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

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.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. Much of our current knowledge about the mechanistic aspects of type IA topoisomerases is derived from the studies of Escherichia coli topoisomerase I, a prototype of this subclass of topoisomerases 2, 3 . According to a well-accepted model, type IA topoisomerases relax DNA using an enzyme-bridged mechanism, which entails four steps in a catalytic cycle 4,5 . The topoisomerase binds non-covalently to one strand in the single-stranded regions of DNA (step 1), and cleaves the strand non-randomly with the concomitant formation of a covalent linkage between the active-site tyrosyl of the enzyme and the scissile phosphate (step 2); the complementary strand passes through the transient opening on the cleaved strand (step 3), followed by the religation of the cleaved strand (step 4).Topoisomerase III from the hyperthermophilic archaeon Sulfolobus solfataricus (Sso topo III) is a type IA topoisomerase. Sso topo III is most active in DNA relaxation at 75 °C, a temperature optimal for the growth of the

Direct Observation of Topoisomerase IA Gate Dynamics

“…The ligation rate of topo III depended strongly on the magnesium concentration ( Fig. 4d, Supplementary Table 3) This is close to previously measured values of the Km for the magnesium dependence of relaxation by type IA enzymes, which range from 2-4 mM 9,41,45 . The fact that the relationship is sigmoidal rather than hyperbolic is consistent with biochemical evidence indicating that the catalytic cycle depends on two coordinating magnesium ions 45,46 .…”

“…If topo III cleavage and ligation rates were stimulated by the presence of a second ss-or ds-DNA strand, then passage of an initial strand could lead to the fast bursts of processive activity previously observed 39 . The requirement of a second strand of DNA would align with kinetic studies of the S. solfatarius topo III in which ligation of cleaved ssDNA was facilitated by annealing of a complementary strand 41 .…”

“…Ensemble measurements report changes in steady state levels of cleaved and religated product, but do not provide direct information on the cleavage or ligation rates. Stopped flow experiments offer a more direct way to measure these kinetics, but to date few such studies have been done on type IA topoisomerases 41 . We determined the effect of magnesium on the cleavage and religation rates by measuring the gate kinetics as a function of magnesium concentration (Fig 4d, Table 3).…”

Direct observation of topoisomerase IA gate dynamics

“…If topo III cleavage and ligation rates are stimulated by the presence of a second ss- or ds-DNA strand, then passage of an initial strand could lead to the fast bursts of processive activity previously observed 36 . The requirement of a second strand of DNA would align with kinetic studies of S. solfatarius topo III in which ligation of cleaved ssDNA was facilitated by annealing of a complementary strand 39 .…”