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J. Biol. Chem., Vol. 278, Issue 28, 26238-26248, July 11, 2003

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Biochemical Analysis of the Yeast Condensin Smc2/4 Complex

AN ATPase THAT PROMOTES KNOTTING OF CIRCULAR DNA^*

James E. Stray  and Janet E. Lindsley 

From the Department of Biochemistry, University of Utah School of Medicine, Salt Lake City, Utah 84132-3201

To better understand the contributions that the structural maintenance of chromosome proteins (SMCs) make to condensin activity, we have tested a number of biochemical, biophysical, and DNA-associated attributes of the Smc2p-Smc4p pair from budding yeast. Smc2p and Smc4p form a stable heterodimer, the "Smc2/4 complex," which upon analysis by sedimentation equilibrium appears to reversibly self-associate to form heterotetramers. Individually, neither Smc2p nor Smc4p hydrolyzes ATP; however, ATPase activity is recovered by equal molar mixing of both purified proteins. Hydrolysis activity is unaffected by the presence of DNA. Smc2/4 binds both linearized and circular plasmids, and the binding appears to be independent of adenylate nucleotide. High mole ratios of Smc2/4 to plasmid promote a geometric change in circular DNA that can be trapped as knots by type II topoisomerases but not as supercoils by a type I topoisomerase. Binding titration analyses reveal that two Smc2/4-DNA-bound states exist, one disrupted by and one resistant to salt challenge. Competition-displacement experiments show that Smc2/4-DNA-bound species formed at even high protein to DNA mole ratios remain reversible. Surprisingly, only linear and supercoiled DNA, not nicked-circular DNA, can completely displace Smc2/4 prebound to a labeled, nicked-circular DNA. To explain this geometry-dependent competition, we present two models of DNA binding by SMCs in which two DNA duplexes are captured within the inter-coil space of an Smc2/4 heterodimer. Based on these models, we propose a DNA displacement mechanism to explain how differences in geometry could affect the competitive potential of DNA.

Received for publication, March 17, 2003 , and in revised form, April 23, 2003.

^* 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. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Supported in part by National Institutes of Health Training Grant 5T32GM07464-24.

To whom correspondence should be addressed: Dept. of Biochemistry, University of Utah Shool of Medicine, 20 North, 1900 East, Salt Lake City, UT 84132-3201. E-mail: Janet.Lindsley{at}hsc.utah.edu.

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