Determining the Molecular Mechanism of a Caulobacter DNA Replication Checkpoint

 描述（由申请方提供）：所有生物体中的细胞增殖需要依次执行DNA复制、染色体分离和细胞分裂。为了协调这些活动，细胞使用监管“检查点”，以确保在尝试下一个步骤之前忠实地完成每个步骤。虽然细胞周期已经在真核生物中得到了很好的研究，但我们对细菌中细胞周期网络和调控的理解仍然是初步的。新月柄杆菌是研究细菌细胞周期控制的一个很好的模型，因为它在遗传上是易处理的，容易同步化，并表现出不同的G1，和G2期。C. crescentus具有DNA复制检查点，因此当DNA复制被阻断时，细胞周期进程停止。以前的工作表明，DNA复制起始以某种方式促进CtrA的激活，CtrA是一种重要的细胞周期调节因子。Ctra 是双组分信号蛋白家族的反应调节因子，当磷酸化时，作为全局转录因子，促进细胞分裂所需基因的表达。在没有DNA复制的情况下，CtrA不会被激活，从而确保CtrA不会过早地尝试细胞分裂。然而，这个关键的细胞周期检查点的分子机制仍然未知，是这个项目的重点。高通量的候选人和公正的遗传方法将被用来确定耦合CtrA调节DNA复制所需的潜在监管机构。在这些筛选中确定的候选调节器将与先前确定的CtrA调节器一起测试它们在将DNA复制与CtrA激活联系起来中的作用。遗传和生物化学的方法将被用来区分这些监管机构如何促进激活的CtrA在DNA复制依赖的方式。因此，这项研究的结果将为细菌中DNA复制与细胞周期进程的调控机制提供重要的见解。对细菌细胞周期的研究很重要，因为它们（1）可以揭示在所有生命领域中保守的调控原则，（2）将揭示可用于开发新抗生素的关键差异。

 DESCRIPTION (provided by applicant): Cellular proliferation in all organisms requires the sequential execution of DNA replication, chromosome segregation, and cell division. To coordinate these activities, cells employ regulatory "checkpoints" that ensure faithful completion of each step before attempting the next. Although the cell cycle has been well studied in eukaryotes, our understanding of cell cycle networks and regulatory controls in bacteria remains rudimentary. Caulobacter crescentus represents an excellent model for studies of cell cycle control in bacteria as it is genetically tractable, easily synchronized, and exhibits distinct G1, , and G2 phases. C. crescentus exhibits a DNA replication checkpoint, such that when DNA replication is blocked, cell cycle progression halts. Previous work demonstrated that DNA replication initiation somehow promotes activation of CtrA, an essential cell cycle regulator. CtrA is a response regulator of the two-component signaling protein family that, when phosphorylated, acts as a global transcription factor, promoting expression of genes necessary for cell division. In the absence of DNA replication, CtrA is not activated, thus ensuring that cels do not attempt cell division prematurely. However, the molecular mechanism underlying this critical cell cycle checkpoint remains unknown and is the focus of this project. High-throughput candidate and unbiased genetic approaches will be used to identify potential regulators necessary for coupling CtrA regulation to DNA replication. The candidate regulators identified in these screens will be tested alongside one previously identified CtrA regulator for their roles in linking DNA replication to CtrA activation. Genetic and biochemical approaches will be used to distinguish how these regulators promote activation of CtrA in a DNA replication-dependent manner. Thus, the results from this study will provide important insights into the regulatory mechanisms coupling DNA replication to cell cycle progression in bacteria. Studies of the bacterial cell cycle are important as they (1) can reveal regulatory principles conserved in all domains of life and (2) will reveal critical differences that could be exploited to develop new antibiotics.