Role of MutS ATPase activity in MutS,L-dependent block of in vitro strand transfer.
In addition to mismatch recognition, Escherichia coli MutS has an associated ATPase activity that is fundamental to repair. Hence, we have characterized two MutS mutant gene products to define the role of ATP hydrolysis in homeologous recombination. These mutants, denoted MutS501 and MutS506, have single point mutations within the Walker A motif, and rate constants for ATP hydrolysis are down 60-100-fold as compared with wild type. Both MutS501 and MutS506 retain mismatch binding and, unlike wild type, fail to relinquish this specificity in the presence of ATP, adenosine 5'-O-(thiotriphosphate), and adenosine 5'-(beta, gamma-imino)triphosphate. Both MutS501 and MutS506 blocked the level of strand transfer between M13 and fd DNAs. The level of inhibition varied between the mutants and corresponded with the relative affinities to a G/T mispair. Neither MutS501 nor MutS506, however, would afford complete block of full-length heteroduplex in the presence of MutL. DNase I footprinting data are consistent with these results, as the region of protection by MutS501 and MutS506 was unchanged in the presence of ATP and MutL. Taken together, these studies suggest that 1) MutS impedes RecA-mediated homeologous exchange as a distinct mismatch-provoked event and 2) the role of MutL is coupled to MutS-dependent ATP hydrolysis. These observations are in good agreement with the present model for E. coli methyl-directed mismatch repair.
Laboratory of Molecular Genetics, NIEHS, National Institutes of Health, P.O. Box 12233, Research Triangle, North Carolina 27709, USA. worth@nieh.nih.gov
L
L
Worth
T
T
Bader
J
J
Yang
S
S
Clark
D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D016172:DNA Fingerprinting; D004260:DNA Repair; D004268:DNA-Binding Proteins; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D006868:Hydrolysis; D008957:Models, Genetic; D000070956:MutL Proteins; D051719:MutS DNA Mismatch-Binding Protein; D017354:Point Mutation; D011485:Protein Binding; D011938:Rec A Recombinases / Q000378:metabolism; D011995:Recombination, Genetic
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C074770:MutL protein, E coli; D000255:Adenosine Triphosphate; D011938:Rec A Recombinases; D000251:Adenosine Triphosphatases; D000070956:MutL Proteins; D051719:MutS DNA Mismatch-Binding Protein; C497175:MutS protein, E coli