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Results demonstrate that DnaA is membrane-bound consistent with the finding that it requires phospholipids in a lipid bilayer to convert from inactive to an active replicative form.

The role of electrostatic forces in the interaction of DnaA protein and lipid in Escherichia coli has been studied through flotation gradient ultracentrifugation and intrinsic tryptophan fluorescence. DNA initiates chromosomal replication in E coli but is influenced by the behavior of the lipids. Results indicate that anionic phospholipids are needed in DnaA-lipid binding but is inhibited by increasing ionic strength. This finding suggests that electrostatic forces have a role to play in DnaA-lipid binding.

The beta subunit of DNA polymerase III in Escherichia coli is essential for negative regulation of the initiator protein, DnaA. This ability to inactivate DnaA depends on the beta subunit's assembly as a sliding clamp on DNA and a partially purified factor IdaB protein. The DnaA inactivation is further stimulated by DNA synthesis, suggesting a close linkage between initiator inactivation and replication. In vivo, DnaA takes on the ADP form. This supports the hypothesis that the initiator is negatively regulated by action of the replicase.

The timing of the replication of the Escherichia coli chromosome depends on methylated form of oriC, SeqA protein, DnaA protein and acidic phospholipids. Methylated oriC stimulates transcription while the SeqA protein inhibits transcription. Inhibition of replication is either due to the binding of SeqA to oriC to stop the formation of an open complex or by controlling the activity of the DnaA protein, an inhibiting factor. The acidic phospholipids and oriC converts the inactive ADP-DnaA form into the active ATP-bound form. Sequestering prevents replication in recently formed fragments.

Research reveals that the inactivation of DnaA component of the homologous to DnaA (Hda) protein is required for controlled initiation of chromosomal replication in Escherichia coli as shown in a strain deficient in hda gene.