DnaA Protein Is Sensitive to a Soluble Factor and Is Specifically Inactivated for Initiation of in Vitro Replication of the Escherichia coli Minichromosome

DnaA protein loses the capacity to initiate chromosomal replication when treated with a soluble cell extract. This inactivation depends upon DNA and hydrolyzable ribonucleoside triphosphate. The extract does not affect the activities of other replicative proteins or the ability of DnaA to initiate replication of single-stranded DNA that contains a DnaA-binding hairpin, indicating that the inhibitory effect is specific for the action of DnaA at oriC. Gel filtration experiments implicate a 150-kDa factor as being responsible. Mutant DnaAcos protein, which causes overinitiation in vivo, is insensitive to the inactivating factor, suggesting a requirement for this negative control in vivo. We propose that a soluble factor controls initiation through down-regulation of DnaA protein.