The interaction of closed circular DNA with intercalative dyes. I. The superhelix density of SV40 DNA in the presence and absence of dye.

Abstract

Abstract The binding of the intercalating dye ethidium bromide to closed circular SV40 DNA causes an unwinding of the duplex structure and a simultaneous and quantitatively equivalent unwinding of the superhelices. The buoyant densities of both intact (I) and singly nicked (II) SV40 DNA's were measured as a function of free dye concentration, and the moles dye bound per mole nucleotide, ν, calculated from the buoyant density shifts. The binding isotherms were determined over a dye concentration range extending from 0 to 600 μg/ml. in 5.8 m -CsCl. At high dye concentrations all of the binding sites in II, but not in I, are saturated. At free dye concentrations less than 5.4 μg/ml., I has a greater affinity for dye than II. At a critical amount of dye bound, ν = νc, I and II have equal affinities, and at higher dye concentration I has a lower affinity than II. At ν = νc, there are 375 molecules of dye bound per molecule of SV40 DNA. The number of superhelical turns, τ, present in I was calculated at each dye concentration using Fuller & Waring's (1964) estimate of the angle of duplex unwinding per intercalation. With this estimate, we have calculated that the duplex has been unwound by 4500 ° at νc, and that SV40 DNA contains about −13 superhelical turns in concentrated salt solutions. The number of superhelical turns in SV40 DNA I has been evaluated as a function of the free dye concentration. The value of τ changes from −13 to zero to +45 as the free dye concentration is increased to 600 μg/ml. dye. The sedimentation coefficient of SV40 I in 1 m -NaCl decreases upon addition of dye at first slowly, and then rapidly, until the sedimentation coefficient becomes equal to that of II at the same dye concentration. At higher dye concentrations the sedimentation coefficient of I rises rapidly and appears to level off. The sedimentation coefficient of II decreases only slightly and linearly over the same dye concentration range. From the free dye concentration at the minimum and the value of the binding constant in 1 m -NaCl given by Le Pecq (1965), we calculate that there are −16.0 ± 3.5 superhelical turns in SV40 I DNA. Our binding results have allowed us to estimate the dependence of the sedimentation coefficient of SV40 I DNA upon the number of superhelical turns in the presence of dye.