ParB-like CTPases mediate the segregation of bacterial chromosomes and low-copy number plasmids. They act as DNA-sliding clamps that are loaded at parS motifs in the centromere of target DNA molecules and spread laterally to form large nucleoprotein complexes serving as docking points for the DNA segregation machinery. Here, we solve crystal structures of ParB in the pre- and post-hydrolysis state and illuminate the catalytic mechanism of nucleotide hydrolysis. Moreover, we identify conformational changes that underlie the CTP- and parS-dependent closure of ParB clamps. The study of CTPase-deficient ParB variants reveals that CTP hydrolysis serves to limit the sliding time of ParB clamps and thus drives the establishment of a well-defined ParB diffusion gradient across the centromere whose dynamics are critical for DNA segregation. These findings clarify the role of the ParB CTPase cycle in partition complex assembly and function and thus advance our understanding of this prototypic CTP-dependent molecular switch.

ParB 样 CTPases 介导细菌染色体和低拷贝数质粒的分离。它们充当 DNA 滑动夹，加载在目标 DNA 分子着丝粒的parS基序上，并横向扩散以形成大的核蛋白复合物，作为 DNA 分离机制的对接点。在这里，我们解决了水解前和水解后状态下 ParB 的晶体结构，并阐明了核苷酸水解的催化机制。此外，我们确定了 CTP- 和parS 的构象变化-ParB 夹子的依赖闭合。对缺乏 CTPase 的 ParB 变体的研究表明，CTP 水解有助于限制 ParB 夹子的滑动时间，从而推动在着丝粒上建立明确的 ParB 扩散梯度，其动力学对 DNA 分离至关重要。这些发现阐明了 ParB CTPase 循环在分割复合体组装和功能中的作用，从而促进了我们对这种原型 CTP 依赖性分子开关的理解。