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

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The major part of the DNA from polyoma virus has been shown to consist of circular base-paired duplex molecules without chain ends.'-' The intertwined circular form accounts for the ease of renaturation4 of this DNA and the failure of the strands to separate in strand-separating solvents. I-' In previous studiesl-3 a minor component, II, observed in variable amounts in sedimentation analyses of preparations of polyoma DNA at neutral pH, was regarded to be a linear form of the viral DNA. Both the major component I (20S) and II (16S) were infective." I In our further investigations of the minor component the following results, which are reported below, have been obtained: (1) The minor component is a ring-shaped duplex molecule. (2) It is generated by introducing one single-chain scission in component I by the action of pancreatic DNAase or chemical-reducing agents. (3) The sedimentation coefficient of II is insensitive to several single-strand scissions. (4) The conversion products, when not excessively attacked, are infective. The foregoing results raised a new problem. Why does the viral DNA, an intact duplex ring, sediment 20 per cent faster than a similar duplex ring containing one or more single-strand scissions? Experiments bearing on this problem, presented below, indicate the presence of a twisted circular structure in polyoma DNA I. A mechanism for the formation of this locked-in twisted structure is proposed. Methods.-Isolation and purification of the virus and extraction of the DNA: Two methods6 7 for purification of the virus were used. The DNA was isolated by Weil's method4 except that the phenol was freshly distilled under argon. Ultracentrifugation: Sedimentation analyses were performed in a Spinco model E ultracentrifuge by band centrifugation.8 Some of the results were recorded with the photoelectric scanning attachment.9, " Sucrose density gradient experiments were performed at 40, 30,000 rpm, and 9 hr. The 3% and 20% sucrose solutions contained SSC (0.15 M NaCl and 0.015 M Na citrate) and 0.05 M Tris chloride pH 8.0. Enzymes: Pancreatic DNAase, 1 X crystallized, was obtained from Worthington Biochemicals Corp. E. coli endonuclease I, 1000 units/ml," and E. coli phosphodiesterase, 2000 units/ml,"2 were gifts from Professor I. R. Lehman. BSA, 30% bovine albumin solution, sterile, was obtained from Armour Pharmaceutical Co. The endonuclease I, 0.12 units/Mg DNA, converted 60% of I into linear molecules in 8 min at 200 in the incubation mixture described by Lehman." Sedimentation velocity-pH titration: Fifteen Al, 40 Mg/nml DNA in SSC10, flowed from the sample well of the type III13 band-forming centerpiece onto an alkaline CsCl bulk-solution. This solution was prepared by titrating 10 ml (Harshaw Chemical Co.) optical grade CsCl, p = 1.35, with 1 M KOH in CsCl, p = 1.35, at 200 under argon, and was transferred to the cell assembly under argon. Usually four samples in a pH series were analyzed simultaneously. A Beckman research model pH meter, a general purpose probe glass electrode, and a calomel r...

The major part of the DNA from polyoma virus has been shown to consist of circular base-paired duplex molecules without chain ends.' -' The intertwined circular form accounts for the ease of renaturation4 of this DNA and the failure of the strands to separate in strand-separating solvents. I-' In previous studiesl-3 a minor component, II, observed in variable amounts in sedimentation analyses of preparations of polyoma DNA at neutral pH, was regarded to be a linear form of the viral DNA. Both the major component I (20S) and II (16S) were infective." I In our further investigations of the minor component the following results, which are reported below, have been obtained: (1) The minor component is a ring-shaped duplex molecule. (2) It is generated by introducing one single-chain scission in component I by the action of pancreatic DNAase or chemical-reducing agents. (3) The sedimentation coefficient of II is insensitive to several single-strand scissions. (4) The conversion products, when not excessively attacked, are infective.The foregoing results raised a new problem. Why does the viral DNA, an intact duplex ring, sediment 20 per cent faster than a similar duplex ring containing one or more single-strand scissions? Experiments bearing on this problem, presented below, indicate the presence of a twisted circular structure in polyoma DNA I. A mechanism for the formation of this locked-in twisted structure is proposed.Methods.-Isolation and purification of the virus and extraction of the DNA: Two methods6 7 for purification of the virus were used. The DNA was isolated by Weil's method4 except that the phenol was freshly distilled under argon.Ultracentrifugation: Sedimentation analyses were performed in a Spinco model E ultracentrifuge by band centrifugation.8 Some of the results were recorded with the photoelectric scanning attachment.9, " Sucrose density gradient experiments were performed at 40, 30,000 rpm, and 9 hr. The 3% and 20% sucrose solutions contained SSC (0.15 M NaCl and 0.015 M Na citrate) and 0.05 M Tris chloride pH 8.0.Enzymes: Pancreatic DNAase, 1 X crystallized, was obtained from Worthington Biochemicals

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