title: DNA strand transfer catalyzed by vaccinia topoisomerase: peroxidolysis and hydroxylaminolysis of the covalent protein-DNA intermediate.
abstract: Vaccinia topoisomerase forms a covalent DNA-(3'-phosphotyrosyl)-enzyme intermediate at sites containing the sequence 5'-CCCTT downward arrow. The covalently bound topoisomerase can religate the CCCTT strand to a 5'-OH-terminated polynucleotide or else transfer the strand to a non-DNA nucleophile such a water or glycerol. Here, we report that vaccinia topoisomerase also catalyzes strand transfer to hydrogen peroxide. The observed alkaline pH-dependence of peroxidolysis is consistent with enzyme-mediated attack by peroxide anion on the covalent intermediate. The reaction displays apparent first-order kinetics. From a double-reciprocal plot of k(obs) versus [H(2)O(2)] at pH 10, we determined a rate constant for peroxidolysis of 6.3 x 10(-)(3) s(-)(1). This rate is slower by a factor of 200 than the rate of topoisomerase-catalyzed strand transfer to a perfectly aligned 5'-OH DNA strand but is comparable to the rate of DNA strand transfer across a 1-nucleotide gap. Strand transfer to 2% hydrogen peroxide is 300 times faster than strand transfer to 20% glycerol and approximately 2000 times faster than topoisomerase-catalyzed hydrolysis of the covalent intermediate. Hydroxylamine is also an effective nucleophile in topoisomerase-mediated strand transfer (k(obs) = 6.4 x 10(-)(4) s(-)(1)). The rates of the peroxidolysis, hydroxylaminolysis, glycerololysis, and hydrolysis reactions catalyzed by the mutant enzyme H265A were reduced by factors of 100-700, in accordance with the 100- to 400-fold rate decrements in DNA cleavage and religation by H265A. We surmise that vaccinia topoisomerase catalyzes strand transfer to DNA and non-DNA nucleophiles via a common reaction pathway in which His-265 stabilizes the scissile phosphate in the transition state rather than acting as a general acid or base.
authors.affiliation: Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10021, USA.
authors.forename: B O
authors.initials: BO
authors.lastname: Krogh
authors.forename: S
authors.initials: S
authors.lastname: Shuman
mesh_terms: D001483:Base Sequence; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D004264:DNA Topoisomerases, Type I / Q000378:metabolism*; D006861:Hydrogen Peroxide / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism*; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology; D013379:Substrate Specificity; D014616:Vaccinia virus / Q000201:enzymology*
chemical_list: D009838:Oligodeoxyribonucleotides; D012967:Sodium Dodecyl Sulfate; D004247:DNA; D006861:Hydrogen Peroxide; D004264:DNA Topoisomerases, Type I