DnaA binding locus datA promotes DnaA-ATP hydrolysis to enable cell cycle-coordinated replication initiation

Abstract

The initiation of chromosomal DNA replication is rigidly regulated to ensure that it occurs in a cell cycle-coordinated manner. To ensure this in Escherichia coli, multiple systems regulate the activity of the replication initiator ATP-DnaA. The level of ATP-DnaA increases before initiation after which it drops via DnaA-ATP hydrolysis, yielding initiation-inactive ADP-DnaA. DnaA-ATP hydrolysis is crucial to regulation of initiation and mainly occurs by a replication-coupled feedback mechanism named RIDA (regulatory inactivation of DnaA). Here, we report a second DnaA-ATP hydrolysis system that occurs at the chromosomal site datA. This locus has been annotated as a reservoir for DnaA that binds many DnaA molecules in a manner dependent upon the nucleoid-associated factor IHF (integration host factor), resulting in repression of untimely initiations; however, there is no direct evidence for the binding of many DnaA molecules at this locus. We reveal that a complex consisting of datA and IHF promotes DnaA-ATP hydrolysis in a manner dependent on specific inter-DnaA interactions. Deletion of datA or the ihf gene increased ATP-DnaA levels to the maximal attainable levels in RIDA-defective cells. Cell-cycle analysis suggested that IHF binds to datA just after replication initiation at a time when RIDA is activated. We propose a model in which cell cycle-coordinated ATP-DnaA inactivation is regulated in a concerted manner by RIDA and datA.AAA+ | bacteria | initiation regulation | in vitro reconstitution | nucleoprotein complex

The Arg Fingers of Key DnaA Protomers Are Oriented Inward within the Replication Origin oriC and Stimulate DnaA Subcomplexes in the Initiation Complex

“…ADP-DnaA is much more abundant than ATP-DnaA during the cell cycle, except at the time of replication initiation (10,55). According to our results (Figs.…”

“…The loaded DnaB triggers the formation of the sister replisomes needed for bidirectional DNA synthesis (8). ATP-DnaA is converted to ADP-DnaA, which is inactive for initiation, during replication (2,7,9,10).…”

The Escherichia coli datA site promotes proper regulation of cell division

“…The datA site has recently been discovered to have (together with the IHF protein) DnaA-inactivating activity (Kasho & Katayama, 2013). In a wild-type scenario, the RIDA system is active.…”

“…The datA site is a 1 kb DNA sequence with five well conserved DnaA binding sites (DnaA-boxes) (Kitagawa et al, 1996) and several weak DnaA-boxes (Hansen et al, 2007). Recently it was shown that the datA site, together with the IHF protein, stimulates hydrolysis of the ATP bound to the DnaA protein and can therefore contribute to the inactivation of the DnaA protein in a process called DDAH (datA-dependent DnaA-ATP hydrolysis) (Kasho & Katayama, 2013 polymerase . This process, called regulatory inactivation of DnaA (RIDA), has been shown to be dependent on ongoing replication (Kurokawa et al, 1999).…”

Random versus Cell Cycle-Regulated Replication Initiation in Bacteria: Insights from Studying Vibrio cholerae Chromosome 2

“…This requires the participation of DNA-loaded clamps (DNA polymerase processivity factor DnaN) and Hda, a homolog of DnaA. A second mechanism also catalyzes the conversion of DnaA-ATP to DnaA-ADP by making use of a cluster of DnaA binding sites in a locus called datA (39).…”