The interaction of closed circular DNA with intercalative dyes: III. Dependence of the buoyant density upon superhelix density and base composition

Abstract

The binding isotherms for the binding of ethidium bromide to closed circular DNA have been calculated for various values of the superhelix density of the dye-free molecule. These calculations are based upon the binding equation for closed DNA, which was derived previously (Bauer & Vinograd, 1970) and which takes into account the free energy associated with superhelix formation. The difference in the binding of dye by the closed and open forms gives rise to a difference in the buoyant densities of these molecules in the presence of dye. The buoyant density difference is shown to be linearly related to the initial superhelix density. This relation forms the basis for a convenient method for determining the superhelix density of an unknown closed DNA in the preparative ultraeentrifuge, to be described by Gray, Upholt & Vinograd (manuscript in preparation). The buoyant densities of the open DNA's, Micrococcus lysodeikticus, Clostridium, perfringens, crab d(A-T), viral φX, and SV40 II, were measured as a function of dye concentration. These data are helpful in the identification of the various DNA's in a mixture, as observed in many applications of the cesium chlorideethidium bromide buoyant density separation procedure (Radioff, Bauer & Vinograd, 1967). The data also make possible the use of any DNA of known base composition as a marker in buoyant density experiments in the presence of dye.