title: Structure and Metal Binding Properties of a Poxvirus Resolvase.
abstract: Poxviruses replicate their linear genomes by forming concatemers that must be resolved into monomeric units to produce new virions. A viral resolvase cleaves DNA four-way junctions extruded at the concatemer junctions to produce monomeric genomes. This cleavage reaction is required for viral replication, so the resolvase is an attractive target for small molecule inhibitors. To provide a platform for understanding resolvase mechanism and designing inhibitors, we have determined the crystal structure of the canarypox virus (CPV) resolvase. CPV resolvase is dimer of RNase H superfamily domains related to Escherichia coli RuvC, with an active site lined by highly conserved acidic residues that bind metal ions. There are several intriguing structural differences between resolvase and RuvC, and a model of the CPV resolvase·Holliday junction complex provides insights into the consequences of these differences, including a plausible explanation for the weak sequence specificity exhibited by the poxvirus enzymes. The model also explains why the poxvirus resolvases are more promiscuous than RuvC, cleaving a variety of branched, bulged, and flap-containing substrates. Based on the unique active site structure observed for CPV resolvase, we have carried out a series of experiments to test divalent ion usage and preferences. We find that the two resolvase metal binding sites have different preferences for Mg(2+) versus Mn(2+) Optimal resolvase activity is maintained with 5 μm Mn(2+) and 100 μm Mg(2+), concentrations that are well below those required for either metal alone. Together, our findings provide biochemical insights and structural models that will facilitate studying poxvirus replication and the search for efficient poxvirus inhibitors.
authors.affiliation: From the Department of Biochemistry & Biophysics, the Graduate Group in Biochemistry and Molecular Biophysics, and.
authors.forename: Huiguang
authors.initials: H
authors.lastname: Li
authors.affiliation: the Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104.
authors.forename: Young
authors.initials: Y
authors.lastname: Hwang
authors.affiliation: the Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York 14850, and the Argonne National Laboratory, Argonne, Illinois 60439.
authors.forename: Kay
authors.initials: K
authors.lastname: Perry
authors.affiliation: the Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, bushman@mail.med.upenn.edu.
authors.forename: Frederic
authors.initials: F
authors.lastname: Bushman
authors.affiliation: From the Department of Biochemistry & Biophysics, vanduyne@mail.med.upenn.edu.
authors.forename: Gregory D
authors.initials: GD
authors.lastname: Van Duyne
mesh_terms: D028121:Canarypox virus / Q000201:enzymology*; D020134:Catalytic Domain / Q000235:genetics; D018360:Crystallography, X-Ray; D004706:Endodeoxyribonucleases / Q000737:chemistry / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism; D045565:Holliday Junction Resolvases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008274:Magnesium / Q000378:metabolism; D008345:Manganese / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D020836:Protein Structure, Quaternary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D040681:Structural Homology, Protein; D013379:Substrate Specificity; D013816:Thermodynamics; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; D014764:Viral Proteins; C069806:ruvC protein, E coli; D008345:Manganese; D004706:Endodeoxyribonucleases; D045565:Holliday Junction Resolvases; D008274:Magnesium
keywords: Holliday junction; enzyme structure; metalloenzyme; poxvirus; resolvase; viral replication