Dissecting the molecular mechanism of directional centromere DNA segregation in Caulobacter Crescentus

在细胞分裂过程中，染色体的忠实分离对于所有细胞中遗传物质的遗传至关重要。在该细菌中，染色体复制和分离同时发生。最初，复制起点附近的两个着丝粒样PAR蛋白复合体定位于一个细胞极。然后，染色体分离是由这些复合体中的一个从一个细胞极点快速移动到另一个细胞极点开始的。尽管以前发现了Caulbacter染色体分离的关键因子，但驱动这一运输过程的力的产生尚不清楚，也不知道定向极向运输的机制。染色体分离已被证明依赖于PAR蛋白系统和已知的极性定位蛋白。然而，三个模型提出了这些蛋白质亚群如何相互作用以调节染色体分离的不同机制。在拟议的项目中，我将测试这些模型，并通过实验确定着丝粒状复合体的运输机制。通过结合无细胞重组技术、遗传扰动分析和单分子显微镜，我将定量地分析着丝粒分离的最低生化要求，并确定极性基质和空间约束如何影响染色体运输。这些项目将为着丝粒运输的定量动力学提供新的见解，并对细菌染色体分离的初始步骤产生深入的分子理解。

The faithful segregation of chromosomes during cell division is of critical importance for the inheritance of genetic material in all cells. In the bacterium Caulobacter crescentus chromosome replication and segregation occur simultaneously. Initially, the two centromere-like Par proteine complexes near the replicated origin of replication are localized at one cell pole. Then, chromosome segregation is initiated by the rapid movement of one of these complexes from one cell pole to the other. Despite previous identification of key players for Caulobacter chromosome segregation in Caulobacter the generation of force to drive this transport process is not known, nor is the mechanism that orients pole directed transport. Chromosome segregation has been shown to depend on the Par protein system and known polar localized proteins. However, three models have proposed different mechanisms for how subgroups of these proteins interact to mediate chromosome segregation. In the proposed project I will test these models and experimentally determine the mechanism for the transport of the centromere-like complex. By using a combination of cell-free reconstitution techniques, genetic perturbation assays and single-molecule microscopy I will quantitatively analyze the minimal biochemical requirements for centromere segregation in Caulobacter and determine how polar matrix and spatial constrains influence chromosome transport. This projects will provide novel insights into the quantitative dynamics of the centromere transport and yield a deep molecular understanding of the initial steps of bacterial chromosome segregation.