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Richard D. Kolodner - One of the best experts on this subject based on the ideXlab platform.
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reconstitution of saccharomyces cerevisiae dna polymerase e dependent mismatch repair with purified proteins
Proceedings of the National Academy of Sciences of the United States of America, 2017Co-Authors: Nikki Bowen, Richard D. KolodnerAbstract:Mammalian and Saccharomyces cerevisiae mismatch repair (MMR) proteins catalyze two MMR reactions in vitro. In one, mispair binding by either the MutS homolog 2 (Msh2)–MutS homolog 6 (Msh6) or the Msh2–MutS homolog 3 (Msh3) stimulates 5′ to 3′ excision by exonuclease 1 (Exo1) from a single-strand break 5′ to the mispair, excising the mispair. In the other, Msh2–Msh6 or Msh2–Msh3 activate the MutL homolog 1 (Mlh1)–postmeiotic segregation 1 (Pms1) endonuclease in the presence of a mispair and a nick 3′ to the mispair, to make nicks 5′ to the mispair, allowing Exo1 to excise the mispair. DNA polymerase δ (Pol δ) is thought to catalyze DNA synthesis to fill in the gaps resulting from mispair excision. However, colocalization of the S. cerevisiae mispair recognition proteins with the replicative DNA polymerases during DNA replication has suggested that DNA polymerase e (Pol e) may also play a role in MMR. Here we describe the reconstitution of Pol e-dependent MMR using S. cerevisiae proteins. A mixture of Msh2–Msh6 (or Msh2–Msh3), Exo1, RPA, RFC-Δ1N, PCNA, and Pol e was found to catalyze both short-patch and long-patch 5′ nick-directed MMR of a substrate containing a +1 (+T) mispair. When the substrate contained a nick 3′ to the mispair, a mixture of Msh2–Msh6 (or Msh2–Msh3), Exo1, RPA, RFC-Δ1N, PCNA, and Pol e was found to catalyze an MMR reaction that required Mlh1–Pms1. These results demonstrate that Pol e can act in eukaryotic MMR in vitro.
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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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Mlh2 Is an Accessory Factor for DNA Mismatch Repair in Saccharomyces cerevisiae
PLoS genetics, 2014Co-Authors: Christopher S Campbell, Christopher D Putnam, Hans Hombauer, Arshad Desai, Anjana Srivatsan, Nikki Bowen, Kerstin Gries, Richard D. KolodnerAbstract: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.
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Mispair-specific recruitment of the Mlh1-Pms1 complex identifies repair substrates of the Saccharomyces cerevisiae Msh2-Msh3 complex.
The Journal of biological chemistry, 2014Co-Authors: Anjana Srivatsan, Nikki Bowen, Richard D. KolodnerAbstract: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.
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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.
Eric Alani - One of the best experts on this subject based on the ideXlab platform.
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mlh1 mlh3 a meiotic crossover and dna mismatch repair factor is a msh2 msh3 stimulated endonuclease
Journal of Biological Chemistry, 2014Co-Authors: Maria V Rogacheva, Carol M Manhart, Jennifer A Surtees, Cheng Chen, Alba Guarne, Eric AlaniAbstract: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.
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Multiple factors insulate Msh2-Msh6 mismatch repair activity from defects in Msh2 domain I.
Journal of molecular biology, 2011Co-Authors: Charanya Kumar, Eric Alani, Sarah C. Piacente, Justin Sibert, Andrew R. Bukata, Jaime O'connor, Jennifer A SurteesAbstract: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.
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Genetic analysis of baker's yeast Msh4-MSH5 reveals a threshold crossover level for meiotic viability.
PLoS genetics, 2010Co-Authors: K. T. Nishant, Cheng Chen, Miki Shinohara, Akira Shinohara, Eric AlaniAbstract:During meiosis, the Msh4-MSH5 complex is thought to stabilize single-end invasion intermediates that form during early stages of recombination and subsequently bind to Holliday junctions to facilitate crossover formation. To analyze Msh4-MSH5 function, we mutagenized 57 residues in Saccharomyces cerevisiae Msh4 and MSH5 that are either conserved across all Msh4/5 family members or are specific to Msh4 and MSH5. The MSH5 subunit appeared more sensitive to mutagenesis. We identified msh4 and MSH5 threshold (msh4/5-t) mutants that showed wild-type spore viability and crossover interference but displayed, compared to wild-type, up to a two-fold decrease in crossing over on large and medium sized chromosomes (XV, VII, VIII). Crossing over on a small chromosome, however, approached wild-type levels. The msh4/5-t mutants also displayed synaptonemal complex assembly defects. A triple mutant containing a msh4/5-t allele and mutations that decreased meiotic double-strand break levels (spo11-HA) and crossover interference (pch2Δ) showed synergistic defects in spore viability. Together these results indicate that the baker's yeast meiotic cell does not require the ∼90 crossovers maintained by crossover homeostasis to form viable spores. They also show that Pch2-mediated crossover interference is important to maintain meiotic viability when crossovers become limiting.
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Saccharomyces cerevisiae MSH2-MSH3 and MSH2-MSH6 complexes display distinct requirements for DNA binding Domain I in mismatch recognition.
Journal of molecular biology, 2006Co-Authors: Susan D. Lee, Jennifer A Surtees, Eric AlaniAbstract: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.
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Competing Crossover Pathways Act During Meiosis in Saccharomyces cerevisiae
Genetics, 2004Co-Authors: Juan Lucas Argueso, Jennifer J. Wanat, Zekeriyya Gemici, Eric AlaniAbstract:In Saccharomyces cerevisiae the MSH4-MSH5, MLH1-MLH3, and MUS81-MMS4 complexes act to promote crossing over during meiosis. MSH4-MSH5, but not MUS81-MMS4, promotes crossovers that display interference. A role for MLH1-MLH3 in crossover control is less clear partly because mlh1Δ mutants retain crossover interference yet display a decrease in crossing over that is only slightly less severe than that seen in msh4Δ and MSH5Δ mutants. We analyzed the effects of MSH5Δ, mlh1Δ, and mms4Δ single, double, and triple mutants on meiotic crossing over at four consecutive genetic intervals on chromosome XV using newly developed computer software. mlh1Δ mms4Δ double mutants displayed the largest decrease in crossing over (13- to 15-fold) of all mutant combinations, yet these strains displayed relatively high spore viability (42%). In contrast, MSH5Δ mms4Δ and MSH5Δ mms4Δ mlh1Δ mutants displayed smaller decreases in crossing over (4- to 6-fold); however, spore viability (18–19%) was lower in these strains than in mlh1Δ mms4Δ strains. These data suggest that meiotic crossing over can occur in yeast through three distinct crossover pathways. In one pathway, MUS81-MMS4 promotes interference-independent crossing over; in a second pathway, both MSH4-MSH5 and MLH1-MLH3 promote interference-dependent crossovers. A third pathway, which appears to be repressed by MSH4-MSH5, yields deleterious crossovers.
K. T. Nishant - One of the best experts on this subject based on the ideXlab platform.
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The bakers’s yeast Msh4-MSH5 associates with double-strand break hotspots and chromosome axis during meiosis to promote crossovers
2020Co-Authors: Krishnaprasad G. Nandanan, Ajith V. Pankajam, Sagar Salim, Miki Shinohara, Lin, Parijat Chakraborty, Lars M. Steinmetz, Akira Shinohara, K. T. NishantAbstract:ABSTRACT Segregation of homologous chromosomes during the first meiotic division requires at least one obligate crossover/exchange event between the homolog pairs. In the baker’s yeast Saccharomyces cerevisiae and mammals, the mismatch repair-related factors, Msh4-MSH5 and Mlh1-Mlh3 generate the majority of the meiotic crossovers from programmed double-strand breaks (DSBs). To understand the mechanistic role of Msh4-MSH5 in meiotic crossing over, we performed genome-wide ChIP-sequencing and cytological analysis of the MSH5 protein in cells synchronized for meiosis. We observe that MSH5 associates with DSB hotspots, chromosome axis, and centromeres. We found that the initial recruitment of Msh4-MSH5 occurs following DSB resection. A two-step MSH5 binding pattern was observed: an early weak binding at DSB hotspots followed by enhanced late binding upon the formation of double Holliday junction structures. MSH5 association with the chromosome axis is Red1 dependent, while MSH5 association with the DSB hotspots and axis is dependent on DSB formation by Spo11. MSH5 binding was enhanced at strong DSB hotspots consistent with a role for DSB frequency in promoting MSH5 binding. These data on the in vivo localization of MSH5 during meiosis have implications for how Msh4-MSH5 may work with other crossover and synapsis promoting factors to ensure Holliday junction resolution at the chromosome axis. AUTHOR SUMMARY During meiosis, crossovers facilitate physical linkages between homologous chromosomes that ensure their accurate segregation. Meiotic crossovers are initiated from programmed DNA double-strand breaks (DSBs). In the baker’s yeast and mammals, DSBs are repaired into crossovers primarily through a pathway involving the highly conserved mismatch repair related Msh4-MSH5 complex along with other crossover promoting factors. In vitro and physical studies suggest that the Msh4-MSH5 heterodimer facilitates meiotic crossover formation by stabilizing Holliday junctions. We investigated the genome-wide in vivo binding sites of MSH5 during meiotic progression. MSH5 was enriched at DSB hotspots, chromosome axis, and centromere sites. Our results suggest MSH5 associates with both DSB sites on the chromosomal loops and with the chromosome axis to promote crossover formation. These results on the in vivo dynamic localization of the MSH5 protein provide novel insights into how the Msh4-MSH5 complex may work with other crossover and synapsis promoting factors to facilitate crossover formation.
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the bakers s yeast msh4 MSH5 associates with double strand break hotspots and chromosome axis during meiosis to promote crossovers
bioRxiv, 2020Co-Authors: Krishnaprasad G. Nandanan, Ajith V. Pankajam, Sagar Salim, Miki Shinohara, Parijat Chakraborty, Lars M. Steinmetz, Akira Shinohara, Gen Lin, K. T. NishantAbstract:ABSTRACT Segregation of homologous chromosomes during the first meiotic division requires at least one obligate crossover/exchange event between the homolog pairs. In the baker’s yeast Saccharomyces cerevisiae and mammals, the mismatch repair-related factors, Msh4-MSH5 and Mlh1-Mlh3 generate the majority of the meiotic crossovers from programmed double-strand breaks (DSBs). To understand the mechanistic role of Msh4-MSH5 in meiotic crossing over, we performed genome-wide ChIP-sequencing and cytological analysis of the MSH5 protein in cells synchronized for meiosis. We observe that MSH5 associates with DSB hotspots, chromosome axis, and centromeres. We found that the initial recruitment of Msh4-MSH5 occurs following DSB resection. A two-step MSH5 binding pattern was observed: an early weak binding at DSB hotspots followed by enhanced late binding upon the formation of double Holliday junction structures. MSH5 association with the chromosome axis is Red1 dependent, while MSH5 association with the DSB hotspots and axis is dependent on DSB formation by Spo11. MSH5 binding was enhanced at strong DSB hotspots consistent with a role for DSB frequency in promoting MSH5 binding. These data on the in vivo localization of MSH5 during meiosis have implications for how Msh4-MSH5 may work with other crossover and synapsis promoting factors to ensure Holliday junction resolution at the chromosome axis. AUTHOR SUMMARY During meiosis, crossovers facilitate physical linkages between homologous chromosomes that ensure their accurate segregation. Meiotic crossovers are initiated from programmed DNA double-strand breaks (DSBs). In the baker’s yeast and mammals, DSBs are repaired into crossovers primarily through a pathway involving the highly conserved mismatch repair related Msh4-MSH5 complex along with other crossover promoting factors. In vitro and physical studies suggest that the Msh4-MSH5 heterodimer facilitates meiotic crossover formation by stabilizing Holliday junctions. We investigated the genome-wide in vivo binding sites of MSH5 during meiotic progression. MSH5 was enriched at DSB hotspots, chromosome axis, and centromere sites. Our results suggest MSH5 associates with both DSB sites on the chromosomal loops and with the chromosome axis to promote crossover formation. These results on the in vivo dynamic localization of the MSH5 protein provide novel insights into how the Msh4-MSH5 complex may work with other crossover and synapsis promoting factors to facilitate crossover formation.
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Structural Insights into Saccharomyces cerevisiae Msh4–MSH5 Complex Function Using Homology Modeling
PloS one, 2013Co-Authors: Ramaswamy Rakshambikai, Narayanaswamy Srinivasan, K. T. NishantAbstract:The Msh4–MSH5 protein complex in eukaryotes is involved in stabilizing Holliday junctions and its progenitors to facilitate crossing over during Meiosis I. These functions of the Msh4–MSH5 complex are essential for proper chromosomal segregation during the first meiotic division. The Msh4/5 proteins are homologous to the bacterial mismatch repair protein MutS and other MutS homologs (Msh2, Msh3, Msh6). Saccharomyces cerevisiae msh4/5 point mutants were identified recently that show two fold reduction in crossing over, compared to wild-type without affecting chromosome segregation. Three distinct classes of msh4/5 point mutations could be sorted based on their meiotic phenotypes. These include msh4/5 mutations that have a) crossover and viability defects similar to msh4/5 null mutants; b) intermediate defects in crossing over and viability and c) defects only in crossing over. The absence of a crystal structure for the Msh4–MSH5 complex has hindered an understanding of the structural aspects of Msh4–MSH5 function as well as molecular explanation for the meiotic defects observed in msh4/5 mutations. To address this problem, we generated a structural model of the S. cerevisiae Msh4–MSH5 complex using homology modeling. Further, structural analysis tailored with evolutionary information is used to predict sites with potentially critical roles in Msh4–MSH5 complex formation, DNA binding and to explain asymmetry within the Msh4–MSH5 complex. We also provide a structural rationale for the meiotic defects observed in the msh4/5 point mutations. The mutations are likely to affect stability of the Msh4/5 proteins and/or interactions with DNA. The Msh4–MSH5 model will facilitate the design and interpretation of new mutational data as well as structural studies of this important complex involved in meiotic chromosome segregation.
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structural insights into saccharomyces cerevisiae msh4 MSH5 complex function using homology modeling
PLOS ONE, 2013Co-Authors: Ramaswamy Rakshambikai, Narayanaswamy Srinivasan, K. T. NishantAbstract:The Msh4–MSH5 protein complex in eukaryotes is involved in stabilizing Holliday junctions and its progenitors to facilitate crossing over during Meiosis I. These functions of the Msh4–MSH5 complex are essential for proper chromosomal segregation during the first meiotic division. The Msh4/5 proteins are homologous to the bacterial mismatch repair protein MutS and other MutS homologs (Msh2, Msh3, Msh6). Saccharomyces cerevisiae msh4/5 point mutants were identified recently that show two fold reduction in crossing over, compared to wild-type without affecting chromosome segregation. Three distinct classes of msh4/5 point mutations could be sorted based on their meiotic phenotypes. These include msh4/5 mutations that have a) crossover and viability defects similar to msh4/5 null mutants; b) intermediate defects in crossing over and viability and c) defects only in crossing over. The absence of a crystal structure for the Msh4–MSH5 complex has hindered an understanding of the structural aspects of Msh4–MSH5 function as well as molecular explanation for the meiotic defects observed in msh4/5 mutations. To address this problem, we generated a structural model of the S. cerevisiae Msh4–MSH5 complex using homology modeling. Further, structural analysis tailored with evolutionary information is used to predict sites with potentially critical roles in Msh4–MSH5 complex formation, DNA binding and to explain asymmetry within the Msh4–MSH5 complex. We also provide a structural rationale for the meiotic defects observed in the msh4/5 point mutations. The mutations are likely to affect stability of the Msh4/5 proteins and/or interactions with DNA. The Msh4–MSH5 model will facilitate the design and interpretation of new mutational data as well as structural studies of this important complex involved in meiotic chromosome segregation.
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Genetic analysis of baker's yeast Msh4-MSH5 reveals a threshold crossover level for meiotic viability.
PLoS genetics, 2010Co-Authors: K. T. Nishant, Cheng Chen, Miki Shinohara, Akira Shinohara, Eric AlaniAbstract:During meiosis, the Msh4-MSH5 complex is thought to stabilize single-end invasion intermediates that form during early stages of recombination and subsequently bind to Holliday junctions to facilitate crossover formation. To analyze Msh4-MSH5 function, we mutagenized 57 residues in Saccharomyces cerevisiae Msh4 and MSH5 that are either conserved across all Msh4/5 family members or are specific to Msh4 and MSH5. The MSH5 subunit appeared more sensitive to mutagenesis. We identified msh4 and MSH5 threshold (msh4/5-t) mutants that showed wild-type spore viability and crossover interference but displayed, compared to wild-type, up to a two-fold decrease in crossing over on large and medium sized chromosomes (XV, VII, VIII). Crossing over on a small chromosome, however, approached wild-type levels. The msh4/5-t mutants also displayed synaptonemal complex assembly defects. A triple mutant containing a msh4/5-t allele and mutations that decreased meiotic double-strand break levels (spo11-HA) and crossover interference (pch2Δ) showed synergistic defects in spore viability. Together these results indicate that the baker's yeast meiotic cell does not require the ∼90 crossovers maintained by crossover homeostasis to form viable spores. They also show that Pch2-mediated crossover interference is important to maintain meiotic viability when crossovers become limiting.
Winfried Edelmann - One of the best experts on this subject based on the ideXlab platform.
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msh2 atpase domain mutation affects ctg cag repeat instability in transgenic mice
PLOS Genetics, 2009Co-Authors: Stéphanie Tomé, Ian Holt, Glenn E Morris, Christopher E. Pearson, Winfried Edelmann, Arnold Munnich, Geneviève GourdonAbstract: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.
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MSH2 ATPase domain mutation affects CTG*CAG repeat instability in transgenic mice.
PLoS genetics, 2009Co-Authors: Stéphanie Tomé, Ian Holt, Glenn E Morris, Christopher E. Pearson, Winfried Edelmann, Arnold Munnich, Geneviève GourdonAbstract: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.
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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, Michael R Liskay, Frank R Jirik, Latha Narayanan, Winfried Edelmann, 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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localization of mmr proteins on meiotic chromosomes in mice indicates distinct functions during prophase i
Journal of Cell Biology, 2005Co-Authors: Nadine K Kolas, Michael R Liskay, Winfried Edelmann, Steven M. Lipkin, Anton Svetlanov, Michelle Lenzi, Frank P Macaluso, John M Greally, Paula E CohenAbstract: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.
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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, Stefan J Scherer, Diana Ronai, Maria D Iglesiasussel, Jonathan U Peled, Philip Bardwell, Min Zhuang, Alberto Martin, Winfried Edelmann, 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.
Ishita Mukerji - One of the best experts on this subject based on the ideXlab platform.
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MutSγ-Induced DNA Conformational Changes Provide Insights into Its Role in Meiotic Recombination.
Biophysical journal, 2018Co-Authors: Sudipta Lahiri, Manju M. Hingorani, Ishita MukerjiAbstract:Abstract In many organisms, MutSγ plays a role in meiotic recombination, facilitating crossover formation between homologous chromosomes. Failure to form crossovers leads to improper segregation of chromosomes and aneuploidy, which in humans result in infertility and birth defects. To improve current understanding of MutSγ function, this study investigates the binding affinities and structures of MutSγ in complex with DNA substrates that model homologous recombination intermediates. For these studies, we overexpressed and isolated from Escherichia coli the yeast MutSγ protein Saccharomyces cerevisiae (Sc) Msh4-MSH5. Sc Msh4-MSH5 binds Holliday junction (HJ)-like substrates, 3′ overhangs, single-stranded (ss) forks, and the displacement loop with nanomolar affinity. The weakest binding affinities are detected for an intact duplex and open-junction construct. Similar to the human protein, Sc Msh4-MSH5 exhibits the highest affinity for the HJ with a Kd
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Binding Dynamics of the Holliday Junction with Yeast MutS Homolog Msh4-MSH5
Biophysical Journal, 2015Co-Authors: Sudipta Lahiri, Manju M. Hingorani, Ishita MukerjiAbstract:MutS homologs (Msh) are conserved in all organisms and they are involved in the process of mismatch recognition and repair. In Saccharomyces cerevisiae there are five MutS Homologs (Msh2 through Msh6) that participate in repair and recombination processes. The heterodimeric protein complexes, Msh2-Msh3 and Msh2-Msh6, play a role in mismatch repair. MutS homolog Msh4 and MSH5 are not involved in mismatch recognition and repair and genetic studies in S. cerevisiae and mouse indicate that Msh4-MSH5 are responsible for gamete viability during meiosis, recombination and chromosomal segregation. Recent studies have shown that human Msh4-MSH5 recognizes and stabilizes Holliday or four-way DNA Junctions (4WJ), an intermediate in double stranded break repair. Our efforts are focused on elucidating structure-function relationships of S. cerevisiae MutS homolog Msh4-MSH5 by specifically studying the binding interaction of Msh4-MSH5 with DNA 4WJs. We have successfully expressed and purified S. cerevisiae Msh4-MSH5 in E. coli cells as both a heterodimeric protein complex as well as Msh4 and MSH5 monomers. Our initial data demonstrates that the purified proteins are active and binds with high affinity to DNA 4WJ. The binding affinity is in the nanomolar range and similar to that previously reported for human Msh4-MSH5. To study the effect of Msh4-MSH5 on junction conformation, we are using Forster resonance energy transfer. These measurements also probe whether Msh4-MSH5 preferentially binds to the open or stacked form of the junction. These measurements will also address whether stabilization of the stacked conformation is functionally related to suppression of recombination.
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Investigation of the Binding Interaction of S. cerevisiae MutS Homologs MSH2-MSH6 and MSH4-MSH5 with Holliday Junctions
Biophysical Journal, 2011Co-Authors: Ishita Mukerji, Manju M. HingoraniAbstract:The MutS homolog family in eukaryotes (MSH2, MSH3, MSH4, MSH5 and MSH6) forms three heterodimeric protein complexes: MSH2-MSH3, MSH2-MSH6 and MSH4-MSH5. The heterodimer, MSH2-MSH6, plays fundamental roles in post replication mismatch repair and specifically recognizes mismatched base pairs and small insertion/deletion loops. Previous studies have indicated that the MSH4-MSH5 heterodimer participates in meiotic crossover and specifically binds to a homologous genetic recombination intermediate, the four-way junction (4WJ) or Holliday junction. The repair protein, MSH2-MSH6 also binds with high affinity to the 4WJ. In this work, we explore the different interactions and complexes formed between these two proteins and DNA 4WJs to potentially shed light on the different roles they play in Holliday junction processing. The protein-DNA interaction is investigated using gel mobility shift assay and fluorescence anisotropy methods to determine binding affinity. Our work supports the previous finding that MSH2-MSH6 binds to the 4WJ with comparable affinity to mismatched DNA. Fluorescence measurements are being used to determine the stoichiometry of protein binding to the junction. Resonance energy transfer measurements are being used to determine the conformational changes induced upon MSH2-MSH6 binding, specifically addressing the open or stacked nature of the junction upon complexation with MSH2-MSH6.To characterize the MSH4-MSH5 binding interactions with Holliday Junctions, our efforts are currently focused on cloning S. cerevisiae msh4 and MSH5 genes for over-expression in E. coli. We have successfully cloned the msh4 gene and MSH5 genes from the wild type yeast genome and inserted these two genes separately into the two multiple cloning sites on the pCDFDuet-1 vector. Optimal conditions for expressing MSH4-MSH5 heterodimers are being explored and will be presented.