Plasmid DNA isolated from bacterial cells consists of three distinct conformations, namely, linear forms resulting from one or more double strand breaks in the original molecule, open circles containing one or more single strand breaks (OC), and covalently closed circles (CCC). Circular DNA forms may also be present as concatemers comprised of two or more interlocked monomeric OC forms, CCC forms, or mixtures of both. A supercoiled structure seems to be a prerequisite for DNA replication; supercoiling of template molecules is required for initiation of DNA synthesis as well as chain elongation in the case of plasmid ColEl (Gellert et al. 1976). Electron microscopic examination of replicative intermediates from a variety of plasmids has shown that replication forks are bracketed on one side by a supercoiled region and on the other by a relaxed region, which represents the newly replicated part of the molecule (Fig. 1A). Introduction of a single strand break into one of the parental DNA strands of the replicative intermediate converts it into an open circle and enables its typical eye-structure (θ-type, see Fig. 1B; in contrast to the σ-type of a rolling circle) to be visualized.
KeywordsVortex Sucrose EDTA DMSO Sedimentation
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