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Your search keyword '"Winterwerp, HHK"' showing total 2 results
Start Over Author "Winterwerp, HHK" Topic escherichia coli proteins
2 results on '"Winterwerp, HHK"'

1. The unstructured linker arms of MutL enable GATC site incision beyond roadblocks during initiation of DNA mismatch repair.

Author

Mardenborough YSN, Nitsenko K, Laffeber C, Duboc C, Sahin E, Quessada-Vial A, Winterwerp HHK, Sixma TK, Kanaar R, Friedhoff P, Strick TR, and Lebbink JHG

Subjects
Base Pair Mismatch genetics, CRISPR-Associated Protein 9 genetics, DNA Repair Enzymes metabolism, DNA-Binding Proteins metabolism, DNA-Directed RNA Polymerases genetics, Endodeoxyribonucleases metabolism, Genomic Instability genetics, MutS DNA Mismatch-Binding Protein metabolism, DNA Mismatch Repair genetics, DNA, Bacterial genetics, Deoxyribonucleases, Type II Site-Specific metabolism, Escherichia coli genetics, Escherichia coli Proteins metabolism, MutL Proteins metabolism
Abstract

DNA mismatch repair (MMR) maintains genome stability through repair of DNA replication errors. In Escherichia coli, initiation of MMR involves recognition of the mismatch by MutS, recruitment of MutL, activation of endonuclease MutH and DNA strand incision at a hemimethylated GATC site. Here, we studied the mechanism of communication that couples mismatch recognition to daughter strand incision. We investigated the effect of catalytically-deficient Cas9 as well as stalled RNA polymerase as roadblocks placed on DNA in between the mismatch and GATC site in ensemble and single molecule nanomanipulation incision assays. The MMR proteins were observed to incise GATC sites beyond a roadblock, albeit with reduced efficiency. This residual incision is completely abolished upon shortening the disordered linker regions of MutL. These results indicate that roadblock bypass can be fully attributed to the long, disordered linker regions in MutL and establish that communication during MMR initiation occurs along the DNA backbone., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Published
2019
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2. Sharp kinking of a coiled-coil in MutS allows DNA binding and release.

Author

Bhairosing-Kok D, Groothuizen FS, Fish A, Dharadhar S, Winterwerp HHK, and Sixma TK

Subjects
Amino Acid Sequence, Apoproteins genetics, Apoproteins metabolism, Binding Sites, Cloning, Molecular, Crystallography, X-Ray, DNA, Bacterial genetics, DNA, Bacterial metabolism, Escherichia coli metabolism, Escherichia coli Proteins genetics, Escherichia coli Proteins metabolism, Gene Expression, Genetic Vectors chemistry, Genetic Vectors metabolism, Kinetics, Models, Molecular, MutS DNA Mismatch-Binding Protein genetics, MutS DNA Mismatch-Binding Protein metabolism, Mutagenesis, Site-Directed, Mutation, Protein Binding, Protein Conformation, alpha-Helical, Protein Conformation, beta-Strand, Protein Interaction Domains and Motifs, Protein Multimerization, Recombinant Proteins chemistry, Recombinant Proteins genetics, Recombinant Proteins metabolism, Sequence Alignment, Structure-Activity Relationship, Apoproteins chemistry, DNA, Bacterial chemistry, Escherichia coli genetics, Escherichia coli Proteins chemistry, MutS DNA Mismatch-Binding Protein chemistry
Abstract

DNA mismatch repair (MMR) corrects mismatches, small insertions and deletions in DNA during DNA replication. While scanning for mismatches, dimers of MutS embrace the DNA helix with their lever and clamp domains. Previous studies indicated generic flexibility of the lever and clamp domains of MutS prior to DNA binding, but whether this was important for MutS function was unknown. Here, we present a novel crystal structure of DNA-free Escherichia coli MutS. In this apo-structure, the clamp domains are repositioned due to kinking at specific sites in the coiled-coil region in the lever domains, suggesting a defined hinge point. We made mutations at the coiled-coil hinge point. The mutants made to disrupt the helical fold at the kink site diminish DNA binding, whereas those made to increase stability of coiled-coil result in stronger DNA binding. These data suggest that the site-specific kinking of the coiled-coil in the lever domain is important for loading of this ABC-ATPase on DNA., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Published
2019
Full Text
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