MutS has within its c-terminal domain a nucleotide binding concensus sequence responsible for ATP hydrolysis. The exact role of ATP in methyl-directed repair and recombination is not known. During a repair event ATP is thought to be the driving force that allows MutS to translocate along the DNA in search of a hemi-methylated GATC site. To help clarify the biochemistry behind MutS function in homologous exchange, two ATPase defective mutants, MutS501 & MutS506, were examined. We wanted to test and see if; 1. mismatch binding alone will inhibit strand exchange and 2.the enhanced block by MutL concomitant with MutS ATP hydrolysis. MutS501 & MutS506, have been cloned and overexpressed with an n-terminal His-tag. MutS501 & 506 maintain a different point mutation within the highly conserved ATP binding domain. Both have been shown to bind a G/T mismatch with MutS501 behaving identical to wt (both with and without His-tag) and the MutS506 having a 2-fold drop in binding. In terms of mismatch repair, both MutS501 and 506 do not complement MutS- extracts. These observations are consistent with loss of ATPase function which is ~10-fold lower for both mutants as compared to wt. In the strand transfer reaction, MutS501 & MutS506 successfully blocked heteroduplex formation between M13 and fd. To our surprise MutS506 was still able to block exchange given its reduced mismatch binding to a G/T mismatch. Current studies are now addressing MutL and its role with these mutants to see if the enhanced block is coupled to ATP hydrolysis.
