The Experts below are selected from a list of 36 Experts worldwide ranked by ideXlab platform
Peter Friedhoff - One of the best experts on this subject based on the ideXlab platform.
-
identifying an interaction site between muth and the c terminal domain of MutL by crosslinking affinity purification chemical coding and mass spectrometry
Nucleic Acids Research, 2006Co-Authors: Robert Ahrends, Jan Kosinski, Dieter Kirsch, Laura Manelyte, Luis Gironmonzon, Lars Hummerich, Oliver Schulz, Bernhard Spengler, Peter FriedhoffAbstract:To investigate Protein-Protein interaction sites in the DNA mismatch repair system we developed a crosslinking/mass spectrometry technique employing a commercially available trifunctional crosslinker with a thiol-specific methanethiosulfonate group, a photoactivatable benzophenone moiety and a biotin affinity tag. The XACM approach combines photocrosslinking (X), in-solution digestion of the crosslinked mixtures, affinity purification via the biotin handle (A), chemical coding of the crosslinked products (C) followed by MALDI-TOF mass spectrometry (M). We illustrate the feasibility of the method using a single-cysteine variant of the homodimeric DNA mismatch repair Protein MutL. Moreover, we successfully applied this method to identify the photocrosslink formed between the single-cysteine MutH variant A223C, labeled with the trifunctional crosslinker in the C-terminal helix and its activator Protein MutL. The identified crosslinked MutL-peptide maps to a conserved surface patch of the MutL C-terminal dimerization domain. These observations are substantiated by additional mutational and chemical crosslinking studies. Our results shed light on the potential structures of the MutL holoenzyme and the MutH-MutL-DNA complex.
-
site specific Protein modification to identify the MutL interface of muth
Nucleic Acids Research, 2003Co-Authors: Grischa H Toedt, Ravi Krishnan, Peter FriedhoffAbstract:We have mapped the region for the Protein interaction site of the Escherichia coli mismatch repair Protein MutH for its activator Protein MutL by a site-specific Protein modification approach. For this purpose we generated a cysteine-free variant of MutH and 12 variants thereof, each containing a single cysteine residue at surface positions selected on the basis of available structural and sequence information for MutH. All MutH variants displayed wild type activity both in vivo and in vitro. These variants were then site-specifically modified at their cysteine residues with thiol-specific reagents and then tested for their ability to be stimulated in their DNA cleavage activity by the activator Protein MutL. Thereby we were able to identify a defined region in the MutH Protein that is important for interaction with MutL, and most likely represents the MutL binding site of MutH.
Salvatore Venuta - One of the best experts on this subject based on the ideXlab platform.
-
human mismatch repair Protein MutL homologue 1 mlh1 interacts with escherichia coli MutL and muts in vivo and in vitro a simple genetic system to assay mlh1 function
Biochemical Journal, 2003Co-Authors: Barbara Quaresima, Pietro Alifano, Pierfrancesco Tassone, Enrico V Avvedimento, Francesco Costanzo, Salvatore VenutaAbstract:A simple genetic system has been developed to test the effect of over-expression of wild-type or mutated human MutL homologue 1 (hMLH1) Proteins on methyl-directed mismatch repair (MMR) in Escherichia coli. The system relies on detection of Lac(+) revertants using MMR-proficient or MMR-deficient E. coli strains carrying a lac +1 frameshift mutation expressing hMLH1 Proteins. We report that expression of wild-type hMLH1 Protein causes an approx. 19-fold increase in mutation rates. The mutator phenotype was due to the ability of hMLH1 Protein to interact with bacterial MutL and MutS Proteins, thereby interfering with the formation of complexes between MMR Proteins and mismatched DNA. Conversely, expression of Proteins encoded by alleles deriving from hereditary-non-polyposis-colon-cancer (HNPCC) families decreases mutation rates, depending on the specific amino acid substitutions. These effects parallel the MutL-and MutS-binding and ATP-binding/hydrolysis activities of the mutated Proteins.
Claire G Cupples - One of the best experts on this subject based on the ideXlab platform.
-
the escherichia coli mismatch repair Protein MutL recruits the vsr and muth endonucleases in response to dna damage
Journal of Bacteriology, 2009Co-Authors: Yaroslava Y Polosina, Justin Mui, Photini Pitsikas, Claire G CupplesAbstract:The activities of the Vsr and MutH endonucleases of Escherichia coli are stimulated by MutL. The interaction of MutL with each enzyme is enhanced in vivo by 2-aminopurine treatment and by inactivation of the mutY gene. We hypothesize that MutL recruits the endonucleases to sites of DNA damage.
Reuben S. Harris - One of the best experts on this subject based on the ideXlab platform.
-
mismatch repair Protein MutL becomes limiting during stationary phase mutation
Genes & Development, 1997Co-Authors: Kimberly J Ross, Roger Sidhu, Carl Thulin, Simonne Longerich, Susan K Szigety, Reuben S. Harris, Gang Feng, Malcolm E WinklerAbstract:Postsynthesis mismatch repair is an important contributor to mutation avoidance and genomic stability in bacteria, yeast, and humans. Regulation of its activity would allow organisms to regulate their ability to evolve. That mismatch repair might be down-regulated in stationary-phase Escherichia coli was suggested by the sequence spectrum of some stationary-phase (“adaptive”) mutations and by the observations that MutS and MutH levels decline during stationary phase. We report that overproduction of MutL inhibits mutation in stationary phase but not during growth. MutS overproduction has no such effect, and MutL overproduction does not prevent stationary-phase decline of either MutS or MutH. These results imply that MutS and MutH decline to levels appropriate for the decreased DNA synthesis in stationary phase, whereas functional MutL is limiting for mismatch repair specifically during stationary phase. Modulation of mutation rate and genetic stability in response to environmental or developmental cues, such as stationary phase and stress, could be important in evolution, development, microbial pathogenicity, and the origins of cancer.
Adam B Robertson - One of the best experts on this subject based on the ideXlab platform.
-
the uvrd helicase and its modulation by the mismatch repair Protein MutL
Nucleic Acids Research, 2006Co-Authors: Steven W Matson, Adam B RobertsonAbstract:UvrD is a superfamily I DNA helicase with well documented roles in excision repair and methyl-directed mismatch repair (MMR) in addition to poorly understood roles in replication and recombination. The MutL Protein is a homodimeric DNA-stimulated ATPase that plays a central role in MMR in Escherichia coli. This Protein has been characterized as the master regulator of mismatch repair since it interacts with and modulates the activity of several other Proteins involved in the mismatch repair pathway including MutS, MutH and UvrD. Here we present a brief summary of recent studies directed toward arriving at a better understanding of the interaction between MutL and UvrD, and the impact of this interaction on the activity of UvrD and its role in mismatch repair.
