Limiting DNA replication in E. coli to once and only once per cell cycle

In E. coli the origin of chromosomal replication is inactivated after initation has occurred. This inactivation, termed “origin sequestration”, is the first line of defense against overinitiation. Sequestration provides a time window during which the origin is unavailable for initiation and during which the initiation potential can be reduced by other means. At this point three different mechanisms have been suggested to participate in lowering the initiation potential. These are: (i) titration of DnaA proteins to newly replicated chromosomal elements, (ii) sequestration of the dnaA gene promoter, and (iii) regulation of the activity of the DnaA initiator protein by an ATP/ADP switch. During our work we have investigated the two latter mechanisms and their contribution to lowering the initiation potential. Our data suggest that non of these mechanisms on their own are sufficient for this process.