Covalently closed circular duplex DNA's are now known to be widespread among living organisms. This DNA structure, originally identified in polyoma viral DNA,1' 2 has been assigned to the mitochondrial DNA's in ox3 and sheep heart,4 in mouse and chicken liver,3 and in unfertilized sea urchin egg.5 The animal viral DNA's-polyoma, SV40, rabbit7 and human8 papilloma-the intracellular forms of the bacterial viral DNA's 4X174,9 10lambda,", 12 1\113,13 and P2214 -and a bacterial plasmid DNA, the colicinogenic factor E2,15 have all been shown to exist as closed circular duplexes. Other mitochondrial DNA's'6' 17 and a portion of the DNA from boar sperm'8 have been reported to be circular, but as yet have not been shown to be covalently closed.The'physicochemical properties of closed circular DNA differ in several respects from those of linear DNA or of circular DNA containing one or more singlestrand scissions."9 The resistance to denaturation,2' 20 the sedimentation velocity in neutral and alkaline solution, and the buoyant density in alkaline solution are all enhanced in the closed circular molecules. These three effects are a direct consequence of the topological requirement that the number of interstrand crossovers must remain constant in the closed molecule.The principal methods currently used for the detection and the isolation of closed circular DNA are based on the first two general properties. In this communication we describe a method based on the buoyant behavior of closed circular DNA in the presence of intercalating dyes.The binding of intercalative dyes has recently been shown to cause a partial unwinding of the duplex structure in closed circular DNA. 22-24 In such molecules any unwinding of the duplex causes a change in the number of superhelical turns, so that the total number of turns in the molecule remains constant. A small and critical amount of dye-binding reduces the number of superhelical turns to zero. Further dye-binding results in the formation of superhelices of the opposite sign or handedness. The creation of these new superhelices introduces mechanical stresses into the duplex and a more ordered conformation into the molecule. These effects increase the free energy of formation of the DNA-dye complex. The maximum amount of dye that can be bound by the closed molecule is therefore smaller than by the linear or nicked circular molecule. Correspondingly, since the buoyant density of the DNA-dye complex23' 25 is inversely related to the amount of dye bound, the buoyant density of the closed circular DNA-dye complex at saturation is greater than that of the linear or nicked circular DNA-dye complex.23 Bauer and Vinograd have shown that the above effect results in a buoyant density difference of approximately 0.04 gm/ml in CsCl containing saturating amounts of ethidium bromide, an intercalating dye extensively studied by Waring26 and Le Pecq.27 1514

Highly purified nicking-closing enzyme from mouse cells in 20-fold enzyme/substrate excess converts closed circular native PM2, ColEl, and Minicol DNA into limit product sets of DNAs. Each set has a mean degree of supercoiling of approximately zero. The individual species in the sets differ by Ar = i1, i42, etc., and the relative masses fit a Boltzmann distribution. It was also demonstrated that "nonsupercoiled" closed circular duplex molecules serve as substrates for the nicking-closing enzyme, and that a distribution of topological isomers is generated. Polynucleotide ligase, acting on nicked circular DNA, forms under the same conditions, the same set of closed DNAs. The latter enzyme freezes the population into sets of molecules otherwise in configurational equilibrium in solution.Nicking-closing (N-C) activities that alter the topological winding number (a) of closed circular DNA occur widely in nature (1-5). The topological winding number is the number of revolutions that one strand makes about the other if the molecule is constrained to lie in a plane. The activities have been demonstrated to be enzymatic with proteins from Escherichia coli (6), mouse (7), and human (8) cells in culture. The N-C enzyme from mouse LA9 cell nuclei, purified to homogeneity in good yield, is a major constituent of chromatin and accounts for about 1% of the total protein (H-P.Vosberg and J. Vinograd, unpublished work). It is similar to other eukaryotic N-C enzymes in its ability to relax both positive and negative superhelical turns. A probable in vivo role for the enzyme is to provide the transient swivels required for DNA replication. Such swivels may also be required in transcription, and in the condensation and decondensation of chromatin.In this study we have examined the limit product of the action of N-C enzyme on several closed circular DNAs by gel electrophoresis. Under appropriate analytical conditions, the limit product separates into a set of species differing in topological winding number. Individual species, isolated from the set, regenerate the original distribution upon incubation with the N-C enzyme. We view the foregoing as the consequence of four necessary events: nicking of the DNA, relaxation, random rotation about the swivel, and closure (Fig. 1). A set of species is also found when E. colh polynucleotide ligase is used to close a nicked circular DNA (9, 10). It is shown here that distribution of products obtained with ligase is indistinguishable from that obtained with N-C enzyme when the incubation conditions are the same for both reactions.The relative masses of the species, when plotted against a, fall on a Gaussian curve. Such a curve is anticipated for a Boltzmann distribution, when the energy of supercoiling is Abbreviations: N-C, nicking-closing; EtdBr, ethidium bromide.proportional to the square (11) of the degree of supercoiling. The similarity of the products obtained with two different enzyme systems and the further similarities of the values of the free energy of supercoiling obtained...

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A fraction of the covalently closed mitochondrial DNA in mouse L cells contains a replicated heavy-strand segment that is hydrogen bonded to the circular light strand. The inserted single strand is dissociable from the circular duplex at an elevated temperature.M1itochondrial DNA from animal cells occurs in the form of closed-circular molecules with a molecular weight of about 10 X 100 (1). In the course of a study of the mechanism of replication of this DNA in two strains of mouse L cells, we have found that about one half of the covalently closed molecules in exponentially growing cells contain a short threestranded DNA region, which we have called a D-loop, or displacement loop (Fig. 1). The D-loop appears to have been formed in the closed DNA by displacement synthesis of a short progeny strand with a specific region of the mitochondrial light strand serving as a template. This communication presents the results of experiments designed to elucidate the -structure and properties of this new kind of closedcircular DNA and considers its role as an intermediate in the replication process. MATERIALS AND METHODSThe two cell lines studied were the LD line (our designation) obtained from C. Schildkraut, Albert Einstein College of Medicine, New York, and the LA9 line isolated by Littlefield (2) and obtained from L. Crawford, Imperial Cancer Research Fund, London. Mitochondrial DNA (mtDNA) in the LD cells occurs in the form of circular dimers with a contour length of 10 Am. The LA9 cells contain monomeric mtDNA.The spinner-adapted cells were grown in Dulbecco's modification of Eagle's phosphate medium, containing 10% calf serum. The generation times of the LA9 and LD cells were 22 and 20 hr, respectively. Exponentially growing cells, about 2 X 105 cells/ml, were labeled with 0.5 MCi/ml of [3H]thymidine (Schwarz/Mann), 15.6 Ci/mmol, for 3 hr before harvest.