Studies of DNA Gyrase at the Single Molecule Level

Abstract

Gyrase, a bacterial type II topoisomerase, is the only topoisomerase capable of introducing negative supercoils into DNA. Negative supercoiling results from the coordinated movement of double stranded DNA and protein domains linked to ATP hydrolysis. Thus, it is important to combine structural data with dynamic information of protein and/or DNA movements to obtain a comprehensive picture of the mechanism of gyrase. Although the structures of several individual domains of different type II enzymes have been elucidated, there are still no structures of the intact gyrase heterotetramer or an intact gyrase/DNA complex precluding the correlation of conformational changes in the protein with movements of DNA. To alleviate this shortcoming, we are combining structural and single molecule studies to investigate the conformational changes that occur in gyrase as it alters DNA topology. To facilitate our research, we have optimized a procedure for purifying stable gyrase/DNA complexes in the amounts required for biophysical studies. This method results in homogeneous complexes that are captured in well-defined states along the catalytic cycle. We are also developing a novel single molecule technique combining magnetic tweezers and fluorescence microscopy to study the movements of both protein and DNA during the DNA supercoiling process. The combination of dynamic single molecule and structural information promises to provide a more comprehensive picture of the mechanism used by this molecular machine to alter DNA topology.