Studies on the initiation of plasmid DNA replication.

Abstract

The replication of the bacterial plasmids is dependent on two chromosome‐determined products (dnaA and dnaC), which are involved in the initiation of the chromosomal DNA synthesis of Escherichia coli. The inhibition of Col E1 DNA synthesis in a dnaA‐mutant (CRT 46) at the restrictive temperature, is eliminated by the addition of low doses of chloramphenicol (3 μg/ml). Under these conditions, a stimulated and relaxed synthesis of this plasmid is observed. The ability of chloramphenicol to overcome the inhibition of Col E1 DNA synthesis is dependent on the extent of the residual plasmid DNA synthesis at the time of addition of the antibiotic. Col E1 DNA synthesized at 43 °C in the presence of chloramphenicol, largely consists of supercoiled DNA circles containing RNA segments as revealed by their sensitivity to ribonuclease H of Escherichia coli and alkali. Addition of rifampicin to chloramphenicol‐treated cells causes first a slight inhibition of the relaxed Col E1 DNA synthesis at 43 °C and then a further stimulation.An immediate increase in plasmid DNA synthesis after the addition of rifampicin to chlor‐amphenicol‐treated cells at 43 °C is observed in the dnaA‐mutant carrying the antibiotic resistance factor R1. Another transmissible plasmid (Hly PM152) does not show this stimulated DNA synthesis. Stimulation of Col E1 and R1 DNA synthesis by rifampicin does not occur in chlor‐amphenicol‐treated cells of the dnaA‐mutant with a rifampicin‐resistant RNA polymerase. Chloramphenicol and/or rifampicin cannot eliminate the inhibition of plasmid replication at the restrictive temperature in a dnaC mutant (PC 2). In the presence of chloramphenicol, however, more complex Col E1 DNA molecules (dimeric and trimeric DNA forms) are synthesized at the restrictive temperature in the dnaC‐mutant.