Sedimentation velocity behavior of closed circular SV40 DNA as a function of superhelix density, ionic strength, counterion and temperature

Abstract

Ten closed SV40 DNA's with superhelix densities (superhelical turns per ten base pairs) 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 at high ionic strength in cesium chloride and sodium chloride solutions. The sedimentation coefficient increases as the absolute value of the superhelix density, ¦ σ o ¦, rises from a low value to 0.019, then decreases to a local minimum at 0.035, and finally increases steadily as ¦ σ o ¦ rises to 0.085. Examination of several of these DNA's in the electron microscope has suggested a plausible explanation for the observed non-monotonic variation of S with σ o . The sedimentation velocity-ethidium bromide titrations of these DNA's (Gray, Upholt & Vinograd, 1971) have been converted from the primary S 20 0.∗ versus c data, in which S 20 0.∗ 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 S 20, w 0 versus σ form, with the aid of the coefficients in the expression for the free energy of superhelix formation developed by Bauer & Vinograd (1970a). The resultant curves form a family that is approximately superimposable on the curve for S 20, w 20 versus σ 0 in the absence of ethidium bromide. Similarly transformed sedimentation velocitydye titrations for viral PM2 DNA (6 × 10 6 daltons) and a high superhelix-density λb 2b 5c DNA (25 × 10 6 daltons) have the same general character and contain both a local maximum and a local minimum. The results of a study of the dependence of the sedimentation coefficient of selected SV40 DNA's upon ionic strength, the nature of the cation and temperature are consistent with the previously reported effects of these variables (Wang, 1969a) on the rotation angle of the base pairs along the helix axis.