Multiple pathways regulating DnaA function in Escherichia coli: Distinct roles for DnaA titration by the datA locus and the regulatory inactivation of DnaA

Escherichia coli DnaA protein forms a multimeric complex at the chromosomal origin of replication (oriC), where a series of initiation reactions occurs and DNA polymerase III holoenzyme is loaded. The ATP-bound form of DnaA, which is active for initiation, is converted to the inactive ADP-bound form through interaction with the sliding clamp, the β subunit of DNA polymerase III holoenzyme loaded on DNA. This negative regulation, termed RIDA, is required for preventing untimely initiations. Here, we asked if RIDA is functionally related to another negative regulation, DnaA titration by the datA site. The datA site can harbor hundreds of DnaA molecules, and is also required for preventing untimely initiations. We reveal here that, in growing cells of the datA⁺ and datA-deleted strains, the ATP-DnaA levels were both maintained in a limited range of about 20-30% of the total ATP- plus ADP-DnaA molecules. This indicates that RIDA functions in the absence of datA. In synchronized datA-deleted cells, the ATP-DnaA level fluctuated in a manner similar to that observed in datA⁺ cells. This suggests that RIDA operates independent from DnaA titration to datA. We suggest that these two mechanisms may play complementary roles during the cell cycle to prevent untimely initiations and thus ensure the scheduled initiation.