Non-Equilibrium Topology Simplification by Type II Topoisomerases: A Test of Kinetic Proofreading

Abstract

Type II topoisomerases are essential enzymes that modify DNA topology. These topoisomerases pass a double stranded segment (T-segment) of DNA though a transient double stranded break in a second segment (G-segment). This strand passage mechanism allows type II topoisomerases to unlink, unknot, and relax supercoiled DNA to below equilibrium levels1. The mechanisms underlying this non-equilibrium topology simplification remain speculative. Several theoretical models have been proposed but experimental data has not been able to distinguish among them. One model developed by Yan and Marko2, postulates that the strand passage mechanism of type II topoisomerases is governed by a kinetic proofreading process. In practice, this model suggests that type II topoisomerases require two collisions between the T- and G-segments prior to strand passage. The first collision of a T-segment with the enzyme-bound G-segment transiently activates the enzyme and the second collision of the T-segment with the activated enzyme results in strand passage. The model predicts that the strand passage probability scales as the square of the collision rate, which has not been tested. We directly tested this prediction of the kinetic proofreading model using a single-crossing DNA unlinking assay. We measured the rate that topoisomerase IV, a bacterial type II topoisomerase, unlinked DNA as a function of the strand collision probability obtained from Monte Carlo simulations of the DNA crossings. The unlinking rate was linearly related to the collision probability, which is inconsistent with the kinetic proofreading model.1.Rybenkov, V. V., Ullsperger, C., Vologodskii, A. V. & Cozzarelli, N. R. Simplification of DNA topology below equilibrium values by type II topoisomerases. Science 277, 690-693 (1997).2.Yan, J., Magnasco, M. O. & Marko, J. F. A kinetic proofreading mechanism for disentanglement of DNA by topoisomerases. Nature 401, 932-935, doi:10.1038/44872 (1999).