A CTP-dependent gating mechanism enables ParB spreading on DNA

Proper chromosome segregation is essential in all living organisms. The ParA-ParB-parS system is widely employed for chromosome segregation in bacteria. Previously, we showed that Caulobacter crescentus ParB requires cytidine triphosphate to escape the nucleation site parS to spread by sliding to the neighboring DNA. Here, we provide the structural basis for this transition from nucleation to spreading by solving co-crystal structures of a C-terminal domain truncated C. crescentus ParB with parS and with a CTP analog. Nucleating ParB is an open clamp, in which parS is captured at the DNA-binding domain (the DNA-gate). Upon binding CTP, the N-terminal domain (NTD) self-dimerizes to close the NTD-gate of the clamp. The DNA-gate also closes, thus driving parS into a compartment between the DNA-gate and the C-terminal domain. CTP hydrolysis and/or the release of hydrolytic products may re-open the gates. Overall, we suggest a CTP-operated gating mechanism that regulates ParB nucleation and spreading. Chromatin-immunoprecipitation with deep sequencing experiments (ChIP-seq) were performed on exponential-growing Caulobacter crescentus (see the supplementary Materials and Methods for the exact treatment that were applied to each strain).

染色体的精准分离对所有生命体而言均至关重要。ParA-ParB-parS系统（ParA-ParB-parS system）广泛参与细菌的染色体分离过程。此前本研究团队已证实，新月柄杆菌（Caulobacter crescentus）的ParB蛋白需依赖胞苷三磷酸（cytidine triphosphate，CTP），方可脱离成核位点parS，并通过滑动至邻近DNA的方式实现扩散。本研究通过解析截短C端结构域的新月柄杆菌ParB分别与parS及CTP类似物结合的共晶结构，阐明了其从成核状态向扩散状态转变的结构基础。处于成核状态的ParB呈开放式夹状结构，parS被捕获于其DNA结合结构域（即DNA门控区）。当结合CTP后，其N端结构域（N-terminal domain，NTD）发生自身二聚化，以闭合夹状结构的NTD门控区；同时DNA门控区也随之闭合，从而将parS推入DNA门控区与C端结构域之间的腔室中。CTP水解反应及/或水解产物的释放可重新打开上述门控区。综上，本研究提出了一种由CTP调控的门控机制，可对ParB的成核与扩散过程进行调控。本研究对处于指数生长期的新月柄杆菌开展了染色质免疫共沉淀联合深度测序（Chromatin-immunoprecipitation with deep sequencing，ChIP-seq）实验，具体处理方案详见补充材料与方法部分。