Site-specific protein modification to identify the MutL interface of MutH.
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.
Toedt
Grischa H
GH
Institut für Biochemie (FB 08), Justus-Liebig-Universität, Heinrich-Buff-Ring 58, D-35392 Giessen, Germany.
Krishnan
Ravi
R
Friedhoff
Peter
P
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D001665:Binding Sites; D003545:Cysteine / Q000235:genetics; D045643:DNA Repair Enzymes; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004706:Endodeoxyribonucleases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000378:metabolism*; D008958:Models, Molecular; D000070956:MutL Proteins; D016297:Mutagenesis, Site-Directed; D025941:Protein Interaction Mapping; D013329:Structure-Activity Relationship
D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C074770:MutL protein, E coli; D004706:Endodeoxyribonucleases; C059682:methyl-directed mismatch repair protein, E coli; D000251:Adenosine Triphosphatases; D000070956:MutL Proteins; D045643:DNA Repair Enzymes; D003545:Cysteine