JBC, Vol. 250, Issue 3, 1061-1070, Feb, 1975

Ethidium binding affinity of circular lambda deoxyribonucleic acid determined fluorometrically

D. M. Hinton and V. C. Bode

Ethidium-binding isotherms for purified circular lambda DNA, isolated from a superinfected lysogen, and for linear lambda DNA, isolated from the purified phage, were constructed from fluorescence measurements of ethidium-DNA MIXTURES. The measurements were made in 0.01 M Tris-HC1-0.001 M EDTA,pH 7.1, buffer at 20 degrees and in the same buffer containing 0.1, 0.4, or 1.0 M NaC1. When NaC1 was present, differences in the binding affinity for supercoiled and linear DNA could be quantitated. As the ethidium concentration was increased, supercoiled lambda DNA molecules bound the intercalating dye first more and then less avidly than nonsupercoiled ones. The number of potential supercoils in a circular lambda DNA molecular in the absence of dye was calculated from the amount of dye bound when it exhibited the same affinity for dye as its linear counterpart. The point of equivalent affinity shifted from 0.053 mol of dye bound per mol of nucleotide in 0.1 M NaC1 to 0.067 mol in 1.0 M NaC1. This corresponds to the removal of 164 and 206 supercoiling turns per molecule and superhelix densities in the absence of dye equal to 0.036 and 0.045 superhelical turns per 10 base pairs. If this difference in the number of supercoils reflects a salt-dependent change in the average rotation angle between base pairs of the Watson-Crick helix the angle differs by 0.32% in the two ionic environments.
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