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J. Biol. Chem., Vol. 281, Issue 9, 6030-6037, March 3, 2006

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Crystal Structure of a Bacterial Type IB DNA Topoisomerase Reveals a Preassembled Active Site in the Absence of DNA^*

Asmita Patel, Stewart Shuman¹, and Alfonso Mondragón²

From the Department of Biochemistry, Molecular and Cell Biology, Northwestern University, Evanston, Illinois 60208 and Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10021

Type IB DNA topoisomerases are found in all eukarya, two families of eukaryotic viruses (poxviruses and mimivirus), and many genera of bacteria. They alter DNA topology by cleaving and resealing one strand of duplex DNA via a covalent DNA-(3-phosphotyrosyl)-enzyme intermediate. Bacterial type IB enzymes were discovered recently and are described as poxvirus-like with respect to their small size, primary structures, and bipartite domain organization. Here we report the 1.75-Å crystal structure of Deinococcus radiodurans topoisomerase IB (DraTopIB), a prototype of the bacterial clade. DraTopIB consists of an amino-terminal (N) -sheet domain (amino acids 1–90) and a predominantly -helical carboxyl-terminal (C) domain (amino acids 91–346) that closely resemble the corresponding domains of vaccinia virus topoisomerase IB. The five amino acids of DraTopIB that comprise the catalytic pentad (Arg-137, Lys-174, Arg-239, Asn-280, and Tyr-289) are preassembled into the active site in the absence of DNA in a manner nearly identical to the pentad configuration in human topoisomerase I bound to DNA. This contrasts with the apoenzyme of vaccinia topoisomerase, in which three of the active site constituents are either displaced or disordered. The N and C domains of DraTopIB are splayed apart in an "open" conformation, in which the surface of the catalytic domain containing the active site is exposed for DNA binding. A comparison with the human topoisomerase I-DNA cocrystal structure suggests how viral and bacterial topoisomerase IB enzymes might bind DNA circumferentially via movement of the N domain into the major groove and clamping of a disordered loop of the C domain around the helix.

Received for publication, November 16, 2005

The atomic coordinates and structure factors (code 2F4Q) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This work was supported by National Institutes of Health Grants GM51350 (to A. M.) and GM46330 (to S. S.). 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.

¹ An American Cancer Society research professor.

² To whom correspondence should be addressed: Dept. of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, 2205 Tech Dr., Evanston, IL 60208. Tel.: 847-491-7726; Fax: 847-467-6489; E-mail: a-mondragon{at}northwestern.edu.

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