DNA gyrase : mechanism of supercoiling and interaction with quinolones

DNA gyrase is unique among topoisomerases in its ability to introduce negative supercoils into closed-circular DNA. Deletion of the C-terminal DNA-binding domain of the A subunit of gyrase gives rise to an enzyme that behaves like a conventional type II topoisomerase, suggesting that the unique properties of DNA gyrase are attributable to the wrapping of DNA around the C-terminal DNA-binding domains of the A subunits. However, these results do not unveil the detailed mechanism by which the transported DNA segment is captured and directed through the DNA gate. This mechanism was addressed by probing the topology of the bound DNA segment at distinct steps of the catalytic cycle. A model is proposed in which gyrase captures a contiguous DNA segment with high probability, irrespective of the superhelical density of the DNA, while the efficiency of strand passage depends on the superhelical free-energy. This mechanism is concerted, in that capture of the transported segment induces opening of the DNA gate, which in turn, stimulates ATP hydrolysis.;Mutation of Glu42 to Ala in the B subunit of DNA gyrase abolishes ATP hydrolysis but not nucleotide binding. Gyrase complexes that contain one wild-type and one Ala42 mutant B protein were formed and the ability of such complexes to hydrolyse ATP was investigated. It was found that ATP hydrolysis was able to proceed only in the wild-type subunit, albeit at a lower rate. With only one ATP molecule hydrolysed at a time, gyrase could still perform supercoiling but the limit of this reaction was lower than that observed when both subunits can hydrolyse the nucleotide.;Limited proteolysis was used to identify conformational changes in DNA gyrase and the proteolytic signatures observed were interpreted in terms of four complexes of gyrase, each representing a particular conformational state. Quinolone binding to the gyrase-DNA complex induces a conformational change that results in the blocking of supercoiling. Under these conditions gyrase is still capable of ATP hydrolysis. The kinetics of this reaction have been studied and found to differ from those of the reaction of the drug-free enzyme. By observing the conversion of the ATPase rate to the quinolone-characteristic rate, the formation and dissociation of the gyrase-DNA-quinolone complex can be monitored. Comparison of the time dependence of the conversion of the gyrase ATPase with that of DNA cleavage reveals that formation of the gyrase-DNA-quinolone complex does not correspond to the formation of cleaved DNA. Quinolone binding and drug-induced DNA cleavage are separate processes constituting two sequential steps in the mechanism of action of quinolones on DNA gyrase.