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J. Biol. Chem., Vol. 281, Issue 9, 5640-5647, March 3, 2006

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Reverse Gyrase Functions as a DNA Renaturase

ANNEALING OF COMPLEMENTARY SINGLE-STRANDED CIRCLES AND POSITIVE SUPERCOILING OF A BUBBLE SUBSTRATE^*

From the Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710

Reverse gyrase is a hyperthermophile-specific enzyme that can positively supercoil DNA concomitant with ATP hydrolysis. However, the DNA supercoiling activity is inefficient and requires an excess amount of enzyme relative to DNA. We report here several activities that reverse gyrase can efficiently mediate with a substoichiometric amount of enzyme. In the presence of a nucleotide cofactor, reverse gyrase can readily relax negative supercoils, but not the positive ones, from a plasmid DNA substrate. Reverse gyrase can completely relax positively supercoiled DNA, provided that the DNA substrate contains a single-stranded bubble. Reverse gyrase efficiently anneals complementary single-stranded circles. A substoichiometric amount of reverse gyrase can insert positive supercoils into DNA with a single-stranded bubble, in contrast to plasmid DNA substrate. We have designed a novel method based on phage-mid DNA vectors to prepare a circular DNA substrate containing a single-stranded bubble with defined length and sequence. With these bubble DNA substrates, we demonstrated that efficient positive supercoiling by reverse gyrase requires a bubble size larger than 20 nucleotides. The activities of annealing single-stranded DNA circles and positive supercoiling of bubble substrate demonstrate that reverse gyrase can function as a DNA renaturase. These biochemical activities also suggest that reverse gyrase can have an important biological function in sensing and eliminating unpaired regions in the genome of a hyperthermophilic organism.

Received for publication, December 13, 2005 , and in revised form, December 27, 2005.

^* This work was supported by National Institutes of Health Grant GM29006. 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1.

¹ To whom correspondence should be addressed. Tel.: 919-684-6501; Fax: 919-684-8885; E-mail: hsieh{at}biochem.duke.edu.

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