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J. Biol. Chem., Vol. 282, Issue 48, 34644-34652, November 30, 2007

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DNA Branch Nuclease Activity of Vaccinia A22 Resolvase^*

From the Department of Microbiology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104

DNA replication, recombination, and repair can result in formation of diverse branched DNA structures. Many large DNA viruses are known to encode DNA branch nucleases, but several of the expected activities have not previously been found among poxvirus enzymes. Vaccinia encodes an enzyme, A22 resolvase, which is known to be active on four-stranded DNA junctions (Holliday junctions) or Holliday junction-like structures containing three of the four strands. Here we report that A22 resolvase in fact has a much wider substrate specificity than previously appreciated. A22 resolvase cleaves Y-junctions, single-stranded DNA flaps, transitions from double strands to unpaired single strands ("splayed duplexes"), and DNA bulges in vitro. We also report site-directed mutagenesis studies of candidate active site residues. The results identify the likely active site and support a model in which a single active site is responsible for cleavage on Holliday junctions and splayed duplexes. Lastly, we describe possible roles for the A22 resolvase DNA-branch nuclease activity in DNA replication and repair.

Received for publication, June 28, 2007 , and in revised form, September 7, 2007.

^* This work was supported in part by Grant U54 AI057168 from the NIAID, National Institutes of Health Mid-Atlantic Regional Center of Excellence for Biodefense Research. 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 Table S1.

¹ Supported by National Institutes of Health Training Grant T32 AI 07324.

² To whom correspondence should be addressed: Dept. of Microbiology, University of Pennsylvania School of Medicine, 402C Johnson Pavilion, 3610 Hamilton Walk, Philadelphia, PA 19104-6076. Tel.: 215-573-8732; Fax: 215-573-4856; E-mail: bushman{at}mail.med.upenn.edu.

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