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

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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.