E. Coli Gyrase Fails to Negatively Supercoil Diaminopurine-Substituted DNA

Abstract

Type II topoisomerases modify DNA supercoiling, and crystal structures suggest that they sharply bend the gate segment DNA in the process. Bacterial gyrases are a class of type II topoisomerases which introduce negative supercoiling by also tightly wrapping DNA flanking the gate segment before strand passage. To reveal whether topoisomerases can bend or wrap artificially stiffened DNA, E. coli gyrase and human topoisomerase IIα were challenged with normal DNA or stiffer DNA produced by PCR reactions in which diaminopurine (DAP) replaced adenine deoxyribonucleotidetriphosphates. The rates or pauses during relaxation of positive supercoils produced with magnetic tweezers in single molecules of DAP-substituted versus normal DNA distinguished the enzymes. Gyrase struggled to bend or perhaps open a gap in DAP-substituted DNA, and segments of wider DAP DNA may have fit poorly into the N-gate of the human topoisomerase IIα. Pauses during processive activity on both types of DNA exhibited ATP-dependence consistent with two pathways leading to the strand-passage competent state with a bent gate segment and a transfer segment trapped by an ATP-loaded and latched N-gate. However, E. coli DNA gyrase did not negatively supercoil 35% stiffer DAP DNA most likely by failing to wrap DNA flanking the gate segment before strand passage.