Year-end Sale % OFF 
Abstract
The bacterial replication cycle is driven by the DnaA protein which cycles between the active ATP-bound form and the inactive ADP-bound form. It has been suggested that DnaA also is the main controller of initiation frequency. Initiation is thought to occur when enough ATP-DnaA has accumulated. In this work we have performed cell cycle analysis of cells that contain a surplus of ATP-DnaA and asked whether initiation then occurs earlier. It does not. Cells with more than a 50% increase in the concentration of ATP-DnaA showed no changes in timing of replication. We suggest that although ATP-DnaA is the main actor in initiation of replication, its accumulation does not control the time of initiation. ATP-DnaA is the motor that drives the initiation process, but other factors will be required for the exact timing of initiation in response to the cell’s environment. We also investigated the in vivo roles of datA dependent DnaA inactivation (DDAH) and the DnaA-binding protein DiaA. Loss of DDAH affected the cell cycle machinery only during slow growth and made it sensitive to the concentration of DiaA protein. The result indicates that compromised cell cycle machines perform in a less robust manner.
Highlights
The ORC- and CDC6-like prokaryotic initiator protein DnaA has been studied extensively for many years, but it is still not clear whether the protein contributes to actual regulation of the initiation of replication or whether it works as a cell cycle motor which “licenses” initiation at regular intervals
We suggest that cell cycle regulation in E. coli has similarities to that of eukaryotes in that origins are “licensed” to initiate by a cell cycle motor and that the precise timing depends on other signaling
A two-fold increase in DnaA concentration does not affect the timing of replication initiation
Summary
The ORC- and CDC6-like prokaryotic initiator protein DnaA has been studied extensively for many years, but it is still not clear whether the protein contributes to actual regulation of the initiation of replication or whether it works as a cell cycle motor which “licenses” initiation at regular intervals. The high-affinity boxes are most likely bound by DnaA throughout the cell cycle [8], while binding to the “last” low-affinity sites has been suggested to trigger the initiation process at a time when the ATP-DnaA level has reached a threshold concentration [9]. Formation of a DnaA oligomer in the origin region causes the unwinding of the DNA in the AT-rich region and formation of the open complex [1,3] This process is probably facilitated by transcription by RNA polymerase [10,11,12,13] and by DiaA, a DnaA-binding protein that has been shown to promote formation of ATP-DnaA complexes at oriC and stimulate oriC unwinding in vitro [14,15,16]
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
