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J. Biol. Chem., Vol. 280, Issue 23, 22245-22257, June 10, 2005

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Analysis of the Interaction between the Saccharomyces cerevisiae MSH2-MSH6 and MLH1-PMS1 Complexes with DNA Using a Reversible DNA End-blocking System^*

Marc L. Mendillo, Dan J. Mazur, and Richard D. Kolodner

From the Ludwig Institute for Cancer Research, Departments of Medicine and Cellular and Molecular Medicine, and Cancer Center, University of California, San Diego School of Medicine, La Jolla, California 92093-0669

The Lac repressor-operator interaction was used as a reversible DNA end-blocking system in conjunction with an IAsys biosensor instrument (Thermo Affinity Sensors), which detects total internal reflectance and allows monitoring of binding and dissociation in real time, in order to develop a system for studying the ability of mismatch repair proteins to move along the DNA. The MSH2-MSH6 complex bound to a mispaired base was found to be converted by ATP binding to a form that showed rapid sliding along the DNA and dissociation via the DNA ends and also showed slow, direct dissociation from the DNA. In contrast, the MSH2-MSH6 complex bound to a base pair containing DNA only showed direct dissociation from the DNA. The MLH1-PMS1 complex formed both mispair-dependent and mispair-independent ternary complexes with the MSH2-MSH6 complex on DNA. The mispair-independent ternary complexes were formed most efficiently on DNA molecules with free ends under conditions where ATP hydrolysis did not occur, and only exhibited direct dissociation from the DNA. The mispair-dependent ternary complexes were formed in the highest yield on DNA molecules with blocked ends, required ATP and magnesium for formation, and showed both dissociation via the DNA ends and direct dissociation from the DNA.

Received for publication, July 7, 2004 , and in revised form, March 29, 2005.

^* This work was supported in part by National Institutes of Health Grants GM50006 and CA92584 (to R. D. K.). 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.

The on-line version of this article (available at http://www.jbc.org) contains Fig. S1.

Recipient of a postdoctoral fellowship from the American Cancer Society.

To whom correspondence should be addressed: Ludwig Institute for Cancer Research, Depts. of Medicine and Cellular and Molecular Medicine, and Cancer Center, University of California San Diego School of Medicine, 9500 Gilman Dr., La Jolla, CA 92093-0669. Tel.: 858-534-4920 (Ext. 7804); Fax: 858-534-7750; E-mail: rkolodner{at}ucsd.edu.

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