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Nucleic Acids Research 2005 33(17):5415-5425; doi:10.1093/nar/gki846
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Published online 28 September 2005

© The Author 2005. Published by Oxford University Press. All rights reserved
The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact journals.permissions{at}oxfordjournals.org

Article

Bacillus cereus DNA topoisomerase I and III{alpha}: purification, characterization and complementation of Escherichia coli TopoIII activity

Zhiyu Li, Hiroshi Hiasa1 and Russell DiGate*

Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland Baltimore, MA, USA 1Department of Pharmacology, University of Minnesota Medical School MN, USA

*To whom correspondence should be addressed. Tel: +1 215 596 8805; Fax: +1 215 596 8977; Email: r.digate{at}usip.edu

Received June 1, 2005. Revised September 1, 2005. Accepted September 1, 2005.

The Bacillus cereus genome possesses three type IA topoisomerase genes. These genes, encoding DNA topoisomerase I and III{alpha} (bcTopo I, bcTopo III{alpha}), have been cloned into T7 RNA polymerase-regulated plasmid expression vectors and the enzymes have been overexpressed, purified and characterized. The proteins exhibit similar biochemical activity to their Escherichia coli counterparts, DNA topoisomerase I and III (ecTopo I, ecTopo III). bcTopo I is capable of efficiently relaxing negatively supercoiled DNA in the presence of Mg2+ but does not possess an efficient DNA decatenation activity. bcTopo III{alpha} is an active topoisomerase that is capable of relaxing supercoiled DNA at a broad range of Mg2+ concentrations; however, its DNA relaxation activity is not as efficient as that of bcTopo I. In addition, bcTopo III is a potent DNA decatenase that resolves oriC-based plasmid replication intermediates in vitro. Interestingly, bcTopo I and bcTopo III{alpha} are both able to compensate for the loss of ecTopo III in E.coli cells that lack ecTopo I. In contrast, ecTopo I cannot substitute for ecTopo III under these conditions.

Present address: Russell DiGate, University of the Sciences in Philadelphia, 600 South 43rd Street, Philadelphia, PA 19104-4495, USA


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