Studies of DNA Gyrase at the Single Molecule Level

Abstract

Topoisomerases are proteins that manipulate the topology of DNA. Unique among topoisomerases is DNA gyrase, a Type II bacterial topoisomerase, which is the only one capable of introducing negative supercoils. In addition, DNA gyrase can relax positive supercoils, an ability shared by other members of both Type I and II topoisomerases. To generate negative supercoils, DNA gyrase forms a double stranded break in DNA and passes a second strand of DNA through the resulting break, a process that is linked to ATP hydrolysis. To elucidate the conformational changes necessary for this action and to obtain a comprehensive picture of the mechanism of gyrase, we will utilize a novel single molecule technique, which combines both magnetic tweezers and TIRF microscopy to simultaneously observe protein and DNA movement during the DNA supercoiling process. Incorporating information from both dynamic single molecule and static structural studies promises to provide a more comprehensive picture of the mechanism used by this molecular machine to alter DNA topology.