DNA strand transfer catalyzed by vaccinia topoisomerase: Peroxidolysis and hydroxylaminolysis of the covalent protein-DNA intermediate Journal Article
| Authors: | Krogh, B. O.; Shuman, S. |
| Article 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↓. 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 [H2O2] 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 ~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. |
| Keywords: | nonhuman; protein dna binding; ph; dna; molecular sequence data; kinetics; enzyme analysis; substrate specificity; vaccinia virus; base sequence; hydrogen peroxide; dna determination; chemical reaction; hydrogen-ion concentration; dna cleavage; dna topoisomerase; dna topoisomerases, type i; oligodeoxyribonucleotides; vaccinia; dna protein complex; priority journal; article; sodium dodecyl sulfate |
| Journal Title: | Biochemistry |
| Volume: | 39 |
| Issue: | 21 |
| ISSN: | 0006-2960 |
| Publisher: | American Chemical Society |
| Date Published: | 2000-05-30 |
| Start Page: | 6422 |
| End Page: | 6432 |
| Language: | English |
| DOI: | 10.1021/bi000184u |
| PUBMED: | 10828956 |
| PROVIDER: | scopus |
| DOI/URL: | |
| Notes: | Export Date: 18 November 2015 -- Source: Scopus |
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