The Effects of Nucleotides on MutS-DNA Binding Kinetics Clarify the Role of MutS ATPase Activity in Mismatch Repair

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MutS protein initiates mismatch repair with recognition of a non-Watson–Crick base-pair or base insertion/deletion site in DNA, and its interactions with DNA are modulated by ATPase activity. Here, we present a kinetic analysis of these interactions, including the effects of ATP binding and hydrolysis, reported directly from the mismatch site by 2-aminopurine fluorescence. When free of nucleotides, the Thermus aquaticus MutS dimer binds a mismatch rapidly (kON=3×106 M−1 s−1) and forms a stable complex with a half-life of 10 s (kOFF=0.07 s−1). When one or both nucleotide-binding sites on the MutS•mismatch complex are occupied by ATP, the complex remains fairly stable, with a half-life of 5–7 s (kOFF=0.1–0.14 s−1), although MutSATP becomes incapable of (re-)binding the mismatch. When one or both nucleotide-binding sites on the MutS dimer are occupied by ADP, the MutS•mismatch complex forms rapidly (kON=7.3×106 M−1 s−1) and also dissociates rapidly, with a half-life of 0.4 s (kOFF=1.7 s−1). Integration of these MutS DNA-binding kinetics with previously described ATPase kinetics reveals that: (a) in the absence of a mismatch, MutS in the ADP-bound form engages in highly dynamic interactions with DNA, perhaps probing base-pairs for errors; (b) in the presence of a mismatch, MutS stabilized in the ATP-bound form releases the mismatch slowly, perhaps allowing for onsite interactions with downstream repair proteins; (c) ATP-bound MutS then moves off the mismatch, perhaps as a mobile clamp facilitating repair reactions at distant sites on DNA, until ATP is hydrolyzed (or dissociates) and the protein turns over.