The DNA Gyrase-Quinolone Complex

Abstract

Quinolone binding to the gyrase-DNA complex in- duces a conformational change that results in the block- ing of supercoiling. Under these conditions gyrase is still capable of ATP hydrolysis which now proceeds through an alternative pathway involving two different conformations of the enzyme (Kampranis, S. C., and Maxwell, A. (1998) J. Biol. Chem. 269, 22606–22614). The kinetics of ATP hydrolysis via this pathway have been studied and found to differ from those of the reaction of the drug-free enzyme. The quinolone-characteristic ATPase rate is DNA-dependent and can be induced in the presence ofDNA fragments as small as 20 base pairs. By observing the conversion of the ATPase rate to the quinolone characteristic rate, the formation and disso- ciation of the gyrase-DNA-quinolone complex can be monitored. Comparison of the time dependence of the conversion of the gyrase ATPase with that ofDNA cleav- age reveals that formation of the gyrase-DNA-quinolone complex does not correspond to the formation ofcleaved DNA. Quinolone-induced DNA cleavage proceeds via a mechanism consisting of two cleavage events that is modulated in the presence of a nucleotide cofactor. We demonstrate that quinolone binding and drug-induced DNA cleavage are separate processes constituting two sequential steps in the mechanism of action of quino- lones on DNA gyrase. DNA