Nucleic Acids Research Advance Access published online on July 9, 2008
Nucleic Acids Research, doi:10.1093/nar/gkn418
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Nucleic Acid Enzymes |
Dissection of reverse gyrase activities: insight into the evolution of a thermostable molecular machine
Institute of Protein Biochemistry, Consiglio Nazionale delle Ricerche, Via P. Castellino 111, 80131 Naples, Italy
*To whom correspondence should be addressed. Tel: +39 081 613 2247; Fax: +39 081 613 2248; Email: m.ciaramella{at}ibp.cnr.it
Received May 12, 2008. Revised June 12, 2008. Accepted June 16, 2008.
Reverse gyrase is a peculiar DNA topoisomerase, specific of thermophilic microorganisms, which induces positive supercoiling into DNA molecules in an ATP-dependent reaction. It is a modular enzyme and comprises an N-terminal helicase-like module fused to a C-terminal topoisomerase IA-like domain. The exact molecular mechanism of this unique reaction is not understood, and a fundamental mechanistic question is how its distinct steps are coordinated. We studied the cross-talk between the components of this molecular motor and probed communication between the DNA-binding sites and the different activities (DNA relaxation, ATP hydrolysis and positive supercoiling). We show that the isolated ATPase and topoisomerase domains of reverse gyrase form specific physical interactions, retain their own DNA binding and enzymatic activities, and when combined cooperate to achieve the unique ATP-dependent positive supercoiling activity. Our results indicate a mutual effect of both domains on all individual steps of the reaction. The C-terminal domain shows ATP-independent topoisomerase activity, which is repressed by the N-terminal domain in the full-length enzyme; experiments with the isolated domains showed that the C-terminal domain has stimulatory influence on the ATPase activity of the N-terminal domain. In addition, the two domains showed a striking reciprocal thermostabilization effect.
This paper is dedicated to the memory of our colleague and dear friend Elio Parisi. The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors.