The Escherichia coli MutL protein physically interacts with MutH and stimulates the MutH-associated endonuclease activity

All possible pairwise combinations of UvrD, MutL, MutS, and MutH were teste d using the yeast two-hybrid system to identify potential interactions invo lving mismatch repair proteins. A two-hybrid screen previously identified a physical interaction between MutL and UvrD. Although several other known i nteractions were not observed, a novel interaction between MutL and MutH wa s detected. A series of truncations from the NH2 and COOH termini of MutL d emonstrated that the COOH-terminal 218 amino acids were sufficient for the two hybrid interaction with MutH. Removal of a small number of residues fro m either the NH2 or COOH termini of MutH eliminated the two-hybrid interact ion with MutL. Protein affinity chromatography experiments confirmed that M utL, but not MutS, physically associates with MutH. Furthermore, MutL great ly stimulated the d(GATC)-specific endonuclease activity of MutH in the abs ence of MutS and a mispaired base. Stimulation of the MutH-associated endon uclease activity by MutL was dependent on ATP binding but not ATP hydrolysi s. Further stimulation of this reaction by MutS required the presence of a DNA mismatch and a hydrolyzable form of ATP. These results suggest that Mut L activates the MutH associated endonuclease activity through a physical in teraction during methyl-directed mismatch repair in Escherichia coli.