Modulation of MutS ATP hydrolysis by DNA cofactors

Escherichia coli MutS protein, which is required for mismatch repair, has a slow ATPase activity that obeys Michalelis-Menten kinetics, At 37 degrees C, the steady-state turnover rate for ATP hydrolysis is 1.0 +/- 0.3 min(-1) per monomer equivalent with a K-m of 33 +/- 6 mu M Hydrolysis is competiti vely inhibited by the ATP analogues AMPPNP and ATP gamma S, with Ki, values of 4 mu M in both cases, and by ADP with a. K-i of 40 mu M. The rate of AT P hydrolysis is stimulated 2-5-fold by short hetero- and homoduplex DNAs. T he concentration of DNA cofactor that yields half-maximal stimulation is lo west, for oligodeoxynucleotide duplexes that contain a mismatched base pair . Pre-steady-state chemical quench analysis has demonstrated a substoichiom etric initial burst of ADP formation by free MutS that is governed by a rat e constant of 78 min(-1), indicating that the rate-limiting step for the st eady-state reaction occurs after hydrolysis. Prebinding of MutS to homodupl ex DNA does not alter the burst kinetics or amplitude but only increases th e s t.eadystate rate. In contrast, binding of the protein to heteroduplex D NA abolishes the burst of ADP formation, indicating that the rate-limiting step now occurs before hydrolysis. Gel filtration analysis indicates that t he MutS dimer assembles into higher order oligomers in a concentration-depe ndent. manner, and that ATP binding shifts this equilibrium to favor assemb ly. nonequivalence of subunits within a MutS oligomer These results, togeth er with kinetic findings, indicate with respect to ATP hydrolysis and DNA b inding.