Initiation of chromosomal replication is a critical and highly regulated step in the E. coli cell cycle. DNA replication is controlled in such a way that each origin of replication, oriC, is initiated once and only once each cell cycle at a fairly constant cell mass per origin. Regulatory inactivation of the DnaA protein, termed RIDA, is a process believed to reduce the activity of the initiator protein during the sequestration period, by converting DnaA-ATP, the form active for initiation, to inactive DnaA-ADP, by hydrolysis. This prevents origins from being re-initiated at the end of the sequestration period. The conversion of DnaA-ATP to DnaA-ADP is facilitated by a complex of DNA loaded β-clamps, the DnaA protein, and the Hda protein, the latter being instrumental to RIDA. We have evaluated the contribution of RIDA to timely once-per-cell-cycle initiation of replication. This was done by altering the cellular concentration of the Hda protein. Hda deficient cells rarely re-initiated an origin within the same cell cycle, and when re-initiation occurred it did not coincide with the end of sequestration. Therefore Hda mediated conversion of DnaA-ATP to DnaA-ADP is not involved in preventing immediate re-initiation from oriC. Likewise overproduction of the Hda protein only had modest effects on cell cycle control. The level of Hda did however affect dnaA gene expression in such a way that a direct relationship between Hda and DnaA levels in the cell was observed: Hda depletion led to reduced dnaA gene expression, and overproduction of Hda resulted in DnaA overproduction. Finally, Hda deficient cells were highly sensitive to the cellular DnaA protein level, and DnaA overproduction led to uncontrolled replication from oriC and growth impairment. Based on these observations we suggest that RIDA and dnaA gene autoregulation act as homeostatic mechanisms to ensure timely initiation in wildtype cells.
|24 aug. 2006
|Cell cycle and cytoskeletal elements in bacteria