Circular duplex DNA

Bacterial plasmids are small, circular, duplex DNA molecules whose natural function is to confer antibiotic resistance to the host cell. Plasmids have several properties that make them extremely useful as cloning vectors. They exist as single or multiple copies within the bacterium and replicate independently from the bacterial DNA. The complete DNA sequence of many plasmids is known hence, the precise location of restriction enzyme... 

A closed circular duplex DNA has a 90 base-pair segment of alternating G and C residues. Upon transfer to a solution containing a high salt concentration, this segment undergoes a transition from the B conformation to the Z conformation. 

Figure 27-24 Structures of RecA protein spiral filaments. (A) RecA protein filament formed on circular duplex DNA in the presence of ATP(Y-S), shadowed with Pt and seen by electron microscopy. (B) Diagram of RecA bound to duplex DNA in the presence of ATP(y-S), as determined by electron microscopy. RecA monomers are shown as spheres, but their exact shape is unknown. (C) Diagram of RecA spiral filament in crystals of RecA protein free of DNA, based on X-ray crystallographic data. Arrows indicated alignment of monomers. From Howard-Flanders, West, and Stasiak.536...

Supercoiling of circular duplex DNA is quantitatively considered in terms of the linking number (L), an integer that specifies the number of complete turns made by one strand around the other. The linking number can change only if a covalent linkage in the DNA backbone is broken and reformed. Enzymes called topoisomerases (see chapter... 

Electrophoretic patterns of highly supercoiled or partially supercoiled DNA. Strip A represents a sample of circular duplex DNA obtained by deproteinization of the animal virus SV40. In strips B and C the DNA has been exposed for increasing times to an enzyme (topoisomerase) that catalyzes relaxation. Adjacent bands differ by 1 in linking number. (From W. Keller, Characterization of purified DNA-relaxing enzyme from human tissue culture cells, Proc. Natl. Acad. Sci. USA 72 2553, 1975.)... 

Reactions catalyzed by purified recA protein in vitro. RecA catalyzes a number of different reactions between DNA strands, all of them involving the unwinding and winding of base-paired structures, (a) D-loop formation by interaction between supercoiled circular duplex DNA and single-stranded DNA. (b) Strand exchange between a gapped circular duplex structure and a linear duplex structure, (c) Complex formation between two helices, one of which is gapped. 

Why does circular duplex DNA renature more rapidly than linear duplex DNA ... 

Many plasmids and bacteriophages have been ingeniously modified to enhance the delivery of recombinant DNA molecules into bacteria and to facilitate the selection of bacteria harboring these vectors. Plasmids are circular duplex DNA molecules occurring naturally in some bacteria and ranging in size from 2 to several hundred kilobases. They carry genes for the inactivation of antibiotics, the production of toxins, and the breakdown of natural products. These accessory chromosomes can replicate independently of the host chromosome. In contrast with the host genome, they are dispensable under certain conditions. A bacterial cell may have no plasmids at all or it may house as many as 20 copies of a plasmid. 

Figure 6.13. Genetic Map of the Plasmid PBR322. This plasmid carries two genes for antibiotic resistance. Like all other plasmids, it is a circular duplex DNA.

The ability to supercoil, relax, knot, and catenate closed-circular duplex DNA. 

Retroviruses of vertebrates are, perhaps, the most widely studied class of eukaryotic transposable elements. These RNA viruses use reverse transcriptase to synthesize a circular duplex DNA, which can integrate into many sites of the host cell chromosome. The integrated retroviral genome bears remarkable resemblance to a bacterial composite transposon (compare Figure 25.38 with Figure 25.35). 

A synthetic oligodeoxynucleotide mismatched at a single nucleotide to a specific complementary site on a wild-type circular < >X174 DNA can be used to produce a defined point mutation, thus in vitro incorporation into a closed circular duplex DNA by elongation with DNA polymerase and ligation followed by transfection... 

RadlofF, R., Bauer, W., and Vinograd, J. (1967) A dye-buoyant-density method for the detection and isolation of closed circular duplex DNA in FleLa cells. Proc. Natl. Acad. Sci. U.S.A., 57, 1514-1521. 

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