Mutations in the signature motif in MutS affect ATP-induced clamp formation and mismatch repair.
MutS protein dimer recognizes and co-ordinates repair of DNA mismatches. Mismatch recognition by the N-terminal mismatch recognition domain and subsequent downstream signalling by MutS appear coupled to the C-terminal ATP catalytic site, Walker box, through nucleotide-mediated conformational transitions. Details of this co-ordination are not understood. The focus of this study is a conserved loop in Escherichia coli MutS that is predicted to mediate cross-talk between the two ATP catalytic sites in MutS homodimer. Mutagenesis was employed to assess the role of this loop in regulating MutS function. All mutants displayed mismatch repair defects in vivo. Biochemical characterization further revealed defects in ATP binding, ATP hydrolysis as well as effective mismatch recognition. The kinetics of initial burst of ATP hydrolysis was similar to wild type but the magnitude of the burst was reduced for the mutants. Given its proximity to the ATP bound in the opposing monomer in the crystal and its potential analogy with signature motif of ABC transporters, the results strongly suggest that the loop co-ordinates ATP binding/hydrolysis in trans by the two catalytic sites. Importantly, our data reveal that the loop plays a direct role in co-ordinating conformational changes involved in long-range communication between Walker box and mismatch recognition domains.
Acharya
Samir
S
Department of Molecular Virology, Immunology and Medical Genetics, and Comprehensive Cancer Center, Ohio State University, Columbus, OH 43210, USA. samir.acharya@osumc.edu
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000595:Amino Acid Sequence; D020134:Catalytic Domain; D053843:DNA Mismatch Repair; D019281:Dimerization; D004926:Escherichia coli / Q000502:physiology*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D051719:MutS DNA Mismatch-Binding Protein / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism*; D020125:Mutation, Missense; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment
D029968:Escherichia coli Proteins; D050505:Mutant Proteins; D000255:Adenosine Triphosphate; D051719:MutS DNA Mismatch-Binding Protein; C497175:MutS protein, E coli