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

  • the properties of MSH2 msh6 atp binding mutants suggest a signal amplification mechanism in dna mismatch repair
    Journal of Biological Chemistry, 2018
    Co-Authors: William J Graham, Christopher D Putnam, Richard D. Kolodner
    Abstract:

    : 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.

  • The properties of MSH2–Msh6 ATP binding mutants suggest a signal amplification mechanism in DNA mismatch repair
    The Journal of biological chemistry, 2018
    Co-Authors: William J Graham, Christopher D Putnam, Richard D. Kolodner
    Abstract:

    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.

  • Activation of Saccharomyces cerevisiae Mlh1-Pms1 Endonuclease in a Reconstituted Mismatch Repair System
    The Journal of biological chemistry, 2015
    Co-Authors: Catherine E. Smith, Eva M Goellner, William J Graham, Anjana Srivatsan, Nikki Bowen, Richard D. Kolodner
    Abstract:

    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.

  • 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.

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

  • 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.

  • Multiple factors insulate MSH2-Msh6 mismatch repair activity from defects in MSH2 domain I.
    Journal of molecular biology, 2011
    Co-Authors: Charanya Kumar, Eric Alani, Sarah C. Piacente, Justin Sibert, Andrew R. Bukata, Jaime O'connor, Jennifer A Surtees
    Abstract:

    Abstract DNA mismatch repair (MMR) is a highly conserved mutation avoidance mechanism that corrects DNA polymerase misincorporation errors. In initial steps in MMR, MSH2–Msh6 binds mispairs and small insertion/deletion loops, and MSH2–Msh3 binds larger insertion/deletion loops. The MSH2Δ1 mutation, which deletes the conserved DNA-binding domain I of MSH2, does not dramatically affect MSH2–Msh6-dependent repair. In contrast, MSH2Δ1 mutants show strong defects in MSH2–Msh3 functions. Interestingly, several mutations identified in patients with hereditary non-polyposis colorectal cancer map to domain I of MSH2; none have been found in MSH3 . To understand the role of MSH2 domain I in MMR, we examined the consequences of combining the MSH2Δ1 mutation with mutations in two distinct regions of MSH6 and those that increase cellular mutational load ( pol3-01 and rad27 ). These experiments reveal MSH2Δ1 -specific phenotypes in MSH2–Msh6 repair, with significant effects on mutation rates. In vitro assays demonstrate that MSH2Δ1–Msh6 DNA binding is less specific for DNA mismatches and produces an altered footprint on a mismatch DNA substrate. Together, these results provide evidence that, in vivo , multiple factors insulate MMR from defects in domain I of MSH2 and provide insights into how mutations in MSH2 domain I may cause hereditary non-polyposis colorectal cancer.

  • 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.

  • Heteroduplex rejection during single-strand annealing requires Sgs1 helicase and mismatch repair proteins MSH2 and Msh6 but not Pms1
    Proceedings of the National Academy of Sciences of the United States of America, 2004
    Co-Authors: Neal Sugawara, Eric Alani, Barbara Studamire, Tamara Goldfarb, James E. Haber
    Abstract:

    Recombination between moderately divergent DNA sequences is impaired compared with identical sequences. In yeast, an HO endonuclease-induced double-strand break can be repaired by single-strand annealing (SSA) between flanking homologous sequences. A 3% sequence divergence between 205-bp sequences flanking the double-strand break caused a 6-fold reduction in repair compared with identical sequences. This reduction in heteroduplex rejection was suppressed in a mismatch repair-defective msh6 Delta strain and partially suppressed in an MSH2 separation-of-function mutant. In mlh1 Delta strains, heteroduplex rejection was greater than in msh6 Delta strains but less than in wild type. Deleting PMS1, MLH2,or MLH3 had no effect on heteroduplex rejection, but a pms1 Delta mlh2 Delta mlh3 Delta triple mutant resembled mlh1 Delta. However, correction of the mismatches within heteroduplex SSA intermediates required PMS1 and MLH1 to the same extent as MSH2 and MSH6. An SSA competition assay in which either diverged or identical repeats can be used for repair showed that heteroduplex DNA is likely to be unwound rather than degraded. This conclusion is supported by the finding that deleting the SGS1 helicase also suppressed heteroduplex rejection.

  • MSH2 separation of function mutations confer defects in the initiation steps of mismatch repair.
    Journal of molecular biology, 2003
    Co-Authors: Amanda W. Kijas, Barbara Studamire, Eric Alani
    Abstract:

    In eukaryotes the MSH2-MSH3 and MSH2-MSH6 heterodimers initiate mismatch repair (MMR) by recognizing and binding to DNA mismatches. The MLH1-PMS1 heterodimer then interacts with the MSH proteins at or near the mismatch site and is thought to act as a mediator to recruit downstream repair proteins. Here we analyzed five MSH2 mutants that are functional in removing 3' non-homologous tails during double-strand break repair but are completely defective in MMR. Because non-homologous tail removal does not require MSH6, MLH1, or PMS1 functions, a characterization of the MSH2 separation of function alleles should provide insights into early steps in MMR. Using the Taq MutS crystal structure as a model, three of the MSH2 mutations, MSH2-S561P, MSH2-K564E, MSH2-G566D, were found to map to a domain in MutS involved in stabilizing mismatch binding. Gel mobility shift and DNase I footprinting assays showed that two of these mutations conferred strong defects on MSH2-MSH6 mismatch binding. The other two mutations, MSH2-S656P and MSH2-R730W, mapped to the ATPase domain. DNase I footprinting, ATP hydrolysis, ATP binding, and MLH1-PMS1 interaction assays indicated that the MSH2-S656P mutation caused defects in ATP-dependent dissociation of MSH2-MSH6 from mismatch DNA and in interactions between MSH2-MSH6 and MLH1-PMS1. In contrast, the MSH2-R730W mutation disrupted MSH2-MSH6 ATPase activity but did not strongly affect ATP binding or interactions with MLH1-PMS1. These results support a model in which MMR can be dissected into discrete steps: stable mismatch binding and sensing, MLH1-PMS1 recruitment, and recycling of MMR components.

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.

  • 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.

  • 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.

Manju Hingorani - One of the best experts on this subject based on the ideXlab platform.

  • Enhanced Dynamics of Mismatched Base Pairs Associated with MSH2-Msh6 Recognition
    Biophysical Journal, 2015
    Co-Authors: Yan Li, Manju Hingorani, Ishita Mukerji
    Abstract:

    The DNA mismatch repair (MMR) system guards the integrity of genetic material by scanning and correcting errors in a post-replicative manner. In eukaryotic cells, the initiation of MMR is achieved by recognition by MutS homologs (Msh). The MSH2-Msh6 heterodimer plays this important initial role in recognizing single base mismatches and small insertion/deletion loops (IDL) in MMR. Although MSH2-Msh6 recognizes mismatched DNA with high affinity, the exact mechanism by which MSH2-Msh6 distinguishes different types of mismatched base pairs from a large excess of canonical Watson-Crick base pairs is still unknown. In this study, we use the intrinsic fluorescent probe 6-methylisoxanthopterin (6-MI, guanosine analog) in the context of the ATFAA (F = 6-MI) pentamer sequence where it exhibits enhanced fluorescence, to measure the binding affinity of S. cerevisiae MSH2-Msh6 to different single base pair mismatches. Fluorescence anisotropy measurements reveal the following order for MSH2-Msh6 mismatch bp binding affinity: A:A ≈ A:G > A:C ≈ G:T ≈ +T > T:T ≈ G:G > T:C ≈ G:C. Fluorescence intensity measurements suggest a greater degree of DNA distortion accompanies binding to well-recognized mismatches. We employ Forster resonance energy transfer to measure the bending angle and compare with that observed in MutS/MSH2-Msh6 co-crystal structures. We have also investigated DNA dynamics upon MSH2-Msh6 binding using time-resolved fluorescence spectroscopy. Specific placement of the probe at the mismatch site or adjacent to it reveals significant local motion prior to protein binding. We observe that high affinity binding is associated with those mismatches that exhibit the greatest amount of motion. Protein binding stabilizes mismatch local motion, which is consistent with Phe intercalation at the site, as observed in MSH2-Msh6-DNA co-crystal structures.

  • Application of Stopped-flow Kinetics Methods to Investigate the Mechanism of Action of a DNA Repair Protein
    Journal of Visualized Experiments, 2010
    Co-Authors: F. Noah Biro, Jie Zhai, Christopher William Doucette, Manju Hingorani
    Abstract:

    Transient kinetic analysis is indispensable for understanding the workings of biological macromolecules, since this approach yields mechanistic information including active site concentrations and intrinsic rate constants that govern macromolecular function. In case of enzymes, for example, transient or pre-steady state measurements identify and characterize individual events in the reaction pathway, whereas steady state measurements only yield overall catalytic efficiency and specificity. Individual events such as protein-protein or protein-ligand interactions and rate-limiting conformational changes often occur in the millisecond timescale, and can be measured directly by stopped-flow and chemical-quench flow methods. Given an optical signal such as fluorescence, stopped-flow serves as a powerful and accessible tool for monitoring reaction progress from substrate binding to product release and catalytic turnover1,2. Here, we report application of stopped-flow kinetics to probe the mechanism of action of MSH2-Msh6, a eukaryotic DNA repair protein that recognizes base-pair mismatches and insertion/deletion loops in DNA and signals mismatch repair (MMR)3-5. In doing so, MSH2-Msh6 increases the accuracy of DNA replication by three orders of magnitude (error frequency decreases from ~10-6 to10-9 bases), and thus helps preserve genomic integrity. Not surprisingly, defective human MSH2-Msh6 function is associated with hereditary non-polyposis colon cancer and other sporadic cancers6-8. In order to understand the mechanism of action of this critical DNA metabolic protein, we are probing the dynamics of MSH2-Msh6 interaction with mismatched DNA as well as the ATPase activity that fuels its actions in MMR. DNA binding is measured by rapidly mixing MSH2-Msh6 with DNA containing a 2-aminopurine (2-Ap) fluorophore adjacent to a G:T mismatch and monitoring the resulting increase in 2-aminopurine fluorescence in real time. DNA dissociation is measured by mixing pre-formed MSH2-Msh6 G:T(2-Ap) mismatch complex with unlabeled trap DNA and monitoring decrease in fluorescence over time9. Pre-steady state ATPase kinetics are measured by the change in fluorescence of 7-diethylamino-3-((((2-maleimidyl)ethyl)amino)carbonyl) coumarin)-labeled Phosphate Binding Protein (MDCC-PBP) on binding phosphate (Pi) released by MSH2-Msh6 following ATP hydrolysis9,10. The data reveal rapid binding of MSH2-Msh6 to a G:T mismatch and formation of a long-lived MSH2-Msh6 G:T complex, which in turn results in suppression of ATP hydrolysis and stabilization of the protein in an ATP-bound form. The reaction kinetics provide clear support for the hypothesis that ATP-bound MSH2-Msh6 signals DNA repair on binding a mismatched base pair in the double helix. F. Noah Biro and Jie Zhai contributed to this paper equally.

  • saccharomyces cerevisiae MSH2 msh6 dna binding kinetics reveal a mechanism of targeting sites for dna mismatch repair
    Proceedings of the National Academy of Sciences of the United States of America, 2010
    Co-Authors: Jie Zhai, Manju Hingorani
    Abstract:

    The DNA mismatch repair system (MMR) identifies replication errors and damaged bases in DNA and functions to preserve genomic integrity. MutS performs the task of locating mismatched base pairs, loops and lesions and initiating MMR, and the fundamental question of how this protein targets specific sites in DNA is unresolved. To address this question, we examined the interactions between Saccharomyces cerevisiae MSH2-Msh6, a eukaryotic MutS homolog, and DNA in real time. The reaction kinetics reveal that MSH2-Msh6 binds a variety of sites at similarly fast rates (kON ∼ 107 M-1 s-1), and its selectivity manifests in differential dissociation rates; e.g., the protein releases a 2-Aminopurine:T base pair approximately 90-fold faster than a G:T mismatch. On releasing the 2-Ap:T site, MSH2-Msh6 is able to move laterally on DNA to locate a nearby G:T site. The long-lived MSH2-Msh6·G:T complex triggers the next step in MMR—formation of an ATP-bound clamp—more effectively than the short-lived MSH2-Msh6·2-Ap:T complex. Mutation of Glu in the conserved Phe-X-Glu DNA binding motif stabilizes MSH2-Msh6E339A·2-Ap:T complex, and the mutant can signal 2-Ap:T repair as effectively as wild-type MSH2-Msh6 signals G:T repair. These findings suggest a targeting mechanism whereby MSH2-Msh6 scans DNA, interrogating base pairs by transient contacts and pausing at potential target sites, and the longer the pause the greater the likelihood of MMR.

  • Mechanism of cadmium-mediated inhibition of MSH2-Msh6 function in DNA mismatch repair.
    Biochemistry, 2009
    Co-Authors: Markus Wieland, Mikhail K. Levin, Karan S. Hingorani, F. Noah Biro, Manju Hingorani
    Abstract:

    The observation that Cadmium (Cd2+) inhibits MSH2-Msh6, which is responsible for identifying base pair mismatches and other discrepancies in DNA, has led to the proposal that selective targeting of this protein and consequent suppression of DNA repair or apoptosis promote the carcinogenic effects of the heavy metal toxin. It has been suggested that Cd2+ binding to specific sites on MSH2-Msh6 blocks its DNA binding and ATPase activities. To investigate the mechanism of inhibition, we measured Cd2+ binding to MSH2-Msh6, directly and by monitoring changes in protein structure and enzymatic activity. Global fitting of the data to a multiligand binding model revealed that binding of about 100 Cd2+ ions per MSH2-Msh6 results in its inactivation. This finding indicates that the inhibitory effect of Cd2+ occurs via a nonspecific mechanism. Cd2+ and MSH2-Msh6 interactions involve cysteine sulfhydryl groups, and the high Cd2+:MSH2-Msh6 ratio implicates other ligands such as histidine, aspartate, glutamate, and the ...

  • contribution of MSH2 and msh6 subunits to the asymmetric atpase and dna mismatch binding activities of saccharomyces cerevisiae MSH2 msh6 mismatch repair protein
    DNA Repair, 2006
    Co-Authors: Edwin Antony, Sapna Khubchandani, Siying Chen, Manju Hingorani
    Abstract:

    Previous analyses of both Thermus aquaticus MutS homodimer and Saccharomyces cerevisiae MSH2–Msh6 heterodimer have revealed that the subunits in these protein complexes bind and hydrolyze ATP asymmetrically, emulating their asymmetric DNA binding properties. In the MutS homodimer, one subunit (S1) binds ATP with high affinity and hydrolyzes it rapidly, while the other subunit (S2) binds ATP with lower affinity and hydrolyzes it at an apparently slower rate. Interaction of MutS with mismatched DNA results in suppression of ATP hydrolysis at S1—but which of these subunits, S1 or S2, makes specific contact with the mismatch (e.g., base stacking by a conserved phenylalanine residue) remains unknown. In order to answer this question and to clarify the links between the DNA binding and ATPase activities of each subunit in the dimer, we made mutations in the ATPase sites of MSH2 and Msh6 and assessed their impact on the activity of the MSH2–Msh6 heterodimer (in MSH2–Msh6, only Msh6 makes base specific contact with the mismatch). The key findings are: (a) Msh6 hydrolyzes ATP rapidly, and thus resembles the S1 subunit of the MutS homodimer, (b) MSH2 hydrolyzes ATP at a slower rate, and thus resembles the S2 subunit of MutS, (c) though itself an apparently weak ATPase, MSH2 has a strong influence on the ATPase activity of Msh6, (d) Msh6 binding to mismatched DNA results in suppression of rapid ATP hydrolysis, revealing a “cis” linkage between its mismatch recognition and ATPase activities, (e) the resultant MSH2–Msh6 complex, with both subunits in the ATP-bound state, exhibits altered interactions with the mismatch.

Ursula Storb - One of the best experts on this subject based on the ideXlab platform.

  • somatic hypermutation and class switch recombination in msh6 ung double knockout mice
    Journal of Immunology, 2006
    Co-Authors: Hong Ming Shen, Grazyna Bozek, Dan L Nicolae, Atsushi Tanaka, Ursula Storb
    Abstract:

    Somatic hypermutation (SHM) and class switch recombination (CSR) are initiated by activation-induced cytosine deaminase (AID). The uracil, and potentially neighboring bases, are processed by error-prone base excision repair and mismatch repair. Deficiencies in Ung, MSH2, or Msh6 affect SHM and CSR. To determine whether MSH2/Msh6 complexes which recognize single-base mismatches and loops were the only mismatch-recognition complexes required for SHM and CSR, we analyzed these processes in Msh6 −/− Ung −/− mice. SHM and CSR were affected in the same degree and fashion as in MSH2 −/− Ung −/− mice; mutations were mostly C,G transitions and CSR was greatly reduced, making MSH2/Msh3 contributions unlikely. Inactivating Ung alone reduced mutations from A and T, suggesting that, depending on the DNA sequence, varying proportions of A,T mutations arise by error-prone long-patch base excision repair. Further, in Msh6 −/− Ung −/− mice the 5′ end and the 3′ region of Ig genes was spared from mutations as in wild-type mice, confirming that AID does not act in these regions. Finally, because in the absence of both Ung and Msh6, transition mutations from C and G likely are “footprints” of AID, the data show that the activity of AID is restricted drastically in vivo compared with AID in cell-free assays.

  • Somatic Hypermutation and Class Switch Recombination in Msh6−/−Ung−/− Double-Knockout Mice
    Journal of Immunology, 2006
    Co-Authors: Hong Ming Shen, Grazyna Bozek, Dan L Nicolae, Atsushi Tanaka, Ursula Storb
    Abstract:

    Somatic hypermutation (SHM) and class switch recombination (CSR) are initiated by activation-induced cytosine deaminase (AID). The uracil, and potentially neighboring bases, are processed by error-prone base excision repair and mismatch repair. Deficiencies in Ung, MSH2, or Msh6 affect SHM and CSR. To determine whether MSH2/Msh6 complexes which recognize single-base mismatches and loops were the only mismatch-recognition complexes required for SHM and CSR, we analyzed these processes in Msh6 −/− Ung −/− mice. SHM and CSR were affected in the same degree and fashion as in MSH2 −/− Ung −/− mice; mutations were mostly C,G transitions and CSR was greatly reduced, making MSH2/Msh3 contributions unlikely. Inactivating Ung alone reduced mutations from A and T, suggesting that, depending on the DNA sequence, varying proportions of A,T mutations arise by error-prone long-patch base excision repair. Further, in Msh6 −/− Ung −/− mice the 5′ end and the 3′ region of Ig genes was spared from mutations as in wild-type mice, confirming that AID does not act in these regions. Finally, because in the absence of both Ung and Msh6, transition mutations from C and G likely are “footprints” of AID, the data show that the activity of AID is restricted drastically in vivo compared with AID in cell-free assays.