STUDIES OF FUNCTIONS INVOLVED IN GENETIC RECOMBINATION

F质粒蛋白CcdB能杀死E.大肠杆菌细胞表达它，除非 它们还表达其抑制剂CcdA。CcdB通过抑制DNA促旋酶发挥作用 并使其切割染色体DNA。我们研究了 CcdB与DNA促旋酶相互作用。我们已经证实，CcdB可以 通过促旋酶诱导DNA切割并显示该切割反应 当底物是线性DNA时，需要ATP水解，但 当使用负超螺旋DNA时不依赖于水解。的 促旋酶A蛋白的64 kDa结构域，可催化DNA切割 在B蛋白和喹诺酮类药物存在下， 在CcdB存在下切割DNA，除非C-末端33 kDa结构域 促旋酶A蛋白也存在。 CcdB诱导的DNA切割 促旋酶需要最小长度的DNA（>160 bp），而在 喹诺酮类药物促旋酶的存在可以切割更短的DNA 分子。 CcdB与喹诺酮类一样，与促旋酶形成复合物， 通过RNA聚合酶阻断转录。一个模型的相互作用 CcdB与涉及捕获后链通道的促旋酶 建议中间体。 因此，CcdB可以稳定切割复合物 在DNA促旋酶和DNA之间以不同于喹诺酮的方式， 与喹诺酮诱导的裂解复合物一样，CcdB稳定的复合物 也可以形成聚合酶通过的屏障。

The F-plasmid protein CcdB kills E. coli cells that express it, unless they also express its inhibitor CcdA. CcdB acts by inhibiting DNA gyrase and causing it to cleave chromosomal DNA. We have studied the interaction of CcdB with DNA gyrase. We have confirmed that CcdB can induce DNA cleavage by gyrase and show that this cleavage reaction requires ATP hydrolysis when the substrate is linear DNA, but is independent of hydrolysis when negatively-supercoiled DNA is used. The 64 kDa domain of the gyrase A protein, which can catalyse DNA cleavage in the presence of the B protein and quinolone drugs, is unable to cleave DNA in the presence of CcdB unless the C-terminal 33 kDa domain of the gyrase A protein is also present. CcdB-induced DNA cleavage by gyrase requires a minimum length of DNA (>160 bp) whereas in the presence of quinolone drugs gyrase can cleave much shorter DNA molecules. CcdB, like quinolones, forms a complex with gyrase which can block transcription by RNA polymerase. A model for the interaction of CcdB with gyrase involving the trapping of a post-strand-passage intermediate is suggested. Thus, CcdB can stabilise a cleavage complex between DNA gyrase and DNA in a manner distinct from quinolones but, like the quinolone-induced cleavage complex, the CcdB-stabilised complex can also form a barrier to the passage of polymerases.