mlh3 ATPase mutants. In contrast to previous work, our data suggest that ATP hydrolysis by both Mlh1 and Mlh3 is important for both meiotic and MMR functions. In meiotic assays, these mutants showed a roughly linear relationship between spore viability and genetic map distance. To further understand the relationship between crossing over and meiotic viability, we analyzed crossing over on four chromosomes of varying lengths in mlh3Δ mms4Δ strains and observed strong decreases (6- to 17-fold) in crossing over in all intervals. Curiously, mlh3Δ mms4Δ double mutants displayed spore viability levels that were greater than observed in mms4Δ strains that show modest defects in crossing over. The viability in double mutants also appeared greater than would be expected for strains that show such severe defects in crossing over. Together, these observations provide insights for how Mlh1-Mlh3 acts in crossover resolution and MMR and for how chromosome segregation in Meiosis I can occur in the absence of crossing over.

  • Saccharomyces cerevisiae MSH2-MSH3 and MSH2-MSH6 complexes display distinct requirements for DNA binding Domain I in mismatch recognition.
    Journal of molecular biology, 2006
    Co-Authors: Susan D. Lee, Jennifer A Surtees, Eric Alani
    Abstract:

    In eukaryotic mismatch repair (MMR) MSH2-MSH6 initiates the repair of base-base and small insertion/deletion mismatches while MSH2-MSH3 repairs larger insertion/deletion mismatches. In this study we showed that the msh2Δ1 mutation, containing a complete deletion of the conserved mismatch recognition Domain I of MSH2, conferred a separation of function phenotype with respect to MSH2-MSH3 and MSH2-MSH6 functions. Strains bearing the msh2Δ1 mutation were nearly wild-type in MSH2-MSH6-mediated MMR and in suppressing recombination between DNA sequences predicted to form mismatches recognized by MSH2-MSH6. However, these strains were completely defective in MSH2-MSH3-mediated MMR and recombination functions. This information encouraged us to analyze the contributions of Domain I to the mismatch binding specificity of MSH2-MSH3 in genetic and biochemical assays. We found that Domain I in MSH2 contributed a non-specific DNA binding activity while Domain I of MSH3 appeared important for mismatch binding specificity and for suppressing non-specific DNA-binding. These observations reveal distinct requirements for the MSH2 DNA binding Domain I in the repair of DNA mismatches and suggest that the binding of MSH2-MSH3 to mismatch DNA involves protein-DNA contacts that appear very different from those required for MSH2-MSH6 mismatch binding.

Winfried Edelmann - One of the best experts on this subject based on the ideXlab platform.

  • msh2 atpase domain mutation affects ctg cag repeat instability in transgenic mice
    PLOS Genetics, 2009
    Co-Authors: Stéphanie Tomé, Ian Holt, Glenn E Morris, Christopher E. Pearson, Winfried Edelmann, Arnold Munnich, Geneviève Gourdon
    Abstract:

    Myotonic dystrophy type 1 (DM1) is associated with one of the most highly unstable CTG•CAG repeat expansions. The formation of further repeat expansions in transgenic mice carrying expanded CTG•CAG tracts requires the mismatch repair (MMR) proteins MSH2 and MSH3, forming the MutSβ complex. It has been proposed that binding of MutSβ to CAG hairpins blocks its ATPase activity compromising hairpin repair, thereby causing expansions. This would suggest that binding, but not ATP hydrolysis, by MutSβ is critical for trinucleotide expansions. However, it is unknown if the MSH2 ATPase activity is dispensible for instability. To get insight into the mechanism by which MSH2 generates trinucleotide expansions, we crossed DM1 transgenic mice carrying a highly unstable >(CTG)300 repeat tract with mice carrying the G674A mutation in the MSH2 ATPase domain. This mutation impairs MSH2 ATPase activity and ablates base–base MMR, but does not affect the ability of MSH2 (associated with MSH6) to bind DNA mismatches. We found that the ATPase domain mutation of MSH2 strongly affects the formation of CTG expansions and leads instead to transmitted contractions, similar to a Msh2-null or MSH3-null deficiency. While a decrease in MSH2 protein level was observed in tissues from Msh2G674 mice, the dramatic reduction of expansions suggests that the expansion-biased trinucleotide repeat instability requires a functional MSH2 ATPase domain and probably a functional MMR system.

  • differing patterns of genetic instability in mice deficient in the mismatch repair genes pms2 mlh1 msh2 MSH3 and msh6
    Carcinogenesis, 2006
    Co-Authors: Denise C Hegan, Michael R Liskay, Frank R Jirik, Latha Narayanan, Winfried Edelmann, Peter M. Glazer
    Abstract:

    Defects in genes associated with DNA mismatch repair (MMR) have been linked to hereditary colon cancer. Because the MMR pathway includes multiple factors with both overlapping and divergent functions, we sought to compare the impact of deficiencies in each of several MMR genes on genetic instability using a collection of knock-out mouse models. We investigated mutation frequencies and patterns in MMR-deficient mice using two transgenic reporter genes, supFG1 and cII, in the context of mice deficient for Pms2, Mlh1, Msh2, MSH3 or Msh6 or both Msh2 and MSH3 or both MSH3 and Msh6. We found that the mean mutation frequencies of all of the MMR-deficient mice were significantly higher than the mean mutation frequencies of wild-type mice. Mlh1-deficient mice and Msh2-deficient mice had the highest mutation frequencies in a comparison of the single nullizygous mice. Of all the mice studied, mice nullizygous for both Msh2 and MSH3 and those nullizygous for both MSH3 and Msh6 displayed the greatest overall increases in mutation frequencies compared with wild-type mice. Sequence analysis of the mutated reporter genes revealed significant differences between the individual groups of MMR-deficient mice. Taken together, our results further characterize the functions of the MMR factors in mutation avoidance and provide in vivo correlation to biochemical models of the MMR pathway.

  • localization of mmr proteins on meiotic chromosomes in mice indicates distinct functions during prophase i
    Journal of Cell Biology, 2005
    Co-Authors: Nadine K Kolas, Michael R Liskay, Winfried Edelmann, Steven M. Lipkin, Anton Svetlanov, Michelle Lenzi, Frank P Macaluso, John M Greally, Paula E Cohen
    Abstract:

    Mammalian MutL homologues function in DNA mismatch repair (MMR) after replication errors and in meiotic recombination. Both functions are initiated by a heterodimer of MutS homologues specific to either MMR (MSH2–MSH3 or MSH2–MSH6) or crossing over (MSH4–MSH5). Mutations of three of the four MutL homologues (Mlh1, Mlh3, and Pms2) result in meiotic defects. We show herein that two distinct complexes involving MLH3 are formed during murine meiosis. The first is a stable association between MLH3 and MLH1 and is involved in promoting crossing over in conjunction with MSH4–MSH5. The second complex involves MLH3 together with MSH2–MSH3 and localizes to repetitive sequences at centromeres and the Y chromosome. This complex is up-regulated in Pms2−/− males, but not females, providing an explanation for the sexual dimorphism seen in Pms2−/− mice. The association of MLH3 with repetitive DNA sequences is coincident with MSH2–MSH3 and is decreased in Msh2−/− and MSH3−/− mice, suggesting a novel role for the MMR family in the maintenance of repeat unit integrity during mammalian meiosis.

  • correction corrigendum cag n hairpin dna binds to msh2 MSH3 and changes properties of mismatch recognition
    Nature Structural & Molecular Biology, 2005
    Co-Authors: Barbara A L Owen, Teresa M Wilson, Raju Kucherlapati, Winfried Edelmann, Zungyoon Yang, Maoyi Lai, Maciez Gajek, Jeffrey J Hayes, John Badger, Cynthia T Mcmurray
    Abstract:

    Corrigendum: (CAG) n -hairpin DNA binds to Msh2–MSH3 and changes properties of mismatch recognition

  • cag n hairpin dna binds to msh2 MSH3 and changes properties of mismatch recognition
    Nature Structural & Molecular Biology, 2005
    Co-Authors: Barbara A L Owen, Teresa M Wilson, Raju Kucherlapati, Winfried Edelmann, Zungyoon Yang, Maoyi Lai, Maciez Gajek, John D Badger, Jeffrey J Hayes, Cynthia T Mcmurray
    Abstract:

    Cells have evolved sophisticated DNA repair systems to correct damaged DNA. However, the human DNA mismatch repair protein Msh2-MSH3 is involved in the process of trinucleotide (CNG) DNA expansion rather than repair. Using purified protein and synthetic DNA substrates, we show that Msh2-MSH3 binds to CAG-hairpin DNA, a prime candidate for an expansion intermediate. CAG-hairpin binding inhibits the ATPase activity of Msh2-MSH3 and alters both nucleotide (ADP and ATP) affinity and binding interfaces between protein and DNA. These changes in Msh2-MSH3 function depend on the presence of A.A mispaired bases in the stem of the hairpin and on the hairpin DNA structure per se. These studies identify critical functional defects in the Msh2-MSH3-CAG hairpin complex that could misdirect the DNA repair process.

Satya Prakash - One of the best experts on this subject based on the ideXlab platform.

  • Enhancement of MSH2-MSH3-mediated mismatch recognition by the yeast MLH1-PMS1 complex.
    Current biology : CB, 1997
    Co-Authors: Yvette Habraken, Louise Prakash, Patrick Sung, Satya Prakash
    Abstract:

    DNA mismatch repair has a key role in maintaining genomic stability. Defects in mismatch repair cause elevated spontaneous mutation rates and increased instability of simple repetitive sequences, while mutations in human mismatch repair genes result in hereditary nonpolyposis colorectal cancers. Mismatch recognition represents the first critical step of mismatch repair. Genetic and biochemical studies in yeast and humans have indicated a requirement for MSH2-MSH3 and MSH2-MSH6 heterodimers in mismatch recognition. These complexes have, to some extent, overlapping mismatch binding specificities. MLH1 and PMS1 are the other essential components of mismatch repair, but how they function in this process is not known. We have purified the yeast MLH1-PMS1 heterodimer to near homogeneity, and examined its effect on MSH2-MSH3 binding to DNA mismatches. By itself, the MLH1-PMS1 complex shows no affinity for mismatched DNA, but it greatly enhances the mismatch binding ability of MSH2-MSH3.

  • binding of insertion deletion dna mismatches by the heterodimer of yeast mismatch repair proteins msh2 and MSH3
    Current Biology, 1996
    Co-Authors: Yvette Habraken, Louise Prakash, Patrick Sung, Satya Prakash
    Abstract:

    Abstract DNA-mismatch repair removes mismatches from the newly replicated DNA strand. In humans, mutations in the mismatch repair genes hMSH2 , hMLH1 , hPMS1 and hPMS2 result in hereditary non-polyposis colorectal cancer (HNPCC) [1–8]. The hMSH2 (MSH for MutS homologue) protein forms a complex with a 160 kDa protein, and this heterodimer, hMutS α , has high affinity for a G/T mismatch [9,10]. Cell lines in which the 160 kDa subunit of hMutS α is mutated are specifically defective in the repair of base–base and single-nucleotide insertion/deletion mismatches [9,11]. Genetic studies in S. cerevisiae have suggested that MSH2 functions with either MSH3 or MSH6 in mismatch repair, and, in the absence of the latter two genes, MSH2 is inactive [12,13]. MSH6 encodes the yeast counterpart of the 160 kDa subunit of hMutS α [12,13]. As in humans, yeast MSH6 forms a complex with MSH2, and the MSH2–MSH6 heterodimer binds a G/T mismatch [14]. Here, we find that MSH2 and MSH3 form another stable heterodimer, and we purify this heterodimer to near homogeneity. We show that MSH2–MSH3 has low affinity for a G/T mismatch but binds to insertion/deletion mismatches with high specificity, unlike MSH2–MSH6.

  • requirement of the yeast MSH3 and msh6 genes for msh2 dependent genomic stability
    Journal of Biological Chemistry, 1996
    Co-Authors: R.e. Johnson, Gopala K Kovvali, Louise Prakash, Satya Prakash
    Abstract:

    Abstract Defects in DNA mismatch repair result in instability of simple repetitive DNA sequences and elevated levels of spontaneous mutability. The human G/T mismatch binding protein, GTBP/p160, has been suggested to have a role in the repair of base-base and single nucleotide insertion-deletion mismatches. Here we examine the role of the yeast GTBP homolog, MSH6, in mismatch repair. We show that both MSH6 and MSH3 genes are essential for normal genomic stability. Interestingly, although mutations in either MSH3 or MSH6 do not cause the extreme microsatellite instability and spontaneous mutability observed in the msh2 mutant, yeast cells harboring null mutations in both the MSH3 and MSH6 genes exhibit microsatellite instability and mutability similar to that in the msh2 mutant. Results from epistasis analyses indicate that MSH2 functions in mismatch repair in conjunction with MSH3 or MSH6 and that MSH3 and MSH6 constitute alternate pathways of MSH2-dependent mismatch repair.

  • requirement of mismatch repair genes msh2 and MSH3 in the rad1 rad10 pathway of mitotic recombination in saccharomyces cerevisiae
    Genetics, 1996
    Co-Authors: Murat Saparbaev, Louise Prakash, Satya Prakash
    Abstract:

    The RAD1 and RAD10 genes of Saccharomyces cerevisiae are required for nucleotide excision repair and they also act in mitotic recombination. The Rad1-Rad10 complex has a single-stranded DNA endonuclease activity. Here, we show that the mismatch repair genes MSH2 and MSH3 function in mitotic recombination. For both his3 and his4 duplications, and for homologous integration of a linear DNA fragment into the genome, the MSH3Δ mutation has an effect on recombination similar to that of the rad1Δ and rad10Δ mutations. The msh2Δ mutation also reduces the rate of recombination of the his3 duplication and lowers the incidence of homologous integration of a linear DNA fragment. Epistasis analyses indicate that MSH2 and MSH3 function in the RAD1-RAD10 recombination pathway, and studies presented here suggest an involvement of the RAD1-RAD10 pathway in reciprocal recombination. The possible roles of Msh2, MSH3, Rad1, and Rad10 proteins in genetic recombination are discussed. Coupling of mismatch binding proteins with the recombinational machinery could be important for ensuring genetic fidelity in the recombination process.

Cynthia T Mcmurray - One of the best experts on this subject based on the ideXlab platform.

  • crosstalk between msh2 MSH3 and polβ promotes trinucleotide repeat expansion during base excision repair
    Nature Communications, 2016
    Co-Authors: Yanhao Lai, Jill M. Beaver, Helen Budworth, Nelson L S Chan, Zunzhen Zhang, Cynthia T Mcmurray, Yuan Liu
    Abstract:

    Studies in knockout mice provide evidence that MSH2-MSH3 and the BER machinery promote trinucleotide repeat (TNR) expansion, yet how these two different repair pathways cause the mutation is unknown. Here we report the first molecular crosstalk mechanism, in which MSH2-MSH3 is used as a component of the BER machinery to cause expansion. On its own, pol β fails to copy TNRs during DNA synthesis, and bypasses them on the template strand to cause deletion. Remarkably, MSH2-MSH3 not only stimulates pol β to copy through the repeats but also enhances formation of the flap precursor for expansion. Our results provide direct evidence that MMR and BER, operating together, form a novel hybrid pathway that changes the outcome of TNR instability from deletion to expansion during the removal of oxidized bases. We propose that cells implement crosstalk strategies and share machinery when a canonical pathway is ineffective in removing a difficult lesion.

  • conformational trapping of mismatch recognition complex msh2 MSH3 on repair resistant dna loops
    Proceedings of the National Academy of Sciences of the United States of America, 2011
    Co-Authors: Walter H Lang, Julie E Coats, Jerzy Majka, Greg Hura, Yuyen Lin, Ivan Rasnik, Cynthia T Mcmurray
    Abstract:

    Insertion and deletion of small heteroduplex loops are common mutations in DNA, but why some loops are prone to mutation and others are efficiently repaired is unknown. Here we report that the mismatch recognition complex, MSH2/MSH3, discriminates between a repair-competent and a repair-resistant loop by sensing the conformational dynamics of their junctions. MSH2/MSH3 binds, bends, and dissociates from repair-competent loops to signal downstream repair. Repair-resistant Cytosine-Adenine-Guanine (CAG) loops adopt a unique DNA junction that traps nucleotide-bound MSH2/MSH3, and inhibits its dissociation from the DNA. We envision that junction dynamics is an active participant and a conformational regulator of repair signaling, and governs whether a loop is removed by MSH2/MSH3 or escapes to become a precursor for mutation.

  • erratum the nucleotide binding dynamics of human msh2 MSH3 are lesion dependent
    Nature Structural & Molecular Biology, 2009
    Co-Authors: Barbara A L Owen, Walter H Lang, Cynthia T Mcmurray
    Abstract:

    Nat. Struct. Mol. Biol. 16, 550–557 (2009); published online 19 April 2009; corrected after print 18 May 2009 In the version of this article initially published, the first sentence referring to Figure 2c (page 552, first paragraph) was incorrect. It should read: “To further test the stochastic binding of nucleotides, we simultaneously added a constant amount (5 μM) of labeled ADP(+Mg2+) and increasing concentrations of unlabeled AMP-PNP(+Mg2+) to MSH2–MSH3 (Fig.

  • the nucleotide binding dynamics of human msh2 MSH3 are lesion dependent
    Nature Structural & Molecular Biology, 2009
    Co-Authors: Barbara A L Owen, Walter H Lang, Cynthia T Mcmurray
    Abstract:

    The MSH2–MSH3 complex recognizes DNA mismatch lesions, with stronger affinity for small insertion and deletion loops. Now the nucleotide binding properties of MSH2–MSH3 are studied, revealing the changes upon binding to DNA molecules with a loop lesion, indicating how this mismatch sensor can signal the repair machinery.

  • correction corrigendum cag n hairpin dna binds to msh2 MSH3 and changes properties of mismatch recognition
    Nature Structural & Molecular Biology, 2005
    Co-Authors: Barbara A L Owen, Teresa M Wilson, Raju Kucherlapati, Winfried Edelmann, Zungyoon Yang, Maoyi Lai, Maciez Gajek, Jeffrey J Hayes, John Badger, Cynthia T Mcmurray
    Abstract:

    Corrigendum: (CAG) n -hairpin DNA binds to Msh2–MSH3 and changes properties of mismatch recognition