Partially supercoiled replication intermediates of R plasmid DNA are resistant to relaxation

Abstract

A NUMBER of bacterial plasmids, including colicinogenic plasmids, F plasmids, and R plasmids have been isolated from Escherichia coli in the form of covalently closed circular (CCC) or supercoiled DNA–protein relaxation complexes which can be converted to a nicked circular (relaxed) DNA form by treatment with such agents as SDS, Pronase, or ethidium bromide^1,2. The relaxed molecules contain a single nick in a unique strand and at a unique site of the DNA duplex^3,4. These nicks occur at the origin of replication of Col E1 and R6K (refs 5, 6). We report here that the R plasmid ROR12 in Proteus mirabilis is replicated as a partially supercoiled DNA molecule, as has been observed for replicating molecules of SV40 and polyoma viral DNA⁷, mitochondrial DNA⁷, and the DNA of several plasmids^5,6,8–10. Replicating ROR12 molecules have a broad distribution of buoyant densities between the values of the CCC DNA and nicked circular DNA in an ethidium bromide–caesium chloride (EB–CsCl) density gradient. In addition, a substantial fraction of replicating ROR12 DNA was found to have a density which was less than the value of the nicked circular form. These partially supercoiled replication intermediates of ROR12 DNA retain their supercoiled characteristics even when the DNA is isolated using lysis conditions which yield all of the non-replicating R plasmid DNA as relaxed molecules. This suggests that either replicating ROR12 molecules do not exist in the form of DNA–protein relaxation complexes as do the non-replicating plasmids or, if they do, the replicating molecules are not susceptible to relaxation in these conditions.