title: Quaternary structure and cleavage specificity of a poxvirus holliday junction resolvase.
abstract: 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.
authors.affiliation: Laboratory of Viral Diseases, NIAID, NIH, Bethesda, MD 20892, USA.
authors.forename: Alonzo D
authors.initials: AD
authors.lastname: Garcia
authors.forename: Joel
authors.initials: J
authors.lastname: Otero
authors.forename: Jacob
authors.initials: J
authors.lastname: Lebowitz
authors.forename: Peter
authors.initials: P
authors.lastname: Schuck
authors.forename: Bernard
authors.initials: B
authors.lastname: Moss
mesh_terms: D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D017931:DNA Primers / Q000737:chemistry; D045566:DNA, Cruciform / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019281:Dimerization; D045565:Holliday Junction Resolvases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D058619:Maltose-Binding Proteins; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D011212:Poxviridae / Q000201:enzymology*; D020836:Protein Structure, Quaternary; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
chemical_list: D002352:Carrier Proteins; D017931:DNA Primers; D045566:DNA, Cruciform; D058619:Maltose-Binding Proteins; D011993:Recombinant Fusion Proteins; D045565:Holliday Junction Resolvases