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J. Biol. Chem., Vol. 279, Issue 27, 28402-28410, July 2, 2004

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Differential Specificities and Simultaneous Occupancy of Human MutS Nucleotide Binding Sites^*

Diana Martik, Celia Baitinger, and Paul Modrich, An Investigator of the Howard Hughes Medical Institute¶

From the Department of Biochemistry and Howard Hughes Medical Institute, Duke University Medical Center, Durham, North Carolina 27710

We have examined the permissible nucleotide occupancy states of human MutS. The MSH2·MSH6 heterodimer binds 1 mol of ADP and 1 mol of adenosine 5'-O-(thiotriphosphate) (ATPS), with a K_d for each nucleotide of about 1 µM. Anisotropy measurements using BODIPY TR and BODIPY FL fluorescent derivatives of ADP and 5'-adenylyl-,-imidodiphosphate (AMPPNP) also indicate an interaction stoichiometry of 1 mol of ADP and 1 mol of triphosphate analogue per MutS heterodimer. Di- and triphosphate sites can be simultaneously occupied as judged by sequential filling of the two binding site classes with differentially radiolabeled ADP and ATPS and by fluorescence resonance energy transfer between BODIPY TR- and BODIPY FL-labeled ADP and AMPPNP. ATP hydrolysis by MutS is accompanied by a pre-steady-state burst of ADP formation, and analysis of MutS-bound nucleotide during the first turnover has demonstrated the presence of both ADP and ATP. Simultaneous presence of ADP and a nonhydrolyzable ATP analogue modulates MutS·heteroduplex interaction in a manner that is distinct from that observed in the presence of ADP or nonhydrolyzable triphosphate alone, and it is unlikely that this effect is due to the presence of a mixed population of binary complexes between MutS and ADP or a triphosphate analogue. These findings imply that MutS has two nucleotide binding sites with differential specificities for ADP and ATP and suggest that the ADP·MutS·ATP ternary complex has an important role in mismatch repair.

Received for publication, November 5, 2003 , and in revised form, April 5, 2004.

^* This work was supported in part by Grant GM45190 from the NIGMS, National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed: Dept. of Biochemistry and Howard Hughes Medical Institute, Box 3711, Duke University Medical Center, Durham, NC 27710. Tel.: 919-684-2775; Fax: 919-681-7874; E-mail: modrich{at}biochem.duke.edu.

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