Interaction between DNA gyrase and its cleavage site on DNA.

DNA gyrase negatively supercoils DNA. When the requisite breaking and resealing of the DNA are uncoupled by gyrase inhibitors, the DNA becomes cleaved at specific sites. ATP, the cofactor for supercoiling, changes the sites of DNA cleavage. The mechanism of this effect was studied at sites in ColEl and @X174 DNA at which ATP strikingly enhanced cleavage. The following results showed that this increase in cleavage did not require movement of gyrase to these sites. 1) Stimulation of cleavage was unaffected by excess competitor DNA and therefore did not require recruitment of free gyrase. 2) Movement of gyrase from other sites along the DNA was ruled out since stimulation of cleavage still occurred in small DNA fragments containing only one prominent cleavage site and in a DNA fragment large enough (176 base pairs) to bind only a single gyrase molecule. 3) ATP increased cleavage of small DNA fragments without increasing the amount of gyrase bound to the DNA. We conclude that gyrase remains stationarily bound at the discrete locations where it cleaves the DNA and that ATP changes the conformational state of the enzyme, thus altering the proportion of bound gyrase that cleaves. The stability of gyrase binding to DNA varied sitespecifically, depended on DNA tertiary structure, and decreased with diminishing DNA size. Of three DNA fragments containing the same cleavage site, a 509 base pair fragment bound gyrase much more stably than a 176 base pair fragment and a 77 base pair fragment showed no detectable interaction. Gyrase bound very tightly at some sites on linear or relaxed DNA, dissociating with half-lives of several hours at 30°C. However, gyrase bound much less well to negatively supercoiled DNA. Thus, addition of ATP to the complex between gyrase and relaxed, circular DNA triggered processive cycles of supercoiling followed by rapid release of the enzyme from the DNA.