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J. Biol. Chem., Vol. 281, Issue 33, 23395-23404, August 18, 2006

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Interdomain Communication in DNA Topoisomerase II

DNA BINDING AND ENZYME ACTIVATION^*

From the Department of Biochemistry, School of Medicine, Stanford University, Stanford, California 94305-5307

Topoisomerase II catalyzes the ATP-dependent transport of a DNA segment (T-DNA) through a transient double strand break in another DNA segment (G-DNA). A fundamental mechanistic question is how the individual steps in this process are coordinated. We probed communication between the DNA binding sites and the individual enzymatic activities, ATP hydrolysis, and DNA cleavage. We employed short DNA duplexes to control occupancy at the two binding sites of wild-type enzyme and a variant with a G-DNA site mutation. The DNA concentration dependence of ATP hydrolysis and a fluorescence anisotropy assay provided thermodynamic information about DNA binding. The results suggest that G-DNA binds with higher affinity than T-DNA. Enzyme with only G-DNA bound is competent to cleave DNA, indicating that T-DNA is dispensable for DNA cleavage. The ATPase activity of enzyme bound solely to G-DNA is partially stimulated. Full stimulation requires binding of T-DNA. Both DNA binding sites therefore signal to the ATPase domains. The results support and extend current mechanistic models for topoisomerase II-catalyzed DNA transport and provide a framework for future mechanistic dissection.

Received for publication, May 1, 2006 , and in revised form, June 16, 2006.

^* This work was supported by Boehringer Ingelheim Fonds (to F. M.-P.) and by National Institutes of Health Grant GM64798 (to D. H.). 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 supplemental Schemes 1–4, Figs. S1–S7, and Table S1.

¹ To whom correspondence should be addressed: Stanford University, School of Medicine, Beckman Center B400, Dept. of Biochemistry, 279 Campus Dr., Stanford, CA 94305. Tel.: 650-723-9442; Fax: 650-723-6783; E-mail: herschla{at}cmgm.stanford.edu.

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