MutL involvement in two DNA repair pathways: methyl-directed mismatch repair and very short patch repair

MutL is an essential component of the Escherichia coli mismatch repair (MMR) pathway and has been shown to interact with nearly every protein involved in the MMR pathway including MutS, MutH and UvrD. The DNA binding properties of MutL in MMR have been controversial for nearly two decades. The DNA binding requirement by MutL was addressed using a point mutant of MutL—MutL-R266E—which binds DNA poorly. Our results demonstrate, using data obtained from biochemical and genetic analyses with MutL-R266E, that MutL must bind DNA to function in MMR. Therefore, we have attempted to resolve the controversial issue regarding the DNA binding properties of MutL. MutL is thought to be the master coordinator of MMR, accomplishing this regulatory role through its ATP binding, hydrolysis, and release cycle. Using a point mutant of MutL that cannot bind ATP—MutL-D58A, we show that this mutant is unable to interact with MutS, and MutH and UvrD. Additionally, we have identified the step in MMR that MutL must hydrolyze its bound ATP in order to continue its normal function in MMR. We accomplished this using another point mutant of MutL that cannot hydrolyze ATP—MutL-E29A. These results lend support for the hypothesis of temporal regulation of MMR by the MutL ATPase. Using MutL-E29A and MutL-D58A in biochemical and genetic assays, we have attempted to identify a role for the MutL ATPase in MMR. These data suggest that MutL must be bound to ATP to function in MMR prior to the loading of the UvrD helicase. Additionally, these data implicate ATP hydrolysis by MutL at a step in the MMR pathway after the UvrD helicase is loaded onto the DNA by MutL. Previous studies have shown that MutL serves to augment the repair efficiency of the very short patch (VSP) repair pathway. We have reconstituted the minimal VSP repair pathway in vitro. We suggest a biochemical role for MutL in VSP repair. The reconstitution of this system will allow further studies addressing the function of MutL in VSP repair