Mismatch repair proteins correct errors in DNA via an ATP-driven process. In eukaryotes, the Msh2-Msh6 complex recognizes base pair mismatches and small insertion/deletions in DNA and initiates repair. Both Msh2 and Msh6 proteins contain Walker ATP-binding motifs that are necessary for repair activity. To understand how these proteins couple ATP binding and hydrolysis to DNA binding/mismatch recognition, the ATPase activity of Saccharomyces cerevisiae Msh2-Msh6 was examined under pre-steady-state conditions. Acid-quench experiments revealed that in the absence of DNA, Msh2-Msh6 hydrolyzes ATP rapidly (burst rate = 3 s(-1) at 20 degrees C) and then undergoes a slow step in the pathway that limits catalytic turnover (k(cat) = 0.1 s(-1)). ATP is hydrolyzed similarly in the presence of fully matched duplex DNA; however, in the presence of a G:T mismatch or +T insertion-containing DNA, ATP hydrolysis is severely suppressed (rate = 0.1 s(-1)). Pulse-chase experiments revealed that Msh2-Msh6 binds ATP rapidly in the absence or in the presence of DNA (rate = 0.1 microM(-1) s(-1)), indicating that for the Msh2-Msh6.mismatched DNA complex, a step after ATP binding but before or at ATP hydrolysis is the rate-limiting step in the pathway. Thus, mismatch recognition is coupled to a dramatic increase in the residence time of ATP on Msh2-Msh6. This mismatch-induced, stable ATP-bound state of Msh2-Msh6 likely signals downstream events in the repair pathway.