MutS functions in mismatch repair (MMR) to scan DNA for errors, identify a target site and trigger subsequent events in the pathway leading to error removal and DNA re-synthesis. These actions, enabled by the ATPase activity of MutS, are now beginning to be analyzed from the perspective of the protein itself. This study provides the first ensemble transient kinetic data on MutS conformational dynamics as it works with DNA and ATP in MMR. Using a combination of fluorescence probes (on T. aquaticus MutS and DNA) and signals (intensity, anisotropy and resonance energy transfer), we have monitored the timing of key conformational changes in MutS that are coupled to mismatch binding and recognition, ATP binding and hydrolysis, as well as sliding clamp formation and signaling of repair. Significant findings include: (a) a slow step that follows weak initial interaction between MutS and DNA, in which concerted conformational changes in both macromolecules control mismatch recognition, (b) rapid, binary switching of MutS conformations that is concerted with ATP binding and hydrolysis, and (c) is stalled after mismatch recognition to control formation of the ATP-bound MutS sliding clamp. These rate-limiting pre- and post-mismatch recognition events outline the mechanism of action of MutS on DNA during initiation of MMR.

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Transient kinetic analysis is indispensable for understanding the workings of biological macromolecules, since this approach yields mechanistic information including active site concentrations and intrinsic rate constants that govern macromolecular function. In case of enzymes, for example, transient or pre-steady state measurements identify and characterize individual events in the reaction pathway, whereas steady state measurements only yield overall catalytic efficiency and specificity. Individual events such as protein-protein or protein-ligand interactions and rate-limiting conformational changes often occur in the millisecond timescale, and can be measured directly by stopped-flow and chemical-quench flow methods. Given an optical signal such as fluorescence, stopped-flow serves as a powerful and accessible tool for monitoring reaction progress from substrate binding to product release and catalytic turnover 1,2 .Here, we report application of stopped-flow kinetics to probe the mechanism of action of Msh2-Msh6, a eukaryotic DNA repair protein that recognizes base-pair mismatches and insertion/deletion loops in DNA and signals mismatch repair (MMR) [3][4][5] . In doing so, Msh2-Msh6 increases the accuracy of DNA replication by three orders of magnitude (error frequency decreases from~10 -6 to10 -9 bases), and thus helps preserve genomic integrity. Not surprisingly, defective human Msh2-Msh6 function is associated with hereditary non-polyposis colon cancer and other sporadic cancers [6][7][8] . In order to understand the mechanism of action of this critical DNA metabolic protein, we are probing the dynamics of Msh2-Msh6 interaction with mismatched DNA as well as the ATPase activity that fuels its actions in MMR. DNA binding is measured by rapidly mixing Msh2-Msh6 with DNA containing a 2-aminopurine (2-Ap) fluorophore adjacent to a G:T mismatch and monitoring the resulting increase in 2-aminopurine fluorescence in real time. Preparation of reagents for a fluorescence-based kinetic DNA binding experiment on a stopped-flow is similar to that for an equilibrium experiment on a fluorometer. Indeed equilibrium binding analysis should be performed first to estimate the dissociation constant (KD) for the interaction in order to optimize reaction conditions for kinetic analysis. Stopped-flow experiments require larger quantities of biological materials compared with equilibrium or steady-state experiments; therefore, the approach is most feasible when low milligram amounts of protein are available 11,12 and similar amounts of ligands can be prepared or purchased.

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DNA mismatch repair (MMR) is an evolutionarily conserved process that locates and repairs post‐replicative errors in nascent DNA and is critical for maintaining the genome integrity. The first step in the MMR pathway is recognition of the base‐base mismatch/insertion deletion loop (IDL) by MutS in prokaryotes and the MutS homologues, Msh2‐Msh6 and Msh2‐Msh3, in eukaryotes. While there is a significant body of literature concerning DNA‐binding and ATPase activities of MutS, information on MutS conformational changes involved in recognizing an array of non‐Watson‐Crick DNA structures is lacking. Here we present two fluorescence reporter assays developed to examine T. aquaticus MutS conformational changes and better understand the relationship between its structure/dynamics and function. Two mutant proteins were prepared: 1) F39, which contacts DNA, was replaced with tryptophan to monitor intrinsic protein fluorescence during the reaction; 2) M88 in the DNA binding domain was replaced by cysteine and labeled with a thiol reactive fluorophore that is sensitive to protein conformation. We are currently measuring changes in T. aquaticus MutS conformation on mismatch/IDL binding in equilibrium and in real time by transient kinetic analysis.