Properties of Closed Circular DNA

Abstract

A method for the measurement of the superhelix density, σ0, of a closed circular DNA from separation between fluorescent buoyant bands of nicked and closed circular forms in an ethidium bromide-CsCl equilibrium gradient is presented. Nicked and closed circular DNA's are banded together in a gradient formed by centrifuging a CsCl solution of density 1.56 gm/ml. containing 330 µg/ml ethidium bromide at 20°C for 48 hours in the preparative ultracentrifuge. The separation between the bands, normalized by the separation between the nicked and closed circular forms of a DNA of known superhelix density, is shown to be linearly related to the difference in superhelix density between the DNA's of known and unknown superhelix densities according to the equation Δσ0 = (0.115 ± 0.005)(Ωc - 1) where Δσ0 = σ0 - σ0* and Ωc = Δr/Δr* r/r (v1θ*-1)2/(v1θ-1)2 Δr is the separation between the bands, r is the average distance of the two bands from the center of rotation, θ is the buoyant density of the DNA and v1 is the partial specific volume of water. The asterisk refers to the reference DNA. A molecular weight dependence of the intercept is seen when λb2b5c DNA (molecular weight 25 x 106) is banded against a SV40 DNA (3 x 106 standard. The relationship was obtained by measuring the separations for DNA's whose superhelix densities were determined by sedimentation velocity-dye titrations. Native SV40 viral DNA with a superhelix density of -0.039 was used as a standard in all cases. DNA's with altered superhelix densities were prepared by closing nicked circular DNA's with polynucleotide ligase under various conditions. Ten closed SV40 DNA's with superhelix densities ranging from -0.007 to -0.085 have been prepared. This family of DNA's has been used to examine the effects of superhelix density on the sedimentation velocity behavior of closed SV40 DNA. The sedimentation coefficient increases as the absolute value of the superhelix density rises from a low value to 0.019, then decreases to a local minimum at 0.035 and finally increases steadily as |σ0| rises to 0.085. The sedimentation velocity-ethidium bromide titrations of these DNA's have been converted from the primary s020,* versus c data, in which s020,* is the standard sedimentation coefficient still uncorrected for the buoyant effect of bound ethidium chloride and c is the free ethidium bromide concentration, to the more meaningful s020,w versus σ0 form, with the aid of coefficients in the expression for the free energy of superhelix formation. The resultant curves form a family that is approximately superimposable on the curve for s020,w versus σ0 in the absence of ethidium bromide. The dependence of the sedimentation coefficient of selected SV40 DNA's upon ionic strength, the nature of the cation, and temperature is consistent with the previously reported effects of these variables on the rotation angle of the base pairs along the helix axis. Separations between open and closed circular DNA's in buoyant CsCl gradients containing the ethidium bromide analogue, propidium di-diodide, are shown to be 1.8 times larger than in ethidium bromide.