Abstract The MutS protein controls genomic stability by coordinating recognition and repair of DNA mismatches with ATP utilization. The nature of this coordination is unclear. This study demonstrates the importance of a highly conserved flexible loop found in Escherichia coli MutS (residues 658–670) in DNA mismatch repair. This loop is speculated to be analogous to the ABC signature motif of drug transporters based on its proximity to the ATP catalytic site in crystal structures. Our studies show that amino acid residues G666 and S668 control MutS functions subsequent to mismatch recognition by MutS, i.e., nucleotide-mediated exchange and ATP-dependent dissociation from mismatch. G666V mutation affects mismatch-provoked ADP–ATP exchange and results in slower dissociation kinetics of MutS from the mismatch while S668A mutation affects stable clamp formation and dissociation kinetics but does not affect nucleotide exchange. Both mutants harbor defects in ATP hydrolysis and cause a significant mutator phenotype in vivo. The mutator effect of S668A is indistinguishable from that of a MutS-deficient background and is similar to that seen with G658A. Neither mutations affect protein stability or cause a dominant mutator effect. Together with our studies on G658, D661 and F670 , this study implicates the signature motif as a primary regulator of MutS function and suggests concerted action of the individual amino acid residues within this motif in mediating communication between the Walker and mismatch recognition domains.