Vaccinia DNA topoisomerase I: Evidence supporting a free rotation mechanism for DNA supercoil relaxation

James T. Stivers, Thomas K. Harris, Albert S. Mildvan

Research output: Contribution to journalArticle

86 Scopus citations

Abstract

The Vaccinia type I topoisomerase catalyzes site-specific DNA strand cleavage and religation by forming a transient phosphotyrosyl linkage between the DNA and Tyr-274, resulting in the release of DNA supercoils. For type I topoisomerases, two mechanisms have been proposed for supercoil release: (1) a coupled mechanism termed strand passage, in which a single supercoil is removed per cleavage/religation cycle, resulting in multiple topoisomer intermediates and late product formation, or (2) an uncoupled mechanism termed free rotation, where multiple supercoils are removed per cleavage/religation cycle, resulting in few intermediates and early product formation. To determine the mechanism, single-turnover experiments were done with supercoiled plasmid DNA under conditions in which the topoisomerase cleaves predominantly at a single site per DNA molecule. The concentrations of supercoiled substrate, intermediate topoisomers, and relaxed product vs time were measured by fluorescence imaging, and the rate constants for their interconversion were determined by kinetic simulation. Few intermediates and early product formation were observed. From these data, the rate constants for cleavage (0.3 s-1), religation (4 s-1), and the cleavage equilibrium constant on the enzyme (0.075) at 22 °C are in reasonable agreement with those obtained with small oligonucleotide substrates, while the rotation rate of the cleaved DNA strand is fast (~20 rotations/s). Thus, the average number of supercoils removed for each cleavage event greatly exceeds unity (Δn = 5) and depends on kinetic competition between religation and supercoil release, establishing a free rotation mechanism. This free rotation mechanism for a type I topoisomerase differs from the strand passage mechanism proposed for the type II enzymes.

Original languageEnglish (US)
Pages (from-to)5212-5222
Number of pages11
JournalBiochemistry
Volume36
Issue number17
DOIs
StatePublished - Apr 29 1997
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry

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