The Experts below are selected from a list of 2034 Experts worldwide ranked by ideXlab platform
Peggy Hsieh - One of the best experts on this subject based on the ideXlab platform.
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crystal structures of mismatch repair Protein MutS and its complex with a substrate dna
Nature, 2000Co-Authors: Galina Obmolova, Peggy Hsieh, Wei YangAbstract:DNA mismatch repair is critical for increasing replication fidelity in organisms ranging from bacteria to humans. MutS Protein, a member of the ABC ATPase superfamily, recognizes mispaired and unpaired bases in duplex DNA and initiates mismatch repair. Mutations in human MutS genes cause a predisposition to hereditary nonpolyposis colorectal cancer as well as sporadic tumours. Here we report the crystal structures of a MutS Protein and a complex of MutS with a heteroduplex DNA containing an unpaired base. The structures reveal the general architecture of members of the MutS family, an induced-fit mechanism of recognition between four domains of a MutS dimer and a heteroduplex kinked at the mismatch, a composite ATPase active site composed of residues from both MutS subunits, and a transmitter region connecting the mismatch-binding and ATPase domains. The crystal structures also provide a molecular framework for understanding hereditary nonpolyposis colorectal cancer mutations and for postulating testable roles of MutS.
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oligomerization of a MutS mismatch repair Protein from thermus aquaticus
Journal of Biological Chemistry, 1999Co-Authors: Indranil Biswas, Jeffrey W Lary, David A. Yphantis, Karen G. Fleming, Wei Yang, Peggy HsiehAbstract:Abstract The MutS DNA mismatch Protein recognizes heteroduplex DNAs containing mispaired or unpaired bases. We have examined the oligomerization of a MutS Protein from Thermus aquaticus that binds to heteroduplex DNAs at elevated temperatures. Analytical gel filtration, cross-linking of MutS Protein with disuccinimidyl suberate, light scattering, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry establish that the Taq Protein is largely a dimer in free solution. Analytical equilibrium sedimentation showed that the oligomerization ofTaq MutS involves a dimer-tetramer equilibrium in which dimer predominates at concentrations below 10 μm. The ΔG 0 2–4 for the dimer to tetramer transition is approximately −6.9 ± 0.1 kcal/mol of tetramer. Analytical gel filtration of native complexes and gel mobility shift assays of an maltose-binding Protein-MutS fusion Protein bound to a short, 37-base pair heteroduplex DNA reveal that the Protein binds to DNA as a dimer with no change in oligomerization upon DNA binding.
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Oligomerization of a MutS Mismatch Repair Protein from Thermus aquaticus
The Journal of biological chemistry, 1999Co-Authors: Indranil Biswas, Jeffrey W Lary, David A. Yphantis, Karen G. Fleming, Wei Yang, Changill Ban, Jun Qin, Peggy HsiehAbstract:The MutS DNA mismatch Protein recognizes heteroduplex DNAs containing mispaired or unpaired bases. We have examined the oligomerization of a MutS Protein from Thermus aquaticus that binds to heteroduplex DNAs at elevated temperatures. Analytical gel filtration, cross-linking of MutS Protein with disuccinimidyl suberate, light scattering, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry establish that the Taq Protein is largely a dimer in free solution. Analytical equilibrium sedimentation showed that the oligomerization of Taq MutS involves a dimer-tetramer equilibrium in which dimer predominates at concentrations below 10 microM. The DeltaG(0)(2-4) for the dimer to tetramer transition is approximately -6.9 +/- 0.1 kcal/mol of tetramer. Analytical gel filtration of native complexes and gel mobility shift assays of an maltose-binding Protein-MutS fusion Protein bound to a short, 37-base pair heteroduplex DNA reveal that the Protein binds to DNA as a dimer with no change in oligomerization upon DNA binding.
Titia K. Sixma - One of the best experts on this subject based on the ideXlab platform.
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lna modification of single stranded dna oligonucleotides allows subtle gene modification in mismatch repair proficient cells
Proceedings of the National Academy of Sciences of the United States of America, 2016Co-Authors: Thomas W Van Ravesteyn, Alexander Fish, Titia K. Sixma, Marleen Dekker, Astrid Wolters, Rob J Dekker, Hein Te RieleAbstract:Synthetic single-stranded DNA oligonucleotides (ssODNs) can be used to generate subtle genetic modifications in eukaryotic and prokaryotic cells without the requirement for prior generation of DNA double-stranded breaks. However, DNA mismatch repair (MMR) suppresses the efficiency of gene modification by >100-fold. Here we present a commercially available ssODN design that evades MMR and enables subtle gene modification in MMR-proficient cells. The presence of locked nucleic acids (LNAs) in the ssODNs at mismatching bases, or also at directly adjacent bases, allowed 1-, 2-, or 3-bp substitutions in MMR-proficient mouse embryonic stem cells as effectively as in MMR-deficient cells. Additionally, in MMR-proficient Escherichia coli, LNA modification of the ssODNs enabled effective single-base-pair substitution. In vitro, LNA modification of mismatches precluded binding of purified E. coli MMR Protein MutS. These findings make ssODN-directed gene modification particularly well suited for applications that require the evaluation of a large number of sequence variants with an easy selectable phenotype.
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native mass spectrometry provides direct evidence for dna mismatch induced regulation of asymmetric nucleotide binding in mismatch repair Protein MutS
Nucleic Acids Research, 2011Co-Authors: Maria Chiara Monti, Herrie H K Winterwerp, Serge X Cohen, Anastassis Perrakis, Alexander Fish, Arjan Barendregt, Albert J R Heck, Titia K. Sixma, Peter Friedhoff, Robert H H Van Den HeuvelAbstract:The DNA mismatch repair Protein MutS recognizes mispaired bases in DNA and initiates repair in an ATP-dependent manner. Understanding of the allosteric coupling between DNA mismatch recognition and two asymmetric nucleotide binding sites at opposing sides of the MutS dimer requires identification of the relevant MutS.mmDNA.nucleotide species. Here, we use native mass spectrometry to detect simultaneous DNA mismatch binding and asymmetric nucleotide binding to Escherichia coli MutS. To resolve the small differences between macromolecular species bound to different nucleotides, we developed a likelihood based algorithm capable to deconvolute the observed spectra into individual peaks. The obtained mass resolution resolves simultaneous binding of ADP and AMP.PNP to this ABC ATPase in the absence of DNA. Mismatched DNA regulates the asymmetry in the ATPase sites; we observe a stable DNA-bound state containing a single AMP.PNP cofactor. This is the first direct evidence for such a postulated mismatch repair intermediate, and showcases the potential of native MS analysis in detecting mechanistically relevant reaction intermediates.
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magnesium coordination controls the molecular switch function of dna mismatch repair Protein MutS
Journal of Biological Chemistry, 2010Co-Authors: Joyce H G Lebbink, Annet Reumer, G Natrajan, Herrie H K Winterwerp, Alexander Fish, Titia K. SixmaAbstract:The DNA mismatch repair Protein MutS acts as a molecular switch. It toggles between ADP and ATP states and is regulated by mismatched DNA. This is analogous to G-Protein switches and the regulation of their “on” and “off” states by guanine exchange factors. Although GDP release in monomeric GTPases is accelerated by guanine exchange factor-induced removal of magnesium from the catalytic site, we found that release of ADP from MutS is not influenced by the metal ion in this manner. Rather, ADP release is induced by the binding of mismatched DNA at the opposite end of the Protein, a long-range allosteric response resembling the mechanism of activation of heterotrimeric GTPases. Magnesium influences switching in MutS by inducing faster and tighter ATP binding, allowing rapid downstream responses. MutS mutants with decreased affinity for the metal ion are impaired in fast switching and in vivo mismatch repair. Thus, the G-Proteins and MutS conceptually employ the same efficient use of the high energy cofactor: slow hydrolysis in the absence of a signal and fast conversion to the active state when required.
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the crystal structure of dna mismatch repair Protein MutS binding to a g x t mismatch
Nature, 2000Co-Authors: Meindert H Lamers, Herrie H K Winterwerp, Jacqueline H Enzlin, Anastassis Perrakis, Niels De Wind, Titia K. SixmaAbstract:DNA mismatch repair ensures genomic integrity on DNA replication. Recognition of a DNA mismatch by a dimeric MutS Protein initiates a cascade of reactions and results in repair of the newly synthesized strand; however, details of the molecular mechanism remain controversial. Here we present the crystal structure at 2.2 A of MutS from Escherichia coli bound to a G x T mismatch. The two MutS monomers have different conformations and form a heterodimer at the structural level. Only one monomer recognizes the mismatch specifically and has ADP bound. Mismatch recognition occurs by extensive minor groove interactions causing unusual base pairing and kinking of the DNA. Nonspecific major groove DNA-binding domains from both monomers embrace the DNA in a clamp-like structure. The interleaved nucleotide-binding sites are located far from the DNA. Mutations in human MutS alpha (MSH2/MSH6) that lead to hereditary predisposition for cancer, such as hereditary non-polyposis colorectal cancer, can be mapped to this crystal structure.
Wei Yang - One of the best experts on this subject based on the ideXlab platform.
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crystal structures of mismatch repair Protein MutS and its complex with a substrate dna
Nature, 2000Co-Authors: Galina Obmolova, Peggy Hsieh, Wei YangAbstract:DNA mismatch repair is critical for increasing replication fidelity in organisms ranging from bacteria to humans. MutS Protein, a member of the ABC ATPase superfamily, recognizes mispaired and unpaired bases in duplex DNA and initiates mismatch repair. Mutations in human MutS genes cause a predisposition to hereditary nonpolyposis colorectal cancer as well as sporadic tumours. Here we report the crystal structures of a MutS Protein and a complex of MutS with a heteroduplex DNA containing an unpaired base. The structures reveal the general architecture of members of the MutS family, an induced-fit mechanism of recognition between four domains of a MutS dimer and a heteroduplex kinked at the mismatch, a composite ATPase active site composed of residues from both MutS subunits, and a transmitter region connecting the mismatch-binding and ATPase domains. The crystal structures also provide a molecular framework for understanding hereditary nonpolyposis colorectal cancer mutations and for postulating testable roles of MutS.
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oligomerization of a MutS mismatch repair Protein from thermus aquaticus
Journal of Biological Chemistry, 1999Co-Authors: Indranil Biswas, Jeffrey W Lary, David A. Yphantis, Karen G. Fleming, Wei Yang, Peggy HsiehAbstract:Abstract The MutS DNA mismatch Protein recognizes heteroduplex DNAs containing mispaired or unpaired bases. We have examined the oligomerization of a MutS Protein from Thermus aquaticus that binds to heteroduplex DNAs at elevated temperatures. Analytical gel filtration, cross-linking of MutS Protein with disuccinimidyl suberate, light scattering, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry establish that the Taq Protein is largely a dimer in free solution. Analytical equilibrium sedimentation showed that the oligomerization ofTaq MutS involves a dimer-tetramer equilibrium in which dimer predominates at concentrations below 10 μm. The ΔG 0 2–4 for the dimer to tetramer transition is approximately −6.9 ± 0.1 kcal/mol of tetramer. Analytical gel filtration of native complexes and gel mobility shift assays of an maltose-binding Protein-MutS fusion Protein bound to a short, 37-base pair heteroduplex DNA reveal that the Protein binds to DNA as a dimer with no change in oligomerization upon DNA binding.
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Oligomerization of a MutS Mismatch Repair Protein from Thermus aquaticus
The Journal of biological chemistry, 1999Co-Authors: Indranil Biswas, Jeffrey W Lary, David A. Yphantis, Karen G. Fleming, Wei Yang, Changill Ban, Jun Qin, Peggy HsiehAbstract:The MutS DNA mismatch Protein recognizes heteroduplex DNAs containing mispaired or unpaired bases. We have examined the oligomerization of a MutS Protein from Thermus aquaticus that binds to heteroduplex DNAs at elevated temperatures. Analytical gel filtration, cross-linking of MutS Protein with disuccinimidyl suberate, light scattering, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry establish that the Taq Protein is largely a dimer in free solution. Analytical equilibrium sedimentation showed that the oligomerization of Taq MutS involves a dimer-tetramer equilibrium in which dimer predominates at concentrations below 10 microM. The DeltaG(0)(2-4) for the dimer to tetramer transition is approximately -6.9 +/- 0.1 kcal/mol of tetramer. Analytical gel filtration of native complexes and gel mobility shift assays of an maltose-binding Protein-MutS fusion Protein bound to a short, 37-base pair heteroduplex DNA reveal that the Protein binds to DNA as a dimer with no change in oligomerization upon DNA binding.
K. T. Nishant - One of the best experts on this subject based on the ideXlab platform.
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Structural Insights into Saccharomyces cerevisiae Msh4– Msh5 Complex Function Using Homology Modeling
2016Co-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–Msh
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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.
Peter J. Belshaw - One of the best experts on this subject based on the ideXlab platform.
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Correcting errors in synthetic DNA through consensus shuffling. Nucleic Acids Res
2014Co-Authors: Brock F. Binkowski, Kathryn E. Richmond, James Howard Kaysen, Michael R. Sussman, Peter J. BelshawAbstract:Although efficient methods exist to assemble syn-thetic oligonucleotides into genes and genomes, these suffer from the presence of 1–3 random errors/kb of DNA. Here, we introduce a new method termed consensus shuffling and demonstrate its use to significantly reduce random errors in synthetic DNA. In this method, errors are revealed as mis-matches by re-hybridization of the population. The DNA is fragmented, and mismatched fragments are removed upon binding to an immobilized mismatch binding Protein (MutS). PCR assembly of the remain-ing fragments yields a new population of full-length sequences enriched for the consensus sequence of the input population. We show that two iterations of consensus shuffling improved a population of syn-thetic green fluorescent Protein (GFPuv) clones from 60 to.90 % fluorescent, and decreased errors 3.5-to 4.3-fold to final values of 1 error per 3500 bp. In addition, two iterations of consensus shuffling cor-rected a population of GFPuv clones where all mem-bers were non-functional, to a population where 82% of clones were fluorescent. Consensus shuffling should facilitate the rapid and accurate synthesis of long DNA sequences
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Correcting errors in synthetic DNA through consensus shuffling
Nucleic acids research, 2005Co-Authors: Brock F. Binkowski, Kathryn E. Richmond, James Howard Kaysen, Michael R. Sussman, Peter J. BelshawAbstract:Although efficient methods exist to assemble synthetic oligonucleotides into genes and genomes, these suffer from the presence of 1-3 random errors/kb of DNA. Here, we introduce a new method termed consensus shuffling and demonstrate its use to significantly reduce random errors in synthetic DNA. In this method, errors are revealed as mismatches by re-hybridization of the population. The DNA is fragmented, and mismatched fragments are removed upon binding to an immobilized mismatch binding Protein (MutS). PCR assembly of the remaining fragments yields a new population of full-length sequences enriched for the consensus sequence of the input population. We show that two iterations of consensus shuffling improved a population of synthetic green fluorescent Protein (GFPuv) clones from approximately 60 to >90% fluorescent, and decreased errors 3.5- to 4.3-fold to final values of approximately 1 error per 3500 bp. In addition, two iterations of consensus shuffling corrected a population of GFPuv clones where all members were non-functional, to a population where 82% of clones were fluorescent. Consensus shuffling should facilitate the rapid and accurate synthesis of long DNA sequences.
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Correcting errors in synthetic DNA through consensus shuffling
2005Co-Authors: Brock F. Binkowski, Kathryn E. Richmond, James Howard Kaysen, Michael R. Sussman, Peter J. BelshawAbstract:Although efficient methods exist to assemble syn-thetic oligonucleotides into genes and genomes, these suffer from the presence of 1–3 random errors/kb of DNA. Here, we introduce a new method termed consensus shuffling and demonstrate its use to significantly reduce random errors in synthetic DNA. In this method, errors are revealed as mis-matches by re-hybridization of the population. The DNA is fragmented, and mismatched fragments are removed upon binding to an immobilized mismatch binding Protein (MutS). PCR assembly of the remain-ing fragments yields a new population of full-length sequences enriched for the consensus sequence of the input population. We show that two iterations of consensus shuffling improved a population of syn-thetic green fluorescent Protein (GFPuv) clones from 60 to.90 % fluorescent, and decreased errors 3.5-to 4.3-fold to final values of 1 error per 3500 bp. In addition, two iterations of consensus shuffling cor-rected a population of GFPuv clones where all mem-bers were non-functional, to a population where 82% of clones were fluorescent. Consensus shuffling should facilitate the rapid and accurate synthesis of long DNA sequences
