The Twisted Circular Form of Polyoma Viral DNA

Abstract

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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