DNA-DEPENDENT ACTIVATION OF THE HMUTS-ALPHA ATPASE

ATP hydrolysis by MutS homologs is required for function of these prot eins in mismatch repair. However, the function of ATP hydrolysis in th e repair reaction is controversial. In this paper we describe a steady -state kinetic analysis of the DNA-activated ATPase of human MutS alph a. Comparison of salt concentration effects on mismatch repair and mis match-provoked excision in HeLa nuclear extracts with salt effects on the DNA-activated ATPase suggests that ATP hydrolysis by MutS alpha is involved in the rate determining step in the repair pathway. While th e ATPase is activated by homoduplex and heteroduplex DNA, the half-max imal concentration for activation by heteroduplex DNA is significantly lower under physiological salt concentrations. Furthermore, at optima l salt concentration, heteroduplex DNA increases the k(cat) for ATP hy drolysis to a greater extent than does homoduplex DNA, We also demonst rate that the degree of ATPase activation is dependent on DNA chain le ngth, with the k(cat) for hydrolysis increasing significantly with cha in length of the DNA cofactor. These results are discussed in terms of the translocation (Allen, D, J,, Makhov, A., Grilley, M,, Taylor, J,, Thresher, R,, Modrich, P,, and Griffith, J, D, (1997) EMBO J, 16, 446 7-4476) and the molecular switch (Gradia, S,, Acharya, S,, and Fishel, R, (1997) Cell 91, 995-1005) models that invoke distinct roles for AT P hydrolysis in MutS homolog function.