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J. Biol. Chem., Vol. 281, Issue 17, 11618-11626, April 28, 2006

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Quaternary Structure and Cleavage Specificity of a Poxvirus Holliday Junction Resolvase^*

Alonzo D. Garcia¹, Joel Otero², Jacob Lebowitz, Peter Schuck, and Bernard Moss³

From the Laboratory of Viral Diseases, NIAID and the Division of Bioengineering and Physical Science, Office of Research Services, National Institutes of Health, Bethesda, Maryland 20892

Recently, poxviruses were found to encode a protein with signature motifs present in the RuvC family of Holliday junction (HJ) resolvases, which have a key role in homologous recombination in bacteria. The vaccinia virus homolog A22 specifically cleaved synthetic HJ DNA in vitro and was required for the in vivo resolution of viral DNA concatemers into unit-length genomes with hairpin telomeres. It was of interest to further characterize a poxvirus resolvase in view of the low sequence similarity with RuvC, the absence of virus-encoded RuvA and RuvB to interact with, and the different functions of the viral and bacterial resolvases. Because purified A22 aggregated severely, studies were carried out with maltose-binding protein fused to A22 as well as to RuvC. Using gel filtration, chemical cross-linking, analytical ultracentrifugation, and light scattering, we demonstrated that A22 and RuvC are homodimers in solution. Furthermore, the dimeric form of the resolvase associated with HJ DNA, presumably facilitating the symmetrical cleavage of such structures. Like RuvC, A22 symmetrically cleaved fixed HJ junctions as well as junctions allowing strand mobility. Unlike RuvC and other members of the family, however, the poxvirus enzyme exhibited little cleavage sequence specificity. Structural and enzymatic similarities of poxvirus, bacterial, and fungal mitochondrial HJ resolvases are consistent with their predicted evolutionary relationship based on sequence analysis. The absence of a homologous resolvase in mammalian cells makes these microbial enzymes excellent potential therapeutic targets.

Received for publication, January 9, 2006 , and in revised form, February 21, 2006.

^* This research was supported in part by the Intramural Research Program of NIAID, National Institutes of Health. 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 Present address: Laboratory of DNA Viruses, Division of Viral Products, HFM-457, Center for Biologics Evaluation and Research, Food and Drug Administration, 1401 Rockville Pike, Rockville, Bethesda, MD 20892.

² Present address: Cell and Molecular Biology Program, Baylor College of Medicine, One Baylor Plaza, Rm. N1130, Houston, TX 77030.

³ To whom correspondence should be addressed. Tel.: 301-496-9869; Fax: 301-480-1147; E-mail: bmoss{at}nih.gov.

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