Determining the Role DNA of Bending in Topology Simplification by Type II Topoisomerases

Abstract

Type II topoisomerases are essential and universally conserved enzymes that simplify the global topology of DNA by an ATP dependent mechanism that involves passing one double stranded segment (T segment) of DNA through a transient double stranded break in a second segment (G segment) of DNA. This core strand passage mechanism allows type II topoisomerases to decatenate DNA and relax supercoils to below equilibrium levels. Although much is known about the structure and function of these critical proteins, the specific mechanism that drives this topology simplification reaction remains an open question, though several compelling theories have been presented. Some of the more plausible theories postulate that type II topoisomerases achieve non-equilibrium topology simplification by inducing a sharp bend into the G segment. In this study we sought to determine if and to what extent type II topoisomerases impose a bend on DNA. We used Atomic Force Microscopy (AFM) to visualize protein-DNA complexes of three different type II topoisomerases that span the range of topology simplification activity. We directly measured the bend angles imposed on DNA by these proteins to determine if the bend angles could fully account for the observed topology simplification behavior. We found that type II topoisomerases bend DNA, but the measured bend angles were not in accordance with the relative non-equilibrium activity. These findings suggest that bending of DNA could be an important component of the mechanism of topology simplification by type II topoisomerases but it cannot completely account for the observed topology simplification behavior.