Functional studies on the candidate ATPase domains of Saccharomyces cerevisiae MutL alpha

Saccharomyces cerevisiae MutL homologues Mlh1p and Pms1p form a heterodimer , termed MutL alpha, that is required for DNA mismatch repair after mismatc h binding by MutS homologues. Recent sequence and structural studies have p laced the NH2 termini of MutL homologues in a new family of ATPases. To add ress the functional significance of this putative ATPase activity in MutL a lpha, we mutated conserved motifs for ATP hydrolysis and ATP binding in bot h Mlh1p and Pms1p and found that these changes disrupted DNA mismatch repai r in vivo. Limited proteolysis with purified recombinant MutL alpha demonst rated that the NH2 terminus of MutL alpha undergoes conformational changes in the presence of ATP and nonhydrolyzable ATP analogs. Furthermore, two-hy brid analysis suggested that these ATP-binding-induced conformational chang es promote an interaction between the NH2 termini of Mlh1p and Pms1p. Surpr isingly, analysis of specific mutants suggested differential requirements f or the ATPase motifs of Mlh1p and Pms1p during DNA mismatch repair. Taken t ogether, these results suggest that MutL alpha undergoes ATP-dependent conf ormational changes that may serve to coordinate downstream events during ye ast DNA mismatch repair.