DNA Strand Transfer Catalyzed by Vaccinia Topoisomerase: Peroxidolysis and Hydroxylaminolysis of the Covalent Protein−DNA Intermediate

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Abstract
Vaccinia topoisomerase forms a covalent DNA−(3‘-phosphotyrosyl)−enzyme intermediate at sites containing the sequence 5‘-CCCTT↓. 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 kobs versus [H2O2] at pH 10, we determined a rate constant for peroxidolysis of 6.3 × 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 ∼2000 times faster than topoisomerase-catalyzed hydrolysis of the covalent intermediate. Hydroxylamine is also an effective nucleophile in topoisomerase-mediated strand transfer (kobs = 6.4 × 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.