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J Biol Chem, Vol. 275, Issue 4, 2613-2618, January 28, 2000

Steady-state and Rapid Kinetic Analysis of Topoisomerase II Trapped as the Closed-clamp Intermediate by ICRF-193*

Shayne K. MorrisDagger , Cheryl L. BairdDagger , and Janet E. Lindsley§

From the Department of Biochemistry, University of Utah School of Medicine, Salt Lake City, Utah 84132

DNA topoisomerase II uses a complex, sequential mechanism of ATP hydrolysis to catalyze the transport of one DNA duplex through a transient break in another. ICRF-193 is a catalytic inhibitor of topoisomerase II that is known to trap a closed-clamp intermediate form of the enzyme. Using steady-state and rapid kinetic ATPase and DNA transport assays, we have analyzed how trapping this intermediate by the drug perturbs the topoisomerase II mechanism. The drug has no effect on the rate of the first turnover of decatenation but potently inhibits subsequent turnovers with an IC50 of 6.5 ± 1 µM for the Saccharomyces cerevisiae enzyme. This drug inhibits the ATPase activity of topoisomerase II by an unusual, mixed-type mechanism; the drug is not a competitive inhibitor of ATP, and even at saturating concentrations of drug, the enzyme continues to hydrolyze ATP, albeit at a reduced rate. Topoisomerase II that was specifically isolated in the drug-bound, closed-clamp form continues to hydrolyze ATP, indicating that the enzyme clamp does not need to re-open to bind and hydrolyze ATP. When rapid-quench ATPase assays were initiated by the addition of ATP, the drug had no effect on the sequential hydrolysis of either the first or second ATP. By contrast, when the drug was prebound, the enzyme hydrolyzed one labeled ATP at the uninhibited rate but did not hydrolyze a second ATP. These results are interpreted in terms of the catalytic mechanism for topoisomerase II and suggest that ICRF-193 interacts with the enzyme bound to one ADP.

* This work was supported by National Institutes of Health Grant GM51194.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Supported in part by National Institutes of Health Training Grant 5T32GM08753 and the Huntsman Cancer Institute.

§ Supported in part by American Chemical Society Grant JFRA-622. To whom correspondence should be addressed: Dept. of Biochemistry, University of Utah School of Medicine, 50 N. Medical Dr., Salt Lake City, UT 84132. Tel.: 801-581-2797; Fax: 801-581-7959; E-mail: Janet.Lindsley@hsc.utah.edu.

Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
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