THE INTERACTION OF THE F-PLASMID KILLER PROTEIN, CCDB, WITH DNA GYRASE - INDUCTION OF DNA CLEAVAGE AND BLOCKING OF TRANSCRIPTION

We have studied the interaction of the F plasmid killer protein CcdB w ith its intracellular target DNA gyrase. We confirm that CcdB fan indu ce DNA cleavage by gyrase anti show that this cleavage reaction requir es ATP hydrolysis when the substrate is linear DNA, but is independent of hydrolysis when negatively supercoiled DNA is used. The 64 kDa dom ain of the gyrase A protein, which can catalyse DNA cleavage in the pr esence of the B protein and quinolone drugs, is unable to cleave DNA i n the presence of CcdB unless the C-terminal 33 kDa domain of the gyra se A protein is also present. CcdB-induced DNA cleavage by gyrase requ ires a minimum length of DNA (> similar to 160 bp), whereas in the pre sence of quinolone drugs gyrase can cleave much shorter DNA molecules. We show that CcdB, Like quinolones, can form a complex with gyrase wh ich can block transcription by RNA polymerase. A model for the interac tion of CcdB with gyrase involving the trapping of a post-strand-passa ge intermediate is suggested. We conclude that CcdB can stabilise a cl eavage complex between DNA gyrase and DNA in a manner distinct from qu inolones but, like the quinolone-induced cleavage complex, the CcdB-st abilised complex can also form a barrier to the passage of polymerases . (C) 1997 Academic Press Limited.