ATP-Dependent Topology Discrimination by Type IIA Topoisomerases: Implications for below Equilibrium Topology Simplification

Abstract

Type IIA topoisomerases (Topo IIA) are ubiquitous enzymes that play essential roles in maintaining topological homeostasis and in unlinking chromosomes prior to cell division. Topo IIA enzymes, with the exception of DNA gyrase, regulate DNA topology by removing excess DNA supercoils, and inter- and intra-molecular links, corresponding to catenanes and knots respectively. All Topo IIA utilize ATP for their shared catalytic activity in which the enzyme introduces a transient double-strand break in one segment of DNA and transfers a second segment of DNA through this break. The strand passage reaction is coordinated, and made irreversible, through the hydrolysis of ATP. The hydrolysis energy is also coupled to below equilibrium topology simplification activity of Topo IIA that results in the reduction of supercoiling, linking, and knotting to below equilibrium levels. The mechanism of this below equilibrium relaxation remains speculative. In this study, we investigated the correlation between topology-dependent DNA binding and the level of DNA supercoil reduction for two Topo IIA enzymes that exhibit different levels of below equilibrium relaxation: E. coli Topo IV and yeast Topo II. To determine the effect of ATP on the selection of DNA topology, we measured the topology-dependent binding in the presence of two non-hydrolysable ATP analogues: ATPγS and AMPPNP. Topo IV, but not yeast topo II, shows a weak topology-dependent binding in the absence of ATP analogs. Both enzymes exhibit significantly enhanced DNA topology-dependent binding in the presence of ATPγS and AMPPNP. Simulations of Topo IIA activity that incorporate the measured DNA topology dependent binding affinities indicate that DNA topology dependent binding may be a contributing factor explaining non-equilibrium simplification by type IIA topoisomerases.