Abstract
All cellular organisms encode type IA topoisomerases which catalyze DNA topological changes essential for DNA transactions. However, the kinetics of the reaction catalyzed by these enzymes remains poorly characterized. Here we measured the rapid kinetics of template binding, cleavage and religation by Sso topo III, a type IA topoisomerase from the hyperthermophilic archaeon Sulfolobus solfataricus, by using a novel FRET/PIFE-based method in a stopped-flow spectrometer. We show that Sso topo III bound the template rapidly, and the rate of binding was 2–3 orders of magnitudes higher than that of template cleavage at 25 °C. The rate of template cleavage was favored over that of template religation by the enzyme, and was more so at lower temperatures (25–55 °C). Significant template cleavage [(2.23 ± 0.11) × 10−3 s−1] was observed while little religation was detectable at 25 °C. This is consistent with the presence of a higher activation energy for template religation (41 ± 5 kcal·mol−1) than that for template cleavage (32 ± 1 kcal·mol−1). Our results provide a kinetic interpretation for the ability of Sso topo III to relax negatively supercoiled DNA only at higher temperature and offer clues to the adaptation of the reaction mechanisms of thermophilic enzymes to high temperature.
Highlights
DNA topoisomerases are ubiquitous enzymes that catalyze topological changes in DNA essential for DNA transactions, such as DNA replication, transcription, DNA repair and recombination[1]
In order to carry out rapid kinetic analysis of the reaction catalyzed by Sso topo III, we developed a novel assay based on protein induced fluorescence enhancement (PIFE) and fluorescence resonance energy transfer (FRET)
Type IA topoisomerases have been extensively studied, very little is known about the kinetics of individual steps in the reaction cycle catalyzed by these enzymes
Summary
DNA topoisomerases are ubiquitous enzymes that catalyze topological changes in DNA essential for DNA transactions, such as DNA replication, transcription, DNA repair and recombination[1]. Type IA topoisomerases exist in all cellular organisms and include topoisomerases I and III as well as reverse gyrase[1] These enzymes are capable of relaxing negative DNA supercoils, or introducing positive DNA supercoils in the case of reverse gyrase. How the kinetics of the steps in Sso topo III-catalyzed reactions is affected by temperature remains to be determined. We developed a fluorescence-based method for monitoring the rapid kinetics of DNA cleavage and religation by a type IA topoisomerase in real time. Taking advantage of the thermophily of Sso topo III, we determined the rate constants of template cleavage and religation by the enzyme in a temperature range of 25~55 °C. Our results provide a kinetic interpretation for the ability of Sso topo III to relax negatively supercoiled DNA only at higher temperature
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