Mechanistic studies of eukaryotic topoisomerase II: a cysteine footprinting and FRET approach

Abstract

Type II DNA topoisomerases are essential and ubiquitous enzymes that perform important functions in chromosome condensation and segregation, and in regulating intracellular DNA supercoiling. Topoisomerase II (topo II) carries out these DNA transactions by passing one segment of DNA through the other using a reversible, enzyme‐bridged double‐stranded break. The extensive conformational changes that both DNA and topo II undergo during the reaction cycle have been probed with fluorescence resonance energy transfer and cysteine footprinting, respectively. Using fluorescent oligonucleotides, we found that the DNA gate transition has comparable rate constants for both the opening and closing reaction, with an apparent equilibrium constant near unity. To complement the results obtained with fluorescent DNA, cysteine footprinting was used to observe conformational changes in topo II itself. We found contrasting changes in cysteine solvent accessibility in the presence of various drugs and/or cofactors, depending on the domain in which the cysteine is located. Our results provide direct evidence linking opposing movements in the topo II domains; as the ATPase domain closes, the domain directly beneath the ATPase domain opens. This work was supported by NIH grants and a grant from the Department of Defense Breast Cancer Research Program.