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

所有细胞都必须确保DNA复制的正确时间，并与细胞周期事件协调。在模式细菌新月形Caulobacter crescent中，DNA复制由细胞周期主调控因子CtrA和复制起始蛋白DNA的相反活性控制，两者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dna活性振荡的机制仍不清楚。dna的调控主要依赖于dna水平的波动。然而，一些研究结果强烈表明存在控制dna活性的其他机制。本研究旨在了解这些机制，并研究它们如何与茎状杆菌细胞周期协调。我的建议描述了一种新的dna调节因子的遗传筛选，以及一种酵母双杂交筛选，以确定与dna物理相互作用的因素。通过结合遗传、生化和细胞生物学分析，将对新发现的调节因子进行表征，以确定它们如何调节DNA和DNA复制，并阐明它们与已知的CtrA调节回路的联系。除了更好地了解茎状杆菌的细胞周期控制外，这项工作还将为分子基电路的一般设计原则提供见解。

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.