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 asso ciated ATPase activity that is fundamental to repair. Hence, we have c haracterized two MutS mutant gene products to define the role of ATP h ydrolysis in homeologous recombination. These mutants, denoted MutS501 and MutS506, have single point mutations within the Walker A moth, an d rate constants for ATP hydrolysis are down 60-100-fold as compared w ith wild type. Both MutS501 and MutS506 retain mismatch binding and,un like 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 stra nd transfer between M13 and fd DNAs, The level of inhibition varied be tween the mutants and corresponded with the relative affinities to a G /T mispair, Neither MutS501 nor MutS506, however, would afford complet e block of full-length heteroduplex in the presence of MutL. DNase I f ootprinting data are consistent with these results, as the region of p rotection by MutS501 and MutS506 was unchanged in the presence of ATP and MutL, Taken together, these studies suggest that 1) MutS impedes R ecA-mediated homeologous exchange as a distinct mismatch-provoked even t 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.