Temporal sequence of events during the initiation process in Escherichia coli deoxyribonucleic acid replication: roles of the dnaA and dnaC gene products and ribonucleic acid polymerase

Abstract

Three thermosensitive deoxyribonucleic acid (DNA) initiation mutants of E. coli exposed to the restrictive temperature for one to two generations were examined for the ability to reinitiate DNA replication after returning to the permissive temperature in the presence of rifampin, chloramphenicol, or nalidixic acid. Reinitiation in the dnaA mutant was inhibited by rifampin but not by chloramphenicol, whereas reinitiation was not inhibited in two dnaC mutants by either rifampin or chloramphenicol. To observe the rifampin inhibition, the antibiotic must be added at least 10 min before return to the permissive temperature. The rifampin inhibition of reinitiation was not observed when a rifampin-resistant ribonucleic acid (RNA) polymerase gene was introduced into the dnaA mutant, demonstrating that RNA polymerase synthesizes one or more RNA species required for the initiation of DNA replication (origin-RNA). Reinitiation at 30°C was not inhibited by streptolydigin in a streptolydigin-sensitive dnaA mutant. Incubation in the presence of nalidixic acid prevented subsequent reinitiation in the dnaC28 mutant but did not inhibit reinitiation in the dnaA5 mutant. These results demonstrate that the dnaA gene product acts before or during the synthesis of an origin-RNA, RNA polymerase synthesizes this origin-RNA, and the dnaC gene product is involved in a step after this RNA synthesis event. Furthermore, these results suggest that the dnaC gene product is involved in the first deoxyribonucleotide polymerization event whereas the dnaA gene product acts prior to this event. A model is presented describing the temporal sequence of events tht occur during initiation of a round of DNA replication, based on results in this and the accompanying paper.