Kinetic insights into the temperature dependence of DNA strand cleavage and religation by topoisomerase III from the hyperthermophile Sulfolobus solfataricus

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.