Document Type

Article

Publication Date

3-2-2007

Journal or Book Title

Journal of Molecular Biology

Volume

366

Issue

4

Abstract

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 (k(ON)=3 x 10(6) M(-1) s(-1)) and forms a stable complex with a half-life of 10 s (k(OFF)=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 (k(OFF)=0.1-0.14 s(-1)), although MutS(ATP) 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 (k(ON)=7.3 x 10(6) M(-1) s(-1)) and also dissociates rapidly, with a half-life of 0.4 s (k(OFF)=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.

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