Determining the Molecular Mechanism of a Caulobacter DNA Replication Checkpoint

 描述(申请人提供)：所有生物的细胞增殖都需要按顺序进行DNA复制、染色体分离和细胞分裂。为了协调这些活动，细胞采用了监管的“检查点”，以确保在尝试下一步之前忠实地完成每一步。虽然细胞周期在真核生物中已经得到了很好的研究，但我们对细菌中的细胞周期网络和调控控制的理解仍然处于初级阶段。新月形杆菌是研究细菌细胞周期控制的一个很好的模型，因为它在遗传上容易驯化，很容易同步，并且表现出明显的G1和G2期。Crescentus显示出一个DNA复制检查点，当DNA复制被阻止时，细胞周期进程停止。以前的工作表明，DNA复制启动以某种方式促进了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，当被磷酸化时，作为一个全球转录因子，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，从而确保细胞不会过早尝试细胞分裂。然而，这个关键的细胞周期检查点背后的分子机制仍然未知，也是本项目的重点。将使用高通量、候选和无偏见的遗传方法来确定将CtrA调控与DNA复制相结合所需的潜在调控因子。在这些筛选中确定的候选调节器将与先前确定的CtrA调节器一起进行测试，以确定它们在将DNA复制与CtrA激活联系起来方面所扮演的角色。将使用遗传和生化方法来区分这些调节因子如何以依赖于DNA复制的方式促进CtrA的激活。因此，这项研究的结果将为细菌中DNA复制与细胞周期进展的调控机制提供重要的见解。对细菌细胞周期的研究是重要的，因为它们(1)可以揭示在生命的所有领域中保守的调节原则，(2)将揭示关键的差异，可以利用这些差异来开发新的抗生素。

 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.