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Richard D. Kolodner - One of the best experts on this subject based on the ideXlab platform.
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Ligation of newly replicated DNA controls the timing of DNA mismatch repair.
Current biology : CB, 2021Co-Authors: Gloria Reyes, Christopher D Putnam, Richard D. Kolodner, Anna Kolodziejczak, Lovely Jael Paul Solomon Devakumar, Takashi Kubota, Hans HombauerAbstract:Mismatch repair (MMR) safeguards genome stability through recognition and excision of DNA replication errors.1-4 How eukaryotic MMR targets the newly replicated strand in vivo has not been established. MMR reactions reconstituted in vitro are directed to the strand containing a preexisting nick or gap,5-8 suggesting that strand discontinuities could act as discrimination signals. Another candidate is the proliferating cell nuclear antigen (PCNA) that is loaded at replication forks and is required for the activation of Mlh1-PMS1 endonuclease.7-9 Here, we discovered that overexpression of DNA ligase I (Cdc9) in Saccharomyces cerevisiae causes elevated mutation rates and increased chromatin-bound PCNA levels and accumulation of PMS1 foci that are MMR intermediates, suggesting that premature ligation of replication-associated nicks interferes with MMR. We showed that yeast PMS1 expression is mainly restricted to S phase, in agreement with the temporal coupling between MMR and DNA replication.10 Restricting PMS1 expression to the G2/M phase caused a mutator phenotype that was exacerbated in the absence of the exonuclease Exo1. This mutator phenotype was largely suppressed by increasing the lifetime of replication-associated DNA nicks, either by reducing or delaying Cdc9 ligase activity in vivo. Therefore, Cdc9 dictates a window of time for MMR determined by transient DNA nicks that direct the Mlh1-PMS1 in a strand-specific manner. Because DNA nicks occur on both newly synthesized leading and lagging strands,11 these results establish a general mechanism for targeting MMR to the newly synthesized DNA, thus preventing the accumulation of mutations that underlie the development of human cancer.
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the properties of msh2 msh6 atp binding mutants suggest a signal amplification mechanism in dna mismatch repair
Journal of Biological Chemistry, 2018Co-Authors: William J Graham, Christopher D Putnam, Richard D. KolodnerAbstract:: DNA mismatch repair (MMR) corrects mispaired DNA bases and small insertion/deletion loops generated by DNA replication errors. After binding a mispair, the eukaryotic mispair recognition complex Msh2-Msh6 binds ATP in both of its nucleotide-binding sites, which induces a conformational change resulting in the formation of an Msh2-Msh6 sliding clamp that releases from the mispair and slides freely along the DNA. However, the roles that Msh2-Msh6 sliding clamps play in MMR remain poorly understood. Here, using Saccharomyces cerevisiae, we created Msh2 and Msh6 Walker A nucleotide-binding site mutants that have defects in ATP binding in one or both nucleotide-binding sites of the Msh2-Msh6 heterodimer. We found that these mutations cause a complete MMR defect in vivo The mutant Msh2-Msh6 complexes exhibited normal mispair recognition and were proficient at recruiting the MMR endonuclease Mlh1-PMS1 to mispaired DNA. At physiological (2.5 mm) ATP concentration, the mutant complexes displayed modest partial defects in supporting MMR in reconstituted Mlh1-PMS1-independent and Mlh1-PMS1-dependent MMR reactions in vitro and in activation of the Mlh1-PMS1 endonuclease and showed a more severe defect at low (0.1 mm) ATP concentration. In contrast, five of the mutants were completely defective and one was mostly defective for sliding clamp formation at high and low ATP concentrations. These findings suggest that mispair-dependent sliding clamp formation triggers binding of additional Msh2-Msh6 complexes and that further recruitment of additional downstream MMR proteins is required for signal amplification of mispair binding during MMR.
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The properties of Msh2–Msh6 ATP binding mutants suggest a signal amplification mechanism in DNA mismatch repair
The Journal of biological chemistry, 2018Co-Authors: William J Graham, Christopher D Putnam, Richard D. KolodnerAbstract:DNA mismatch repair (MMR) corrects mispaired DNA bases and small insertion/deletion loops generated by DNA replication errors. After binding a mispair, the eukaryotic mispair recognition complex Msh2-Msh6 binds ATP in both of its nucleotide-binding sites, which induces a conformational change resulting in the formation of an Msh2-Msh6 sliding clamp that releases from the mispair and slides freely along the DNA. However, the roles that Msh2-Msh6 sliding clamps play in MMR remain poorly understood. Here, using Saccharomyces cerevisiae, we created Msh2 and Msh6 Walker A nucleotide-binding site mutants that have defects in ATP binding in one or both nucleotide-binding sites of the Msh2-Msh6 heterodimer. We found that these mutations cause a complete MMR defect in vivo The mutant Msh2-Msh6 complexes exhibited normal mispair recognition and were proficient at recruiting the MMR endonuclease Mlh1-PMS1 to mispaired DNA. At physiological (2.5 mm) ATP concentration, the mutant complexes displayed modest partial defects in supporting MMR in reconstituted Mlh1-PMS1-independent and Mlh1-PMS1-dependent MMR reactions in vitro and in activation of the Mlh1-PMS1 endonuclease and showed a more severe defect at low (0.1 mm) ATP concentration. In contrast, five of the mutants were completely defective and one was mostly defective for sliding clamp formation at high and low ATP concentrations. These findings suggest that mispair-dependent sliding clamp formation triggers binding of additional Msh2-Msh6 complexes and that further recruitment of additional downstream MMR proteins is required for signal amplification of mispair binding during MMR.
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Activation of Saccharomyces cerevisiae Mlh1-PMS1 Endonuclease in a Reconstituted Mismatch Repair System
The Journal of biological chemistry, 2015Co-Authors: Catherine E. Smith, Eva M Goellner, William J Graham, Anjana Srivatsan, Nikki Bowen, Richard D. KolodnerAbstract:Previous studies reported the reconstitution of an Mlh1-PMS1-independent 5' nick-directed mismatch repair (MMR) reaction using Saccharomyces cerevisiae proteins. Here we describe the reconstitution of a mispair-dependent Mlh1-PMS1 endonuclease activation reaction requiring Msh2-Msh6 (or Msh2-Msh3), proliferating cell nuclear antigen (PCNA), and replication factor C (RFC) and a reconstituted Mlh1-PMS1-dependent 3' nick-directed MMR reaction requiring Msh2-Msh6 (or Msh2-Msh3), exonuclease 1 (Exo1), replication protein A (RPA), RFC, PCNA, and DNA polymerase δ. Both reactions required Mg(2+) and Mn(2+) for optimal activity. The MMR reaction also required two reaction stages in which the first stage required incubation of Mlh1-PMS1 with substrate DNA, with or without Msh2-Msh6 (or Msh2-Msh3), PCNA, and RFC but did not require nicking of the substrate, followed by a second stage in which other proteins were added. Analysis of different mutant proteins demonstrated that both reactions required a functional Mlh1-PMS1 endonuclease active site, as well as mispair recognition and Mlh1-PMS1 recruitment by Msh2-Msh6 but not sliding clamp formation. Mutant Mlh1-PMS1 and PCNA proteins that were defective for Exo1-independent but not Exo1-dependent MMR in vivo were partially defective in the Mlh1-PMS1 endonuclease and MMR reactions, suggesting that both reactions reflect the activation of Mlh1-PMS1 seen in Exo1-independent MMR in vivo. The availability of this reconstituted MMR reaction should now make it possible to better study both Exo1-independent and Exo1-dependent MMR.
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pcna and msh2 msh6 activate an mlh1 PMS1 endonuclease pathway required for exo1 independent mismatch repair
Molecular Cell, 2014Co-Authors: Eva M Goellner, Christopher D Putnam, Christopher S Campbell, Hans Hombauer, Catherine E. Smith, Arshad Desai, Richard D. KolodnerAbstract:Genetic evidence has implicated multiple pathways in eukaryotic DNA mismatch repair (MMR) downstream of mispair recognition and Mlh1-PMS1 recruitment, including Exonuclease 1 (Exo1)-dependent and -independent pathways. We identified 14 mutations in POL30, which encodes PCNA in Saccharomyces cerevisiae, specific to Exo1-independent MMR. The mutations identified affected amino acids at three distinct sites on the PCNA structure. Multiple mutant PCNA proteins had defects either in trimerization and Msh2-Msh6 binding or in activation of the Mlh1-PMS1 endonuclease that initiates excision during MMR. The latter class of mutations led to hyperaccumulation of repair intermediate Mlh1-PMS1 foci and were enhanced by an msh6 mutation that disrupted the Msh2-Msh6 interaction with PCNA. These results reveal a central role for PCNA in the Exo1-independent MMR pathway and suggest that Msh2-Msh6 localizes PCNA to repair sites after mispair recognition to activate the Mlh1-PMS1 endonuclease for initiating Exo1-dependent repair or for driving progressive excision in Exo1-independent repair.
Eric Alani - One of the best experts on this subject based on the ideXlab platform.
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Handcuffing intrinsically disordered regions in Mlh1-PMS1 disrupts mismatch repair
2021Co-Authors: Christopher M. Furman, Ting-yi Wang, Qiuye Zhao, Kumar Yugandhar, Eric AlaniAbstract:The DNA mismatch repair (MMR) factor Mlh1-PMS1 contains long intrinsically disordered regions (IDRs). While essential for MMR, their exact functions remain elusive. We performed cross-linking mass spectrometry to identify the major interactions within the Mlh1-PMS1 heterodimer and used this information to insert FRB and FKBP dimerization domains into the IDRs of Mlh1 and PMS1. Yeast bearing these constructs were grown with rapamycin to induce dimerization. Strains containing FRB and FKBP domains in the Mlh1 IDR displayed complete MMR defects when grown with rapamycin, but removing rapamycin restored MMR functions. Furthermore, linking the Mlh1 and PMS1 IDRs through FRB-FKBP dimerization disrupted Mlh1-PMS1 binding to DNA, inappropriately activated Mlh1-PMS1, and caused MMR defects in vivo. We conclude that dynamic and coordinated rearrangements of the MLH IDRs regulate how the complex clamps DNA to catalyze MMR. The application of the FRB-FKBP dimerization system to interrogate in vivo functions of a critical repair complex will be useful for probing IDRs in diverse enzymes and to probe transient loss of MMR on demand.
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Intrinsically disordered regions regulate both catalytic and non-catalytic activities of the MutLα mismatch repair complex
Nucleic acids research, 2019Co-Authors: Yoori Kim, Eric Alani, Carol M Manhart, Christopher M. Furman, Ilya J. FinkelsteinAbstract:Intrinsically disordered regions (IDRs) are present in at least 30% of the eukaryotic proteome and are enriched in chromatin-associated proteins. Using a combination of genetics, biochemistry and single-molecule biophysics, we characterize how IDRs regulate the functions of the yeast MutLα (Mlh1-PMS1) mismatch repair (MMR) complex. Shortening or scrambling the IDRs in both subunits ablates MMR in vivo. Mlh1-PMS1 complexes with shorter IDRs that disrupt MMR retain wild-type DNA binding affinity but are impaired for diffusion on both naked and nucleosome-coated DNA. Moreover, the IDRs also regulate the adenosine triphosphate hydrolysis and nuclease activities that are encoded in the structured N- and C-terminal domains of the complex. This combination of phenotypes underlies the catastrophic MMR defect seen with the mutant MutLα in vivo. More broadly, this work highlights an unanticipated multi-functional role for IDRs in regulating both facilitated diffusion on chromatin and nucleolytic processing of a DNA substrate.
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Intrinsically disordered regions regulate both catalytic and non-catalytic activities of the MutLα mismatch repair complex
2018Co-Authors: Yoori Kim, Eric Alani, Carol M Manhart, Christopher M. Furman, Ilya J. FinkelsteinAbstract:AbstractIntrinsically disordered regions (IDRs) are present in at least 30% of the eukaryotic proteome and are enriched in chromatin-associated proteins. Using a combination of genetics, biochemistry, and single-molecule biophysics, we characterize how IDRs regulate the functions of the yeast MutLα (Mlh1-PMS1) mismatch repair (MMR) complex. Shortening or scrambling the IDRs in both subunits ablates MMR in vivo. Mlh1-PMS1 complexes with shorter IDRs that disrupt MMR retain wild-type DNA binding affinity but are impaired for diffusion on both naked and nucleosome-coated DNA. Moreover, the IDRs also regulate the ATP hydrolysis and nuclease activities that are encoded in the structured N- and C-terminal domains of the complex. This combination of phenotypes underlies the catastrophic MMR defect seen with the mutant MutLα in vivo. More broadly, this work highlights an unanticipated multi-functional role for IDRs in regulating both facilitated diffusion on chromatin and nucleolytic processing of a DNA substrate.
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Incompatibilities in Mismatch Repair Genes MLH1-PMS1 Contribute to a Wide Range of Mutation Rates in Human Isolates of Baker's Yeast.
Genetics, 2018Co-Authors: Vandana Raghavan, Najla Al-sweel, Duyen T. Bui, Charles F. Aquadro, Anne Friedrich, Joseph Schacherer, Eric AlaniAbstract:Laboratory baker's yeast strains bearing an incompatible combination of MLH1 and PMS1 mismatch repair alleles are mutators that can adapt more rapidly to stress, but do so at the cost of long-term fitness. We identified 18 baker's yeast isolates from 1011 surveyed that contain the incompatible MLH1-PMS1 genotype in a heterozygous state. Surprisingly, the incompatible combination from two human clinical heterozygous diploid isolates, YJS5845 and YJS5885, contain the exact MLH1 (S288c-derived) and PMS1 (SK1-derived) open reading frames originally shown to confer incompatibility. While these isolates were nonmutators, their meiotic spore clone progeny displayed mutation rates in a DNA slippage assay that varied over a 340-fold range. This range was 30-fold higher than observed between compatible and incompatible combinations of laboratory strains. Genotyping analysis indicated that MLH1-PMS1 incompatibility was the major driver of mutation rate in the isolates. The variation in the mutation rate of incompatible spore clones could be due to background suppressors and enhancers, as well as aneuploidy seen in the spore clones. Our data are consistent with the observed variance in mutation rate contributing to adaptation to stress conditions (e.g., in a human host) through the acquisition of beneficial mutations, with high mutation rates leading to long-term fitness costs that are buffered by mating or eliminated through natural selection.
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Mismatch Repair Incompatibilities in Diverse Yeast Populations
Genetics, 2017Co-Authors: Duyen T. Bui, Najla Al-sweel, Charles F. Aquadro, Anne Friedrich, Joseph Schacherer, Gianni Liti, Eric AlaniAbstract:An elevated mutation rate can provide cells with a source of mutations to adapt to changing environments. We identified a negative epistatic interaction involving naturally occurring variants in the MLH1 and PMS1 mismatch repair (MMR) genes of Saccharomyces cerevisiae We hypothesized that this MMR incompatibility, created through mating between divergent S. cerevisiae, yields mutator progeny that can rapidly but transiently adapt to an environmental stress. Here we analyzed the MLH1 and PMS1 genes across 1010 S. cerevisiae natural isolates spanning a wide range of ecological sources (tree exudates, Drosophila, fruits, and various fermentation and clinical isolates) and geographical sources (Europe, America, Africa, and Asia). We identified one homozygous clinical isolate and 18 heterozygous isolates containing the incompatible MMR genotype. The MLH1-PMS1 gene combination isolated from the homozygous clinical isolate conferred a mutator phenotype when expressed in the S288c laboratory background. Using a novel reporter to measure mutation rates, we showed that the overall mutation rate in the homozygous incompatible background was similar to that seen in compatible strains, indicating the presence of suppressor mutations in the clinical isolate that lowered its mutation rate. This observation and the identification of 18 heterozygous isolates, which can lead to MMR incompatible genotypes in the offspring, are consistent with an elevated mutation rate rapidly but transiently facilitating adaptation. To avoid long-term fitness costs, the incompatibility is apparently buffered by mating or by acquiring suppressors. These observations highlight effective strategies in eukaryotes to avoid long-term fitness costs associated with elevated mutation rates.
Thomas A. Kunkel - One of the best experts on this subject based on the ideXlab platform.
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Modeling of the DNA-binding site of yeast PMS1 by mass spectrometry.
DNA repair, 2011Co-Authors: Allison N. Schorzman, Thomas A. Kunkel, Lalith Perera, Jenny M. Cutalo-patterson, Lars C. Pedersen, Lee G. Pedersen, Kenneth B. TomerAbstract:Mismatch repair (MMR) corrects replication errors that would otherwise lead to mutations and, potentially, various forms of cancer. Among several proteins required for eukaryotic MMR, MutLα is a heterodimer comprised of Mlh1 and PMS1. The two proteins dimerize along their C-terminal domains (CTDs), and the CTD of PMS1 houses a latent endonuclease that is required for MMR. The highly conserved N-terminal domains (NTDs) independently bind DNA and possess ATPase active sites. Here we use two protein footprinting techniques, limited proteolysis and oxidative surface mapping, coupled with mass spectrometry to identify amino acids involved along the DNA-binding surface of the PMS1-NTD. Limited proteolysis experiments elucidated several basic residues that were protected in the presence of DNA, while oxidative surface mapping revealed one residue that is uniquely protected from oxidation. Furthermore, additional amino acids distributed throughout the PMS1-NTD were protected from oxidation either in the presence of a non-hydrolyzable analog of ATP or DNA, indicating that each ligand stabilizes the protein in a similar conformation. Based on the recently published X-ray crystal structure of yeast PMS1-NTD, a model of the PMS1-NTD/DNA complex was generated using the mass spectrometric data as constraints. The proposed model defines the DNA-binding interface along a positively charged groove of the PMS1-NTD and complements prior mutagenesis studies of Escherichia coli and eukaryotic MutL.
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Functional residues on the surface of the N-terminal domain of yeast PMS1.
DNA repair, 2010Co-Authors: Mercedes E. Arana, John M. Fortune, Lars C. Pedersen, Shannon F. Holmes, Andrea F. Moon, Thomas A. KunkelAbstract:Abstract Saccharomyces cerevisiae MutLα is a heterodimer of Mlh1 and PMS1 that participates in DNA mismatch repair (MMR). Both proteins have weakly conserved C-terminal regions (CTDs), with the CTD of PMS1 harboring an essential endonuclease activity. These proteins also have conserved N-terminal domains (NTDs) that bind and hydrolyze ATP and bind to DNA. To better understand PMS1 functions and potential interactions with DNA and/or other proteins, we solved the 2.5 A crystal structure of yeast PMS1 (yPMS1) NTD. The structure is similar to the homologous NTDs of Escherichia coli MutL and human PMS2, including the site involved in ATP binding and hydrolysis. The structure reveals a number of conserved, positively charged surface residues that do not interact with other residues in the NTD and are therefore candidates for interactions with DNA, with the CTD and/or with other proteins. When these were replaced with glutamate, several replacements resulted in yeast strains with elevated mutation rates. Two replacements also resulted in NTDs with decreased DNA binding affinity in vitro, suggesting that these residues contribute to DNA binding that is important for mismatch repair. Elevated mutation rates also resulted from surface residue replacements that did not affect DNA binding, suggesting that these conserved residues serve other functions, possibly involving interactions with other MMR proteins.
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Mapping the dimer interface in the C-terminal domains of the yeast MLH1-PMS1 heterodimer.
Biochemistry, 2006Co-Authors: Jenny M. Cutalo, Thomas A. Kunkel, Thomas A. Darden, Kenneth B. TomerAbstract:Yeast MutLα is a heterodimer of MLH1 and PMS1 that participates in a variety of DNA transactions, including DNA mismatch repair. Formation of the MutLα heterodimer requires that the C-terminal domains of MLH1 and PMS1 interact in a manner that is not yet fully understood. Here we investigate the interactions involved in heterodimerization. Using protein surface modification and mass spectrometry, we identify numerous lysine residues that are exposed to solvent in monomeric MLH1. A corresponding analysis of the MLH1−PMS1 heterodimer reveals that three of these exposed residues, K665, K675, and K704, are no longer solvent accessible in the heterodimer, suggesting that they are within the dimer interface. We refine secondary structure predictions and sequence alignments of C-terminal residues of seven eukaryotic MutL homologues and then develop homology models for the N- and C-terminal domains of MLH1. On the basis of this information, we present a model for interaction of the C-terminal domains of MLH1 and ...
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DNA binding by yeast Mlh1 and PMS1: implications for DNA mismatch repair.
Nucleic acids research, 2003Co-Authors: Mark C. Hall, Polina V. Shcherbakova, John M. Fortune, Christoph H. Borchers, J. Michael Dial, Kenneth B. Tomer, Thomas A. KunkelAbstract:The yeast Mlh1–PMS1 heterodimer required for mismatch repair (MMR) binds to DNA. Here we map DNA binding to N-terminal fragments of Mlh1 and PMS1. We demonstrate that Mlh1 and PMS1 N-terminal domains (NTDs) independently bind to double-stranded and single-stranded DNA, in the absence of dimerization and with different affinities. Full-length Mlh1p alone, which can homodimerize, also binds to DNA. Substituting conserved positively charged amino acids in Mlh1 produces mutator phenotypes in a haploid yeast strain characteristic of reduced MMR. These substitutions strongly reduce DNA binding by the Mlh1 NTD and, to a lesser extent, they also reduce DNA binding by full-length Mlh1 and the Mlh1–PMS1 heterodimer. Replacement of a homologous PMS1 residue has a much smaller effect on mutation rate and does not reduce DNA binding. The results demonstrate that NTDs of yeast Mlh1 and PMS1 contain independent DNA binding sites and they suggest that the C-terminal region of Mlh1p may also contribute to DNA binding. The differential mutator effects and binding properties observed here further suggest that Mlh1 and PMS1 differ in their interactions with DNA. Finally, the results are consistent with the hypothesis that DNA binding by Mlh1 is important for MMR.
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DNA binding properties of the yeast Msh2-Msh6 and Mlh1-PMS1 heterodimers.
Biological Chemistry, 2002Co-Authors: Karin Drotschmann, Polina V. Shcherbakova, Floyd R. Brownewell, Mark C. Hall, Eric T Kool, Dorothy A Erie, Hong Wang, Thomas A. KunkelAbstract:We describe here our recent studies of the DNA binding properties of Msh2-Msh6 and Mlh1-PMS1, two protein complexes required to repair mismatches generated during DNA replication. Mismatched DNA binding by Msh2-Msh6 was probed by mutagenesis based on the crystal structure of the homologous bacterial MutS homodimer bound to DNA. The results suggest that several amino acid side chains inferred to interact with the DNA backbone near the mismatch are critical for repair activity. These contacts, which are different in Msh2 and Msh6, likely facilitate stacking and hydrogen bonding interactions between side chains in Msh6 and the mismatched base, thus stabilizing a kinked DNA conformation that permits subsequent repair steps coordinated by the Mlh1-PMS1 heterodimer. Mlh1-PMS1 also binds to DNA, but independently of a mismatch. Mlh1-PMS1 binds short DNA substrates with low affinity and with a slight preference for single-stranded DNA. It also binds longer duplex DNA molecules, but with a higher affinity indicative of cooperative binding. Indeed, imaging by atomic force microscopy reveals cooperative DNA binding and simultaneous interaction with two DNA duplexes. The novel DNA binding properties of Mlh1-PMS1 may be relevant to signal transduction during DNA mismatch repair and to recombination, meiosis and cellular responses to DNA damage.
Satya Prakash - One of the best experts on this subject based on the ideXlab platform.
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atp dependent assembly of a ternary complex consisting of a dna mismatch and the yeast msh2 msh6 and mlh1 PMS1 protein complexes
Journal of Biological Chemistry, 1998Co-Authors: Yvette Habraken, Louise Prakash, Satya PrakashAbstract:Abstract MSH2 and MSH6 proteins exist as a stable complex, as do the MLH1 and PMS1 proteins. To study the mismatch binding properties of the MSH2-MSH6 complex and to examine its functional interaction with the MLH1-PMS1 complex, these protein complexes were purified to near homogeneity from overproducing yeast strains. As has been reported previously, the purified MSH2-MSH6 complex binds DNA substrates containing a G/T mismatch and insertion/deletion mismatches, but the binding affinity for the latter decreases as the size of the extrahelical loop increases. Addition of ATP or the nonhydrolyzable ATPγS reduces binding of the MSH2-MSH6 complex to the DNA substrates markedly. Here, we show that MSH2-MSH6 forms a ternary complex with MLH1-PMS1 on a mismatch containing DNA substrate. The formation of this ternary complex requires ATP, which can be substituted by ATPγS, suggesting that ATP binding alone is sufficient for ternary complex formation. Thus, it appears that ATP binding by the MSH2-MSH6 complex induces a conformation that is conducive for the interaction with MLH1-PMS1 complex, leading to the formation of the ternary complex.
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Enhancement of MSH2-MSH3-mediated mismatch recognition by the yeast MLH1-PMS1 complex.
Current biology : CB, 1997Co-Authors: Yvette Habraken, Louise Prakash, Patrick Sung, Satya PrakashAbstract: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.
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Evidence for Involvement of Yeast Proliferating Cell Nuclear Antigen in DNA Mismatch Repair
The Journal of biological chemistry, 1996Co-Authors: Robert E. Johnson, Yvette Habraken, Louise Prakash, Gopala K. Kovvali, Sami N. Guzder, Neelam S. Amin, Connie Holm, Patrick Sung, Satya PrakashAbstract: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.
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Requirement of the yeast RTH1 5' to 3' exonuclease for the stability of simple repetitive DNA
Science (New York N.Y.), 1995Co-Authors: Robert E. Johnson, Louise Prakash, Gopala K. Kovvali, Satya PrakashAbstract:Simple repetitive DNA sequences are unstable in human colorectal cancers and a variety of other cancers. Mutations in the DNA mismatch repair genes MSH2, MLH1, and PMS1 result in elevated rates of spontaneous mutation and cause a marked increase in the instability of simple repeats. Compared with the wild type, a null mutation in the yeast RTH1 gene, which encodes a 5' to 3' exonuclease, was shown to increase the rate of instability of simple repetitive DNA by as much as 280 times and to increase the spontaneous mutation rate by 30 times. Epistasis analyses were consistent with the hypothesis that this RTH1-encoded nuclease has a role in the MSH2-MLH-1-PMS1 mismatch repair pathway.
Jürg Kohli - One of the best experts on this subject based on the ideXlab platform.
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Involvement of nucleotide-excision repair in msh2 PMS1 -independent mismatch repair
Nature genetics, 1999Co-Authors: Oliver Fleck, Primo Schär, Elisabeth Lehmann, Jürg KohliAbstract:Nucleotide-excision repair (NER) and mismatch repair (MMR) are prominent examples of highly conserved DNA repair systems which recognize and replace damaged and/or mispaired nucleotides in DNA. In humans, inheritable defects in components of the NER system are associated with severe diseases such as xeroderma pigmentosum (XP) and Cockayne syndrome (CS), whereas inactivation of MMR is accompanied by predisposition to certain types of cancer. In Schizosaccharomyces pombe, the msh2- and PMS1-dependent long-patch MMR system efficiently corrects small insertion/deletion loops and all base-base mismatches, except C/C. Up to 70% of C/C mismatches generated in recombination intermediates, and to a lesser extent also other base-base mismatches, are thought to undergo correction by a minor, short-patch excision repair system. We identify here the NER genes rhpl4, swi10 and rad16 as components of this repair pathway and show that they act independently of msh2 and PMS1.
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Schizosaccharomyces pombe exo1 is involved in the same mismatch repair pathway as msh2 and PMS1
Current genetics, 1998Co-Authors: Claudia Rudolph, Oliver Fleck, Jürg KohliAbstract:Besides the MutLS-like system, Schizosaccharomyces pombe has an additional pathway of mismatch repair. This minor pathway, producing short excision tracts, repairs C/C and, with lower efficiency, other mismatches also. We investigated the involvement of the exo1 +, msh2 + and PMS1 + genes in the two pathways. The exo1 + gene encodes a 5′ to 3′ exonuclease, while msh2 + and PMS1 + are homologs of Escherichia coli mutS and mutL, respectively. Intragenic two-factor crosses showed that exo1 +, msh2 + and PMS1 + are involved in the major, but not in the C/C-correcting, pathway. Post-meiotic segregation frequencies and mitotic mutation rates in single and double mutants supported this finding. Furthermore, msh2Δ was epistatic over exo1Δ, and the ExoI enzyme is likely to be redundant with other exonucleases.
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Mismatch repair in Schizosaccharomyces pombe requires the mutL homologous gene PMS1: molecular cloning and functional analysis.
Genetics, 1997Co-Authors: Primo Schär, Michel Baur, Claudine Schneider, Jürg KohliAbstract:Homologues of the bacterial mutS and mutL genes involved in DNA mismatch repair have been found in organisms from bacteria to humans. Here, we describe the structure and function of a newly identified Schizosaccharomyces pombe that encodes a predicted amino acid sequence of 794 residues with a high degree of homology to MutL related proteins. On the basis of its closer relationship to the eukaryotic "PMS" genes than to the "MLH" genes, we have designated the S. pombe homologue PMS1. Disruption of the PMS1 gene causes a significant increase of spontaneous mutagenesis as documented by reversion rate measurements. Tetrad analyses of crosses homozygous for the PMS1 mutation reveal a reduction of spore viability from > 92% to 80% associated with a low proportion (approximately 50%) of meioses producing four viable spores and a significant, allele-dependent increase of the level of post-meiotic segregation of genetic marker allele pairs. The mutant phenotypes are consistent with a general function of PMS1 in correction of mismatched base pairs arising as a consequence of DNA polymerase errors during DNA synthesis, or of hybrid DNA formation between homologous but not perfectly complementary DNA strands during meiotic recombination.
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mismatch repair in schizosaccharomyces pombe requires the mutl homologous gene PMS1 molecular cloning and functional analysis
Genetics, 1997Co-Authors: Primo Schär, Michel Baur, Claudine Schneider, Jürg KohliAbstract:Homologues of the bacterial mutS and mutL genes involved in DNA mismatch repair have been found in organisms from bacteria to humans. Here, we describe the structure and function of a newly identified Schizosaccharomyces pombe gene that encodes a predicted amino acid sequence of 794 residues with a high degree of homology to MutL related proteins. On the basis of its closer relationship to the eukaryotic ``PMS'' genes than to the ``MLH'' genes, we have designated the S. pombe homologue PMS1. Disruption of the PMS1 gene causes a significant increase of spontaneous mutagenesis as documented by reversion rate measurements. Tetrad analyses of crosses homozygous for the PMS1 mutation reveal a reduction of spore viability from >92% to 80% associated with a low proportion (~50%) of meioses producing four viable spores and a significant, allele-dependent increase of the level of post-meiotic segregation of genetic marker allele pairs. The mutant phenotypes are consistent with a general function of PMS1 in correction of mismatched base pairs arising as a consequence of DNA polymerase errors during DNA synthesis, or of hybrid DNA formation between homologous but not perfectly complementary DNA strands during meiotic recombination.