Novel regulatory mechanisms controlling DnaA and the timing of DNA replication during the Caulobacter crescentus cell cycle

所有细胞必须确保DNA复制的正确时机及其与细胞周期事件的协调。在模式细菌Caulbacter crescentus中，DNA复制是由细胞周期主调控子CtrA和复制起始蛋白Dna A的相反活性控制的，这两个蛋白都在细胞周期进程中振荡，但彼此不同步。尽管对CtrA的时间调控有了详细的了解，但解释DNAA活动振荡的机制仍然不清楚。DNAA的调节被认为主要取决于DNAA水平的波动。然而，一些发现有力地表明，存在控制DNAA活性的额外机制。这项拟议的研究旨在了解这些机制，并研究它们如何与Caulbacter细胞周期相协调。我的建议描述了DNAA的新型调节子的遗传筛选以及酵母双杂交筛选，以识别与DNAA物理上相互作用的因素。通过结合使用遗传、生化和细胞生物学分析，将对新发现的调节器进行表征，以确定它们如何调节DNAA和DNA复制，并阐明它们与已知的CtrA调节电路的联系。除了更好地理解细胞周期控制以外，这项工作还将提供对基于分子的电路的一般设计原则的洞察。

All cells must ensure the correct timing of DNA replication and its coordination with cell cycle events. In the model bacterium Caulobacter crescentus, DNA replication is controlled by the opposing activities of the cell cycle master regulator CtrA and the replication initiation protein DnaA, both of which oscillate over cell cycle progression, but out of phase with one another. Despite a detailed understanding about the temporal regulation of CtrA, the mechanisms that account for the oscillations of DnaA activity remain unclear. The regulation of DnaA was proposed to depend mainly on the fluctuating levels of DnaA. However, several findings strongly indicate the existence of additional mechanisms that control DnaA activity. The proposed study aims to understand these mechanisms and to study how they are coordinated with the Caulobacter cell cycle. My proposal describes a genetic screen for novel regulators of DnaA as well as a yeast two-hybrid screen to identify factors that interact physically with DnaA. By using a combination of genetic, biochemical, and cell biological assays the newly identified regulators will be characterized to determine how they modulate DnaA and DNA replication and to elucidate their connection with the known CtrA regulatory circuit. In addition to developing a better understanding of cell cycle control in Caulobacter, this work will provide insight into the general design principles of molecular based circuits.