DNA cleavage by the A22R resolvase of vaccinia virus.
Vaccinia virus encodes an enzyme, A22R, required during DNA replication for cleaving viral DNA concatamers to yield unit-length viral genomes. The concatamer junctions contain inverted repeat sequences that can be extruded as cruciforms, yielding Holliday junctions. Previous work indicated that A22R can cleave Holliday junctions in vitro. To investigate the mechanism of action of A22R, we have optimized reaction conditions and characterized the sequence specificity of cleavage. We found that addition of 20% dimethylsulfoxide boosted product formation six-fold, resulting in improved sensitivity of cleavage assays. To analyze cleavage specificity, we took advantage of mobile Holliday junctions, in which branch migration allowed sampling of many DNA sequences. We found that A22R weakly favors cleavage at the sequence 5'-(G/C) downward arrow(A/T)-3', and so is much less sequence specific than its Escherichia coli relative, RuvC. Analysis of the reaction products revealed that A22R cleaves to leave a 3' hydroxyl at the cleaved phosphodiester bond.
Culyba
Matthew J
MJ
Department of Microbiology, University of Pennsylvania School of Medicine, 3610 Hamilton Walk, Philadelphia, PA 19104-6076, USA.
Harrison
Jessamina E
JE
Hwang
Young
Y
Bushman
Frederic D
FD
D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002413:Cations, Divalent / Q000378:metabolism; D045566:DNA, Cruciform / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004279:DNA, Viral / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D005814:Genes, Viral; D045565:Holliday Junction Resolvases / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012965:Sodium Chloride; D014616:Vaccinia virus / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*
D002413:Cations, Divalent; D045566:DNA, Cruciform; D004279:DNA, Viral; D011994:Recombinant Proteins; D012965:Sodium Chloride; D045565:Holliday Junction Resolvases