STUDIES OF FUNCTIONS INVOLVED IN GENETIC RECOMBINATION

基因重组相关功能的研究

• 批准号: 6161956
• 负责人: M GELLERT
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6161956
• 关键词: Archaea; DNA gyrase; DNA topoisomerases; adenosinetriphosphatase; bacterial DNA; bacterial proteins; circular DNA; enzyme activity; enzyme reconstitution; genetic recombination; molecular cloning; nucleic acid sequence; recombinant proteins

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.

F-质粒蛋白CcdB会杀死表达它的大肠杆菌细胞，除非 它们还表达其抑制物CCDA。CcdB通过抑制DNA旋转酶发挥作用 并导致它切割染色体DNA。我们已经研究了 CcdB与DNA旋转酶的相互作用。我们已经确认Ccdb可以 通过旋转酶诱导DNA裂解，表明这种裂解反应 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。这个 催化DNA裂解的旋转酶A蛋白的kDa结构域 在B蛋白和喹诺酮类药物存在的情况下，无法 在CcdB存在下切断DNA，除非C-末端33 kDa结构域 也存在旋转酶A蛋白。CcdB诱导的DNA裂解 旋转酶需要最小长度的DNA(&gt；160bp)，而在 喹诺酮类药物旋转酶可以切割更短的DNA 分子。Ccdb与喹诺酮类药物一样，与旋转酶形成复合体，可以 RNA聚合酶阻断转录。一种相互作用的模型 带有回旋酶的Ccdb，涉及捕获后链通道 建议采用中级。因此，CcdB可以稳定切割复合体。 DNA旋转酶和DNA之间的区别于喹诺酮类药物， 与喹诺酮诱导的切割复合体一样，CcdB稳定的复合体 也可以形成聚合酶通过的障碍。