7 results on '"Winterwerp, HHK"'
Search Results
2. Dual daughter strand incision is processive and increases the efficiency of DNA mismatch repair
- Author
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Hermans, Nicolaas, Laffeber, Charlie, Cristovao, Michele, Artola-Boran, M, Mardenborough, Yannicka, Ikpa, Pauline, Jaddoe, A, Winterwerp, HHK, Wyman, C.L., Jiricny, J, Kanaar, Roland, Friedhoff, P, Lebbink, Joyce, Hermans, Nicolaas, Laffeber, Charlie, Cristovao, Michele, Artola-Boran, M, Mardenborough, Yannicka, Ikpa, Pauline, Jaddoe, A, Winterwerp, HHK, Wyman, C.L., Jiricny, J, Kanaar, Roland, Friedhoff, P, and Lebbink, Joyce
- Abstract
DNA mismatch repair (MMR) is an evolutionarily-conserved process responsible for the repair of replication errors. In Escherichia coli, MMR is initiated by MutS and MutL, which activate MutH to incise transiently-hemimethylated GATC sites. MMR efficiency depends on the distribution of these GATC sites. To understand which molecular events determine repair efficiency, we quantitatively studied the effect of strand incision on unwinding and excision activity. The distance between mismatch and GATC site did not influence the strand incision rate, and an increase in the number of sites enhanced incision only to a minor extent. Two GATC sites were incised by the same activated MMR complex in a processive manner, with MutS, the closed form of MutL and MutH displaying different roles. Unwinding and strand excision were more efficient on a substrate with two nicks flanking the mismatch, as compared to substrates containing a single nick or two nicks on the same side of the mismatch. Introduction of multiple nicks by the human MutL alpha endonuclease also contributed to increased repair efficiency. Our data support a general model of prokaryotic and eukaryotic MMR in which, despite mechanistic differences, mismatch-activated complexes facilitate efficient repair by creating multiple daughter strand nicks.
- Published
- 2016
3. Using stable MutS dimers and tetramers to quantitatively analyze DNA mismatch recognition and sliding clamp formation
- Author
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Groothuizen, FS, Fish, A, Petoukhov, MV, Reumer, A, Manelyte, L, Winterwerp, HHK, Marinus, MG, Lebbink, Joyce, Svergun, DI, Friedhoff, P, Sixma, T.K., Groothuizen, FS, Fish, A, Petoukhov, MV, Reumer, A, Manelyte, L, Winterwerp, HHK, Marinus, MG, Lebbink, Joyce, Svergun, DI, Friedhoff, P, and Sixma, T.K.
- Abstract
The process of DNA mismatch repair is initiated when MutS recognizes mismatched DNA bases and starts the repair cascade. The Escherichia coli MutS protein exists in an equilibrium between dimers and tetramers, which has compromised biophysical analysis. To uncouple these states, we have generated stable dimers and tetramers, respectively. These proteins allowed kinetic analysis of DNA recognition and structural analysis of the full-length protein by X-ray crystallography and small angle X-ray scattering. Our structural data reveal that the tetramerization domains are flexible with respect to the body of the protein, resulting in mostly extended structures. Tetrameric MutS has a slow dissociation from DNA, which can be due to occasional bending over and binding DNA in its two binding sites. In contrast, the dimer dissociation is faster, primarily dependent on a combination of the type of mismatch and the flanking sequence. In the presence of ATP, we could distinguish two kinetic groups: DNA sequences where MutS forms sliding clamps and those where sliding clamps are not formed efficiently. Interestingly, this inability to undergo a conformational change rather than mismatch affinity is correlated with mismatch repair.
- Published
- 2013
4. Native mass spectrometry provides direct evidence for DNA mismatch-induced regulation of asymmetric nucleotide binding in mismatch repair protein MutS
- Author
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Monti, MC, Cohen, SX, Fish, A, Winterwerp, HHK, Barendregt, A (Arjan), Friedhoff, P, Perrakis, A (Anastassis), Heck, AJR, Sixma, T.K., van den Heuvel, RHH, Lebbink, Joyce, Monti, MC, Cohen, SX, Fish, A, Winterwerp, HHK, Barendregt, A (Arjan), Friedhoff, P, Perrakis, A (Anastassis), Heck, AJR, Sixma, T.K., van den Heuvel, RHH, and Lebbink, Joyce
- Abstract
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.
- Published
- 2011
5. The unstructured linker arms of MutL enable GATC site incision beyond roadblocks during initiation of DNA mismatch repair.
- Author
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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
- Full Text
- View/download PDF
6. Sharp kinking of a coiled-coil in MutS allows DNA binding and release.
- Author
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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
- View/download PDF
7. USP48 restrains resection by site-specific cleavage of the BRCA1 ubiquitin mark from H2A.
- Author
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Uckelmann M, Densham RM, Baas R, Winterwerp HHK, Fish A, Sixma TK, and Morris JR
- Subjects
- Animals, BRCA1 Protein genetics, Base Sequence, Cell Line, Tumor, Cells, Cultured, DNA Repair, HeLa Cells, Humans, Kinetics, Mice, Knockout, RNA Interference, Tumor Suppressor p53-Binding Protein 1 metabolism, Ubiquitin-Protein Ligases genetics, Ubiquitin-Protein Ligases metabolism, Ubiquitin-Specific Proteases genetics, Ubiquitination, BRCA1 Protein metabolism, Histones metabolism, Ubiquitin metabolism, Ubiquitin-Specific Proteases metabolism
- Abstract
BRCA1-BARD1-catalyzed ubiquitination of histone H2A is an important regulator of the DNA damage response, priming chromatin for repair by homologous recombination. However, no specific deubiquitinating enzymes (DUBs) are known to antagonize this function. Here we identify ubiquitin specific protease-48 (USP48) as a H2A DUB, specific for the C-terminal BRCA1 ubiquitination site. Detailed biochemical analysis shows that an auxiliary ubiquitin, an additional ubiquitin that itself does not get cleaved, modulates USP48 activity, which has possible implications for its regulation in vivo. In cells we reveal that USP48 antagonizes BRCA1 E3 ligase function and in BRCA1-proficient cells loss of USP48 results in positioning 53BP1 further from the break site and in extended resection lengths. USP48 repression confers a survival benefit to cells treated with camptothecin and its activity acts to restrain gene conversion and mutagenic single-strand annealing. We propose that USP48 promotes genome stability by antagonizing BRCA1 E3 ligase function.
- Published
- 2018
- Full Text
- View/download PDF
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