In all organisms, the duplication of the genetic material is a coordinated and precise event. Chromosomes are replicated only once per cell cycle and when over or under replication take place, cell proliferation is severely hindered. In nearly all bacteria, the coordination of the chromosomal replication is dictated by DnaA. In Escherichia coli, DnaA initiates the replication by binding DNA sequences in the origin of replication (oriC). DnaA can only initiate DNA replication when is bound to ATP. Specifically, DnaA-ATP binds DNA elements and stimulates the unwinding of the origin of replication. Once the replication is started, Hda, a DnaA related protein, stimulates the intrinsic ATPase activity of DnaA to convert the active ATP-bound form to the inactive ADP-bound form. Regulation of the DnaA-ATP level in the cell is a crucial but poorly understood event that we plan to characterize in Escherichia coli. Several uncharacterized suppressors of Hda deficiency, in which DnaA-ATP/ DnaA-ADP ratio is affected, have been isolated in Anders Løbner-Olesen’s lab. We plan on identifying and characterizing these mutants. It has been postulated that the ATPase activity of DnaA could constitute a system that senses the metabolic level of the bacterium and coordinates it with DNA replication. One attractive model suggests that the availability of dNTPs during the cell cycle could determine DnaA activity. Our aim is to establish the first evidence of a mechanism linking metabolism with DNA replication, through the control of the cellular pool of dNTPs. In fixed cells, DnaA was shown to localize to the membrane. We will investigate the dynamics of wild type DnaA localization and its known ATPase mutants using a novel fluorescence microscopy technique in living cells. We wish to develop this new approach to localize other essential component of the DnaA regulatory circuit that can otherwise not be examined by conventional fluorescence microscopy techniques.
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