Kinetic analysis of mutant T. aquaticus DNA mismatch repair protein MutS

DNA mismatch repair (MMR) is a highly conserved pathway responsible for recognizing and correcting post‐replicative errors, such as base‐base mismatches and small insertion deletion loops (IDLs) in nascent DNA, and is critical for maintaining genomic stability. MutS is the protein responsible for recognizing these errors, and tightly couples its DNA binding and ATPase activity to signal downstream events that result in excision of the error and resynthesis of the strand with the correct nucleotide. Mutations in a variety of mismatch repair genes, including MutS, that compromise MMR function in vivo lead to a mutator phenotype characterized by carcinogenesis, most notably Hereditary Non‐Polyposis Colorectal Cancer (HNPCC). Here, we have begun to investigate three HNPCC‐associated mutations in Thermus aquaticus MutS. Thus far, three T. aquaticus MutS mutants have been purified (G153R, R286Q, and H696D) and their ATPase and mismatch/IDL binding activities are being measured in vitro . Initial steady‐state ATPase data measured using the Malachite Green assay indicate that the G153R mutant, located in the connector domain, has the same ATPase rate as wild‐type MutS (k cat = 0.2 s −1 ), whereas the R286Q (Lever domain) and H696D (ATPase domain) have a reduced rate (k cat = 0.1s −1 ). 2‐Aminopurine fluorescence‐based DNA binding experiments indicate that R286Q binds +T DNA with a similar affinity to wild type (K D = 17s −1 ), while G153R shows slightly tighter binding (K D = 3s −1 ), and H696D demonstrates weaker binding (K D = 40s −1 ). Future pre‐steady state ATPase and DNA binding measurements are planned determine exactly which step in the MutS‐catalyzed reaction is affected by these mutations. This research is funded by Wesleyan University, the Hughes Program in the Life Sciences, and a National Science Foundation REU grant.