Conformational changes in DNA gyrase and their coordination in DNA supercoiling

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Gyrase is a bacterial DNA topoisomerase that catalyzes the ATP-dependent introduction of negative supercoils into DNA to remove positive supercoils ahead of replication forks. DNA supercoiling is catalyzed via a strand passage mechanism, in which on double-stranded DNA region is cleaved, and a second DNA-region is passed through the gap. Strand passage is guided by coordinated conformational changes of gyrase, but the underlying mechanism is not clear. Here we propose to use single molecule FRET techniques to (1) investigate the kinetics of individual conformational changes in the catalytic cycle of gyrase and their temporal coordination, (2) to directly follow the DNA strand passage event and to dissect its coordination with gyrase conformational changes, and (3) to investigate the coordination of the individual subunits in gyrase in the supercoiling reaction. Gyrase is not present in humans, and is a drug target in the treatment of bacterial infections, but current inhibitors suffer from severe side-effects and increasing resistance. The effect of gyrase inhibitors on the conformational cycle will be tested to identify their limitations and to open up avenues for the design of novel new mechanism-based gyrase inhibitors. Ultimately, understanding the coordination of conformational changes in gyrase with each other, with ATP hydrolysis, and with strand passage and DNA supercoiling, will identify the mechanism of energy coupling in gyrase, and will impact on our understanding of DNA topoisomerase mechanisms and of molecular machines in general.