Covalently closed circular DNA molecules of low superhelix density as intermediate forms in plasmid replication.

Abstract

COVALENTLY closed circular (CCC) duplex extrachromosomal DNAs are widespread in biological systems1. Because of their physical properties and small size, extra chromosomal circular DNAs are useful for studying the biology of genetic material. Their mechanism of replication is of particular interest and has been investigated extensively. Recent studies have shown that the replication of different types of circular DNA, such as plasmids of Escherichia coli, the DNA of polyoma and SV40 viruses, and mitochondrial DNA is basically similar2,3. Non-replicating circular DNA is isolated from cells largely as covalently closed supercoiled molecules (form I DNA). Because of its topological constraints, CCC DNA has a restricted affinity for intercalating dyes such as ethidium bromide and propidium diiodide; its buoyant density in gradients of CsCl containing such dyes is consequently much greater than that of non-circular or nicked circular (form II) DNA species, which can unwind to accommodate maximal amounts of dye4. DNA molecules in the process of replication have been shown to have buoyant densities intermediate to form I and form II DNA (refs 5–12 and L. Katz, P. H. Williams, S. Sato, R. W. Leavitt, and D. R. Helinski, unpublished). In a previous study of replicative forms of a constructed recombinant plasmid (pSC134) of E. coli13, we observed material pulse labelled with radioactive thymidine that had a buoyant density in CsCl–ethidium bromide gradients even greater than that of form I DNA5. Here we report results which suggest that this material, termed “heavy replicative DNA” or hrDNA, is a covalently closed circular DNA species that has a reduced number of supercoils, and is an intermediate in the replicative process.