Replication of plasmid DNA in temperature-sensitive DNA replication mutants of Escherichia coli

Abstract

The replication of extrachromosomal DNA elements has been studied in temperature-sensitive DNA replication mutants of Escherichia coli, in which chromosomal DNA replication immediately ceases on a shift from 30 to 42°. All these mutants carry, in addition to a small plasmid such as the colicinogenic factor E 1 (Col E 1) or minicircular DNA of E. coli 15, a large genetic element such as the colicinogenic factor V ( Col V), the hemolytic factor ( Hly) or the P 1-like plasmid of E. coli 15. At the restrictive temperature the replication of Col E 1 DNA and of minicircular DNA continues, whereas the replication of the larger factors is halted immediately upon a shift to the non-permissive temperature. The supercoiled DNA of these larger factors, synthesized at the permissive temperature, is nicked during incubation at the non-permissive temperature, whereas prelabeled supercoiled Col E 1 DNA and minicircular DNA of E. coli 15 remain intact under these conditions. The replication of Col E 1 DNA and of minicircular DNA at the non-permissive temperature proceeds in a semiconservative manner and does not represent repair. The replication mechanism of minicircular DNA appears to be similar to that of Col E 1 DNA: some DNA copies are replicated at least twice while an equal portion of DNA is not replicated at all during one generation time. This mode of replication is maintained for both plasmids at the restrictive temperature in Dna B − mutants.