MutL-catalyzed ATP hydrolysis is required at a post-UvrD loading step in methyl-directed mismatch repair.
Methyl-directed mismatch repair is a coordinated process that ensures replication fidelity and genome integrity by resolving base pair mismatches and insertion/deletion loops. This post-replicative event involves the activities of several proteins, many of which appear to be regulated by MutL. MutL interacts with and modulates the activities of MutS, MutH, UvrD, and perhaps other proteins. The purified protein catalyzes a slow ATP hydrolysis reaction that is essential for its role in mismatch repair. However, the role of the ATP hydrolysis reaction is not understood. We have begun to address this issue using two point mutants: MutL-E29A, which binds nucleotide but does not catalyze ATP hydrolysis, and MutL-D58A, which does not bind nucleotide. As expected, both mutants failed to complement the loss of MutL in genetic assays. Purified MutL-E29A protein interacted with MutS and stimulated the MutH-catalyzed nicking reaction in a mismatch-dependent manner. Importantly, MutL-E29A stimulated the loading of UvrD on model substrates. In fact, stimulation of UvrD-catalyzed unwinding was more robust with MutL-E29A than the wild-type protein. MutL-D58A, on the other hand, did not interact with MutS, stimulate MutH-catalyzed nicking, or stimulate the loading of UvrD. We conclude that ATP-bound MutL is required for the incision steps associated with mismatch repair and that ATP hydrolysis by MutL is required for a step in the mismatch repair pathway subsequent to the loading of UvrD and may serve to regulate helicase loading.
Robertson
Adam B
AB
Department of Biology, University of North Carolina at Chapel Hill, NC 27599, USA.
Pattishall
Steven R
SR
Gibbons
Erin A
EA
Matson
Steven W
SW
D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D019943:Amino Acid Substitution; D020137:Base Pair Mismatch / Q000235:genetics*; D002151:Calorimetry; D004265:DNA Helicases / Q000235:genetics / Q000378:metabolism*; D019175:DNA Methylation; D004260:DNA Repair / Q000235:genetics*; D004269:DNA, Bacterial / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D005838:Genotype; D007700:Kinetics; D000070956:MutL Proteins; D009154:Mutation; D010957:Plasmids; D015183:Restriction Mapping
D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; C074770:MutL protein, E coli; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; C506746:UvrD protein, E coli; D000070956:MutL Proteins; D004265:DNA Helicases