Novel regulatory mechanisms controlling DnaA and the timing of DNA replication during the Caulobacter crescentus cell cycle
Final Report Abstract
The initiation of DNA replication is a central step in the cell cycle of all organisms. To ensure genome integrity, every initiation of DNA replication has to be precisely coordinated with other cell cycle events. In addition, cells must be able to control the onset of DNA replication in response to changing environmental conditions. In the model bacterium Caulobacter crescentus DNA replication is controlled by the opposing activities of the response regulator CtrA that represses replication initiation and the conserved bacterial replication initiator DnaA that promotes initiation. My work demonstrated that CtrA and DnaA compose two separable control modules that enable the temporal and spatial control DNA replication. While CtrA is required to enforce replicative asymmetry, a characteristic of the Caulobacter cell cycle, DnaA dictates the periodicity of DNA replication and the cell cycle. Importantly, this modularity of replication control is reflected in the evolutionary history of the Caulobacter cell cycle circuit. DnaA is the central component of an ancient and phylogenetically widespread circuit that governs replication periodicity in Caulobacter and most other bacteria. By contrast, CtrA, which is found only in the asymmetrically dividing α-proteobacteria, was integrated later in evolution to enforce replicative asymmetry on daughter cells. While the regulation of CtrA has been studied in detail, the regulatory mechanisms controlling DnaA activity remained poorly understood. My findings showed that in fast-growing Caulobacter DnaA is controlled, like in other bacteria, by switching between an active, ATP-bound form and an inactive, ADP-bound form. The activity switch from DnaA-ATP to DnaA-ADP is stimulated by the protein HdaA, a homolog of E. coli Hda, but additional factors may be involved. Growth of Caulobacter cells in certain stressful conditions, including nutrient starvation and stationary phase, results in a substantial reduction in DnaA levels and a cessation of DNA replication. My work revealed that the Hsp70/DnaK chaperone system is critical for controlling DnaA stability in response to heat shock and likely other stress conditions. A genetic screen revealed that mutations in dnaK, dnaJ, and grpE, encoding the DnaK/J/GrpE chaperone system, restore the viability of a strain overproducing DnaA. I found that the DnaK chaperone is required for DnaA accumulation and the initiation of DNA replication and that cells lacking active DnaK target DnaA for degradation by the protease Lon in two mutually reinforcing ways. (1) Loss of DnaK activity triggers the synthesis of Lon. (2) Surprisingly, unfolded proteins that accumulate following a loss of DnaK activity allosterically activate Lon to degrade DnaA. Collectively, my findings indicate that the Caulobacter cell cycle coordinates DNA replication with the status of intracellular protein folding through the highly conserved Hsp70 chaperone and a AAA+ protease. Adjusting the availability of DnaA likely constitutes an important adaptation to stress by preventing cell cycle progression when cells need to devote their energy and resources to survival strategies rather than proliferation.
Publications
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(2011). Modularity of the bacterial cell cycle enables independent spatial and temporal control of DNA replication. Current Biology. 21:1092-101
Jonas K, Chen YE, Laub MT
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(2011). Spatial gradient of protein phosphorylation underlies replicative asymmetry in a bacterium. Proceedings of the National Academy of Sciences. 108:1052-7
Chen YE, Tropini C, Jonas K, Tsokos CG, Huang KC, Laub MT
