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J. Biol. Chem., Vol. 278, Issue 36, 34667-34673, September 5, 2003

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Assembly and Molecular Activities of the MutS Tetramer^*

Keith P. Bjornson, Leonard J. Blackwell , Harvey Sage, Celia Baitinger , Dwayne Allen  and Paul Modrich  ¶

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

Analytical equilibrium ultracentrifugation indicates that Escherichia coli MutS exists as an equilibrating mixture of dimers and tetramers. The association constant for the dimer-to-tetramer transition is 2.1 x 10⁷ M ^–1, indicating that the protein would consist of both dimers and tetramers at physiological concentrations. The carboxyl terminus of MutS is required for tetramer assembly because a previously described 53-amino acid carboxyl-terminal truncation (MutS800) forms a limiting species of a dimer (Obmolova, G., Ban, C., Hsieh, P., and Yang, W. (2000) Nature 407, 703–710; Lamers, M. H., Perrakis, A., Enzlin, J. H., Winterwerp, H. H., de Wind, N., and Sixma, T. K. (2000) Nature 407, 711–717). MutS800 binds a 20-base pair heteroduplex an order of magnitude more weakly than full-length MutS, and at saturating protein concentrations, the heteroduplex-bound mass observed with MutS800 is only half that observed with the full length protein, indicating that the subunit copy number of heteroduplex-bound MutS is twice that of MutS800. Analytical equilibrium ultracentrifugation using a fluorescein-tagged 20-base pair heteroduplex demonstrated that native MutS forms a tetramer on this single site-sized heteroduplex DNA. Equilibrium fluorescence experiments indicated that dimer-to-tetramer assembly promotes mismatch binding by MutS and that the tetramer can bind only a single heteroduplex molecule, implying nonequivalence of the two dimers within the tetramer. Compared with native MutS, the ability of MutS800 to promote MutL-dependent activation of MutH is substantially reduced.

Received for publication, May 27, 2003

^* This work was supported by Grant GM23719 from 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.

Current address: Amphora Discovery Corp., 800-4 Capitola Dr., Durham, NC 27713.

^¶ Investigator of the Howard Hughes Medical Institute. To whom correspondence should be addressed: Box 3711, Rm. 150 MS1A. Tel.: 919-684-2775; Fax: 919-681-7874; E-mail: modrich{at}biochem.duke.edu.

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