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Richard D Kolodner - One of the best experts on this subject based on the ideXlab platform.
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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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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, Catherine E Smith, Christopher S Campbell, Hans Hombauer, 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.
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engineered disulfide forming amino acid substitutions interfere with a conformational change in the mismatch recognition complex msh2 MSH6 required for mismatch repair
Journal of Biological Chemistry, 2012Co-Authors: Victoria V. Hargreaves, Christopher D Putnam, Richard D KolodnerAbstract:Abstract ATP binding causes mispair-bound Msh2-MSH6 mismatch recognition complex to slide along the DNA away from the mismatch, and ATP is required for the mispair-dependent interaction between Msh2-MSH6 and Mlh1-Pms1. It has been inferred from these observations that ATP induces conformational changes in Msh2-MSH6; however, the nature of these conformational changes and their requirement in mismatch repair are poorly understood. Here we show that ATP induces a conformational change within the C-terminal region of MSH6 that protects the trypsin cleavage site after MSH6 residue R1124. An engineered disulfide bond within this region prevented the ATP-driven conformational change and resulted in an Msh2-MSH6 complex that bound mispaired bases but could not form sliding clamps or bind Mlh1-Pms1. The engineered disulfide bond also reduced mismatch repair efficiency in vivo, indicating that this ATP-driven conformational change plays a role in mismatch repair.
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Interaction between the Msh2 and MSH6 Nucleotide-binding Sites in the Saccharomyces cerevisiae Msh2-MSH6 Complex
Journal of Biological Chemistry, 2010Co-Authors: Victoria V. Hargreaves, Scarlet S. Shell, Dan J. Mazur, Martin T. Hess, Richard D KolodnerAbstract:Indirect evidence has suggested that the Msh2-MSH6 mispair-binding complex undergoes conformational changes upon binding of ATP and mispairs, resulting in the formation of Msh2-MSH6 sliding clamps and licensing the formation of Msh2-MSH6-Mlh1-Pms1 ternary complexes. Here, we have studied eight mutant Msh2-MSH6 complexes with defective responses to nucleotide binding and/or mispair binding and used them to study the conformational changes required for sliding clamp formation and ternary complex assembly. ATP binding to the MSH6 nucleotide-binding site results in a conformational change that allows binding of ATP to the Msh2 nucleotide-binding site, although ATP binding to the two nucleotide-binding sites appears to be uncoupled in some mutant complexes. The formation of Msh2-MSH6-Mlh1-Pms1 ternary complexes requires ATP binding to only the MSH6 nucleotide-binding site, whereas the formation of Msh2-MSH6 sliding clamps requires ATP binding to both the Msh2 and MSH6 nucleotide-binding sites. In addition, the properties of the different mutant complexes suggest that distinct conformational states mediated by communication between the Msh2 and MSH6 nucleotide-binding sites are required for the formation of ternary complexes and sliding clamps.
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chimeric saccharomyces cerevisiae MSH6 protein with an msh3 mispair binding domain combines properties of both proteins
Proceedings of the National Academy of Sciences of the United States of America, 2007Co-Authors: Scarlet S. Shell, Christopher D Putnam, Richard D KolodnerAbstract:Msh2–Msh3 and Msh2–MSH6 are two partially redundant mispair-recognition complexes that initiate mismatch repair in eukaryotes. Crystal structures of the prokaryotic homolog MutS suggest the mechanism by which MSH6 interacts with mispairs because key mispair-contacting residues are conserved in these two proteins. Because Msh3 lacks these conserved residues, we constructed a series of mutants to investigate the requirements for mispair interaction by Msh3. We found that a chimeric protein in which the mispair-binding domain (MBD) of MSH6 was replaced by the equivalent domain of Msh3 was functional for mismatch repair. This chimera possessed the mispair-binding specificity of Msh3 and revealed that communication between the MBD and the ATPase domain is conserved between Msh2–Msh3 and Msh2–MSH6. Further, the chimeric protein retained MSH6-like properties with respect to genetic interactions with the MutL homologs and an Msh2 MBD deletion mutant, indicating that Msh3-like behaviors beyond mispair specificity are not features controlled by the MBD.
Manju Hingorani - One of the best experts on this subject based on the ideXlab platform.
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Mismatch Recognition by Saccharomyces cerevisiae Msh2-MSH6: Role of Structure and Dynamics.
International Journal of Molecular Sciences, 2019Co-Authors: Yan Li, Manju Hingorani, Zane Lombardo, Meera Joshi, Ishita MukerjiAbstract:The mismatch repair (MMR) pathway maintains genome integrity by correcting errors such as mismatched base pairs formed during DNA replication. In MMR, Msh2–MSH6, a heterodimeric protein, targets single base mismatches and small insertion/deletion loops for repair. By incorporating the fluorescent nucleoside base analog 6-methylisoxanthopterin (6-MI) at or adjacent to a mismatch site to probe the structural and dynamic elements of the mismatch, we address how Msh2–MSH6 recognizes these mismatches for repair within the context of matched DNA. Fluorescence quantum yield and rotational correlation time measurements indicate that local base dynamics linearly correlate with Saccharomyces cerevisiae Msh2–MSH6 binding affinity where the protein exhibits a higher affinity (KD ≤ 25 nM) for mismatches that have a significant amount of dynamic motion. Energy transfer measurements measuring global DNA bending find that mismatches that are both well and poorly recognized by Msh2–MSH6 experience the same amount of protein-induced bending. Finally, base-specific dynamics coupled with protein-induced blue shifts in peak emission strongly support the crystallographic model of directional binding, in which Phe 432 of MSH6 intercalates 3′ of the mismatch. These results imply an important role for local base dynamics in the initial recognition step of MMR.
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Enhanced Dynamics of Mismatched Base Pairs Associated with Msh2-MSH6 Recognition
Biophysical Journal, 2015Co-Authors: Yan Li, Manju Hingorani, Ishita MukerjiAbstract: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.
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biochemical analysis of the human mismatch repair proteins hmutsα msh2 g674a MSH6 and msh2 MSH6 t1219d
Journal of Biological Chemistry, 2012Co-Authors: Hui Geng, Manju Hingorani, Dorothy A. Erie, Miho Sakato, Vanessa Derocco, Kazuhiko Yamane, Chunwei Du, Peggy HsiehAbstract:Abstract The heterodimeric human MSH2-MSH6 (hMutSα) protein initiates DNA mismatch repair (MMR) by recognizing mismatched bases that result from replication errors. Msh2 G674A or MSH6 T1217D mice that have mutations in or near the ATP binding site of MSH2 or ATP hydrolysis catalytic site of MSH6 develop cancer and have reduced lifespan due to loss of the MMR pathway (1, 2). Mouse embryonic fibroblasts from these mice retain an apoptotic response to DNA damage. Mutant human MutSα proteins, MSH2G674A-MSH6wt and MSH2wt-MSH6T1219D, are profiled in a variety of functional assays and, as expected, fail to support MMR in vitro, although they retain mismatch recognition activity. Kinetic analyses of DNA binding and ATPase activities and examination of the excision step of MMR reveal that the two mutants differ in their underlying molecular defects. MSH2wt-MSH6T1219D fails to couple nucleotide binding and mismatch recognition; whereas, MSH2G674A-MSH6wt has a partial defect in nucleotide binding. Nevertheless, both mutant proteins remain bound to the mismatch and fail to promote efficient excision thereby inhibiting MMR in vitro in a dominant manner. Implications of these findings for MMR and DNA damage signaling by MMR proteins are discussed.
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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, 2010Co-Authors: Jie Zhai, Manju HingoraniAbstract: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.
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Mechanism of cadmium-mediated inhibition of Msh2-MSH6 function in DNA mismatch repair.
Biochemistry, 2009Co-Authors: Markus Wieland, Mikhail K. Levin, Karan S. Hingorani, F. Noah Biro, Manju HingoraniAbstract: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 ...
Eric Alani - One of the best experts on this subject based on the ideXlab platform.
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dynamic basis for one dimensional dna scanning by the mismatch repair complex msh2 MSH6
Molecular Cell, 2007Co-Authors: Jason Gorman, Eric Alani, Arindam Chowdhury, Jennifer A Surtees, Jun Shimada, David R Reichman, Eric C GreeneAbstract:The ability of proteins to locate specific sites or structures among a vast excess of nonspecific DNA is a fundamental theme in biology. Yet the basic principles that govern these mechanisms remain poorly understood. For example, mismatch repair proteins must scan millions of base pairs to find rare biosynthetic errors, and they then must probe the surrounding region to identify the strand discrimination signals necessary to distinguish the parental and daughter strands. To determine how these proteins might function we used single-molecule optical microscopy to answer the following question: how does the mismatch repair complex Msh2-MSH6 interrogate undamaged DNA? Here we show that Msh2-MSH6 slides along DNA via one-dimensional diffusion. These findings indicate that interactions between Msh2-MSH6 and DNA are dominated by lateral movement of the protein along the helical axis and have implications for how MutS family members travel along DNA at different stages of the repair reaction.
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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, 2004Co-Authors: Neal Sugawara, Eric Alani, Tamara Goldfarb, Barbara Studamire, James E HaberAbstract: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 MSH6Δ strain and partially suppressed in an msh2 separation-of-function mutant. In mlh1Δ strains, heteroduplex rejection was greater than in MSH6Δ strains but less than in wild type. Deleting PMS1, MLH2,or MLH3 had no effect on heteroduplex rejection, but a pms1Δ mlh2Δ mlh3Δ triple mutant resembled mlh1Δ. 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.
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Analysis of yeast MSH2-MSH6 suggests that the initiation of mismatch repair can be separated into discrete steps
Journal of Molecular Biology, 2000Co-Authors: Jayson Bowers, Phuoc T. Tran, R. Michael Liskay, Eric AlaniAbstract:The yeast MSH2-MSH6 complex is required to repair both base-pair and single base insertion/deletion mismatches. MSH2-MSH6 binds to mismatch substrates and displays an ATPase activity that is modulated by mispairs that are repaired in vivo. To understand early steps in mismatch repair, we analyzed mismatch repair (MMR) defective MSH2-MSH6-F337A and MSH2-MSH6-340 complexes that contained amino acid substitutions in the MSH6 mismatch recognition domain. While both heterodimers were defective in forming stable complexes with mismatch substrates, only MSH2-MSH6-340 bound to homoduplex DNA with an affinity that was similar to that observed for MSH2-MSH6. Additional analyses suggested that stable binding to a mispair is not sufficient to initiate recruitment of downstream repair factors. Previously, we observed that MSH2-MSH6 forms a stable complex with a palindromic insertion mismatch that escapes correction by MMR in vivo. Here we show that this binding is not accompanied by either a modulation in MSH2-MSH6 ATPase activity or an ATP-dependent recruitment of the MLH1-PMS1 complex. Together, these observations suggest that early stages in MMR can be divided into distinct recognition, stable binding, and downstream factor recruitment steps.
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a mutation in the MSH6 subunit of the saccharomyces cerevisiae msh2 MSH6 complex disrupts mismatch recognition
Journal of Biological Chemistry, 1999Co-Authors: Jayson Bowers, Tanya Sokolsky, Tony Quach, Eric AlaniAbstract:Abstract In yeast, MSH2 interacts with MSH6 to repair base pair mismatches and single nucleotide insertion/deletion mismatches and with MSH3 to recognize small loop insertion/deletion mismatches. We identified a MSH6 mutation (MSH6-F337A) that when overexpressed in wild type strains conferred a defect in both MSH2-MSH6- and MSH2-MSH3-dependent mismatch repair pathways. Genetic analysis suggested that this phenotype was due to MSH6-F337A sequestering MSH2 and preventing it from interacting with MSH3 and MSH6. In UV cross-linking, filter binding, and gel retardation assays, the MSH2-MSH6-F337A complex displayed a mismatch recognition defect. These observations, in conjunction with ATPase and dissociation rate analysis, suggested that MSH2-MSH6-F337A formed an unproductive complex that was unable to stably bind to mismatch DNA.
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the saccharomyces cerevisiae msh2 and MSH6 proteins form a complex that specifically binds to duplex oligonucleotides containing mismatched dna base pairs
Molecular and Cellular Biology, 1996Co-Authors: Eric AlaniAbstract:: The yeast Saccharomyces cerevisiae encodes six proteins, Msh1p to MSH6p, that show strong amino acid sequence similarity to MutS, a central component of the bacterial mutHLS mismatch repair system. Recent studies with humans and S. cerevisiae suggest that in eukaryotes, specific MutS homolog complexes that display unique DNA mismatch specificities exist. In this study, the S. cerevisiae 109-kDa Msh2 and 140-kDa MSH6 proteins were cooverexpressed in S. cerevisiae and shown to interact in an immunoprecipitation assay and by conventional chromatography. Deletion analysis of MSH2 indicated that the carboxy-terminal 114 amino acids of Msh2p are important for MSH6p interaction. Purified Msh2p-MSH6p selectively bound to duplex oligonucleotide substrates containing a G/T mismatch and a +1 insertion mismatch but did not show specific binding to +2 and +4 insertion mismatches. The mismatch binding specificity of the Msh2p-MSH6p complex, as measured by on-rate and off-rate binding studies, was abolished by ATP. Interestingly, palindromic substrates that are poorly repaired in vivo were specifically recognized by Msh2p-MSH6p; however, the binding of Msh2p-MSH6p to these substrates was not modulated by ATP. Taken together, these studies suggest that the repair of a base pair mismatch by the Msh2p-MSH6p complex is dependent on the ability of the Msh2p-MSH6p-DNA mismatch complex to use ATP hydrolysis to activate downstream events in mismatch repair.
Winfried Edelmann - One of the best experts on this subject based on the ideXlab platform.
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differing patterns of genetic instability in mice deficient in the mismatch repair genes pms2 mlh1 msh2 msh3 and MSH6
Carcinogenesis, 2006Co-Authors: Denise C Hegan, Winfried Edelmann, Latha Narayanan, Frank R Jirik, Michael R Liskay, Peter M GlazerAbstract: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.
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examination of MSH6 and msh3 deficient mice in class switching reveals overlapping and distinct roles of muts homologues in antibody diversification
Journal of Experimental Medicine, 2004Co-Authors: Ziqiang Li, Winfried Edelmann, Stefan J Scherer, Diana Ronai, Maria D Iglesiasussel, Jonathan U Peled, Philip Bardwell, Min Zhuang, Alberto Martin, Matthew D ScharffAbstract:Somatic hypermutation and class switch recombination (CSR) contribute to the somatic diversification of antibodies. It has been shown that MutS homologue (Msh)6 (in conjunction with Msh2) but not Msh3 is involved in generating A/T base substitutions in somatic hypermutation. However, their roles in CSR have not yet been reported. Here we show that MSH6−/− mice have a decrease in CSR, whereas Msh3−/− mice do not. When switch regions were analyzed for mutations, deficiency in MSH6 was associated with an increase in transition mutations at G/C basepairs, mutations at RGYW/WRCY hotspots, and a small increase in the targeting of G/C bases. In addition, MSH6−/− mice exhibited an increase in the targeting of recombination sites to GAGCT/GGGGT consensus repeats and hotspots in Sγ3 but not in Sμ. In contrast to Msh2−/− mice, deficiency in MSH6 surprisingly did not change the characteristics of Sμ-Sγ3 switch junctions. However, MSH6−/− mice exhibited a change in the positioning of Sμ and Sγ3 junctions. Although none of these changes were seen in Msh3−/− mice, they had a higher percentage of large inserts in their switch junctions. Together, our data suggest that MutS homologues Msh2, Msh3, and MSH6 play overlapping and distinct roles during antibody diversification processes.
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the distinct spectra of tumor associated apc mutations in mismatch repair deficient apc1638n mice define the roles of msh3 and MSH6 in dna repair and intestinal tumorigenesis
Cancer Research, 2001Co-Authors: Mari Kuraguchi, Richard D Kolodner, Kan Yang, Edmund Wong, Elena Avdievich, Martin Lipkin, Anthony M C Brown, Raju Kucherlapati, Winfried EdelmannAbstract:In mammalian cells, mismatch recognition has been attributed to two partially redundant heterodimeric protein complexes of MutS homologues, MSH2-MSH3 and MSH2-MSH6. We have conducted a comparative analysis of Msh3 and MSH6 deficiency in mouse intestinal tumorigenesis by generating Apc 1638N mice deficient in Msh3, MSH6 or both. We have found that Apc 1638N mice defective in MSH6 show reduced survival and a 6–7-fold increase in intestinal tumor multiplicity. In contrast, Msh3-deficient Apc 1638N mice showed no difference in survival and intestinal tumor multiplicity as compared with Apc 1638N mice. However, when Msh3 deficiency is combined with MSH6 deficiency ( Msh3 −/− MSH6 −/− Apc 1638N ), the survival rate of the mice was further reduced compared to MSH6 −/− Apc 1638N mice because of a high multiplicity of intestinal tumors at a younger age. Almost 90% of the intestinal tumors from both MSH6 −/− Apc 1638N and Msh3 −/− MSH6 −/− Apc 1638N mice contained truncation mutations in the wild-type Apc allele. Apc mutations in MSH6 −/− Apc 1638N mice consisted predominantly of base substitutions (93%) creating stop codons, consistent with a major role for MSH6 in the repair of base-base mismatches. However, in Msh3 −/− MSH6 −/− Apc 1638N tumors, we observed a mixture of base substitutions (46%) and frameshifts (54%), indicating that in MSH6 −/− Apc 1638N mice frameshift mutations in the Apc gene were suppressed by Msh3. Interestingly, all except one of the Apc mutations detected in mismatch repair-deficient intestinal tumors were located upstream of the third 20-amino acid β-catenin binding repeat and before all of the Ser-Ala-Met-Pro repeats, suggesting that there is selection for loss of multiple domains involved in β-catenin regulation. Our analysis therefore has revealed distinct mutational spectra and clarified the roles of Msh3 and MSH6 in DNA repair and intestinal tumorigenesis.
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the dna mismatch repair genes msh3 and MSH6 cooperate in intestinal tumor suppression
Cancer Research, 2000Co-Authors: Winfried Edelmann, Burkhard Kneitz, Kan Yang, Edmund Wong, Elena Avdievich, Asad Umar, Joerg Heyer, Melanie H Kucherlapati, Gray F Crouse, Thomas A KunkelAbstract:Repair of mismatches in DNA in mammalian cells is mediated by a complex of proteins that are members of two highly conserved families of genes referred to as MutS and MutL homologues. Germline mutations in several members of these families, MSH2 , MSH6 , MLH1 , and PMS2 , but not MSH3 , are responsible for hereditary non-polyposis colorectal cancer. To examine the role of MSH3 , we generated a mouse with a null mutation in this gene. Cells from Msh3−/− mice are defective in repair of insertion/deletion mismatches but can repair base-base mismatches. Msh3−/− mice develop tumors at a late age. When the Msh3−/− and MSH6−/− mutations are combined, the tumor predisposition phenotype is indistinguishable from Msh2−/− or Mlh1−/− mice. These results suggest that MSH3 cooperates with MSH6 in tumor suppression.
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somatic hypermutation in muts homologue msh 3 MSH6 and msh3 MSH6 deficient mice reveals a role for the msh2 MSH6 heterodimer in modulating the base substitution pattern
Journal of Experimental Medicine, 2000Co-Authors: Margrit Wiesendanger, Burkhard Kneitz, Winfried Edelmann, Matthew D ScharffAbstract:Although the primary function of the DNA mismatch repair (MMR) system is to identify and correct base mismatches that have been erroneously introduced during DNA replication, recent studies have further implicated several MMR components in somatic hypermutation of immunoglobulin (Ig) genes. We studied the immune response in mice deficient in MutS homologue (MSH)3 and MSH6, two mutually exclusive partners of MSH2 that have not been examined previously for their role in Ig hypermutation. In MSH6−/− and Msh3−/−/MSH6−/− mice, base substitutions are preferentially targeted to G and C nucleotides and to an RGYW hot spot, as has been shown previously in Msh2−/− mice. In contrast, Msh3−/− mice show no differences from their littermate controls. These findings indicate that the MSH2–MSH6 heterodimer, but not the MSH2–MSH3 complex, is responsible for modulating Ig hypermutation.
Ursula Storb - One of the best experts on this subject based on the ideXlab platform.
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somatic hypermutation and class switch recombination in MSH6 ung double knockout mice
Journal of Immunology, 2006Co-Authors: Hong Ming Shen, Atsushi Tanaka, Grazyna Bozek, Dan L Nicolae, Ursula StorbAbstract: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.
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Somatic Hypermutation and Class Switch Recombination in MSH6−/−Ung−/− Double-Knockout Mice
Journal of Immunology, 2006Co-Authors: Hong Ming Shen, Atsushi Tanaka, Grazyna Bozek, Dan L Nicolae, Ursula StorbAbstract: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.