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J. Biol. Chem., Vol. 283, Issue 25, 17463-17476, June 20, 2008

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Coupling between ATP Binding and DNA Cleavage by DNA Topoisomerase II

A UNIFYING KINETIC AND STRUCTURAL MECHANISM^*

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

DNA topoisomerase II is a molecular machine that couples ATP hydrolysis to the transport of one DNA segment through a transient break in another segment. To learn about the energetic connectivity that underlies this coupling, we investigated how the ATPase domains exert control over DNA cleavage. We dissected the DNA cleavage reaction by measuring rate and equilibrium constants for the individual reaction steps utilizing defined DNA duplexes in the presence and absence of the nonhydrolyzable ATP analog 5'-adenylyl-β,-imidodiphosphate (AMPPNP). Our results revealed the existence of two enzyme conformations whose relative abundance is sensitive to the presence of nucleotides. The predominant species in the absence of nucleotides binds DNA at a diffusion limited rate but cannot efficiently cleave DNA. In the presence of AMPPNP, most of the enzyme is converted to a state in which DNA binding and release is extremely slow but which allows DNA cleavage. A minimal kinetic and thermodynamic framework is established that accounts for the cooperativity of cleavage of the two DNA strands in the presence and absence of bound AMPPNP and includes conformational steps revealed in the kinetic studies. The model unifies available kinetic, thermodynamic, and structural data to provide a description for the reaction in terms of the order and rate of individual reaction steps and the physical nature of the species on the reaction path. Furthermore, this reaction framework provides a foundation for a future in-depth analysis of energy transduction by topoisomerase II, for guiding and interpreting future structural studies, and for analyzing the mechanism of drugs that convert topoisomerase into a cellular poison.

Received for publication, December 7, 2007 , and in revised form, March 10, 2008.

^* This work was supported, in whole or in part, 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 "Experimental Procedures," "Results," additional references, Table S1, Schemes S1–S8, and Figs. S1–S8.

¹ Supported in part by a fellowship from the Boehringer Ingelheim Fonds. Present address: Adolf-Butenandt-Institut, Molekularbiologie, Ludwig-Maximilians-Universität, 80336 München, Germany.

² To whom correspondence should be addressed: Dept. of Biochemistry, Stanford University, School of Medicine, Beckman Center B400, 279 Campus Dr., Stanford, CA 94305. Tel.: 650-723-9442; Fax: 650-723-6783; E-mail: herschla{at}stanford.edu.

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