Metal cofactors in the structure and activity of the fowlpox resolvase.
Poxvirus DNA replication generates linear concatemers containing many copies of the viral genome with inverted repeat sequences at the junctions between monomers. The inverted repeats refold to generate Holliday junctions, which are cleaved by the virus-encoded resolvase enzyme to form unit-length genomes. Here we report studies of the influence of metal cofactors on the activity and structure of the resolvase of fowlpox virus, which provides a tractable model for in vitro studies. Small-molecule inhibitors of related enzymes bind simultaneously to metal cofactors and nearby surface amino acid residues, so understanding enzyme-cofactor interactions is important for the design of antiviral agents. Analysis of inferred active-site residues (D7, E60, K102, D132, and D135) by mutagenesis and metal rescue experiments specified residues that contribute to binding metal ions and that multiple binding sites are probably involved. Differential electrophoretic analysis was used to map the conformation of the DNA junction when bound by resolvase. For the wild-type complex in the presence of EDTA (ethylenediaminetetraacetic acid) or Ca(2+), migration was consistent with the DNA arms arranged in near-tetrahedral geometry. However, the D7N active-site mutant resolvase held the arms in a more planar arrangement in EDTA, Ca(2+), or Mg(2+) conditions, implicating metal-dependent contacts at the active site in the larger architecture of the complex. These data show how divalent metals dictate the conformation of FPV resolvase-DNA complexes and subsequent DNA cleavage.
Culyba
Matthew J
MJ
Department of Microbiology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA.
Hwang
Young
Y
Hu
Jimmy Yan
JY
Minkah
Nana
N
Ocwieja
Karen E
KE
Bushman
Frederic D
FD
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002413:Cations, Divalent; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D045566:DNA, Cruciform / Q000737:chemistry / Q000378:metabolism; D005586:Fowlpox / Q000201:enzymology* / Q000378:metabolism; D008670:Metals / Q000737:chemistry*; D008969:Molecular Sequence Data; D045522:Recombinases / Q000737:chemistry* / Q000378:metabolism; D013379:Substrate Specificity; D014764:Viral Proteins / Q000737:chemistry* / Q000378:metabolism
D002413:Cations, Divalent; D045566:DNA, Cruciform; D008670:Metals; D045522:Recombinases; D014764:Viral Proteins; D004247:DNA