A nucleotide switch as the dominant control mechanism preventing premature re-initiation of chromosomal DNA replication in <i>Escherichia coli</i>
Highly regulated initiation of chromosomal replication is a critical part of cell cycle regulation in prokaryotes and eukaryotes, ensuring that a given cell's genome is duplicated faithfully once, and only once, during the cell cycle. For the bacterium E. coli, it has been proposed that at least three separate, complementary mechanisms contribute to tightly regulated initiation of chromosomal DNA replication from the replicative origin, oriC, by preventing the occurrence of premature re-initiation events. These include: sequestration of oriC from the DnaA replication initiator protein by SeqA, the titration of DnaA away from oriC by the datA chromosomal locus, and the accelerated hydrolysis of the initiator, DnaA, from its active, ATP-bound form to its inactive, ADP-bound form by RIDA (regulatory inactivation of DnaA). This work investigates the physiological relevance of RIDA activity through the construction and analysis of an E. coli strain deficient in Hda protein, which has recently been identified as a necessary component of RIDA. The results demonstrate that the DnaA nucleotide switch, in which the alternating nucleotide-bound forms of the protein perform different functions, is physiologically relevant for maintaining controlled initiation of DNA replication, but that this regulatory mechanism is not essential for cell growth and survival. In addition, contrary to the widely held belief that sequestration of the origin, initiator titration, and RIDA complement one another, genomic DNA microarray analysis reveals that RIDA is the dominant mechanism to inhibit premature re-initiation from oriC in vivo, while origin sequestration by SeqA and initiator titration by datA do not appear to contribute as significantly to the prevention of overinitiation. In addition, the Hda protein, which is necessary for RIDA, appears to be recruited to the RK2 broad-host range plasmid and negatively affects plasmid replication and maintenance in the E. coli host, demonstrating that Hda-mediated RIDA is not unique to regulating initiation from oriC. DnaA is also shown to possibly be a molecular coordinator of DNA replication and gene expression through the action of its alternating nucleotide-bound state.