Light-scattering studies on supercoil unwinding

Abstract

It has been shown previously that supercoiled [unk]X174 bacteriophage intracellular DNA (mol.wt. 3.2×10⁶) with superhelix density, σ=−0.025 (−12 superhelical turns) at 25°C is best represented as a Y shape. In this work two techniques have been used to unwind the supercoil and study the changes in tertiary structure which result from changes in the secondary structure. The molecular weights from all experiments were in the range 3.2×10⁶±0.12×10⁶. In experiments involving temperature change little change in the Y shape was observed between σ=−0.027 (−13 superhelical turns, 14.9°C) and σ=−0.021 (−10 superhelical turns, 53.4°C) as evidenced by the root-mean-square radius and the particle-scattering factor P(θ). However, at σ=−0.0176 (−8 superhelical turns, 74.5°C) the root-mean-square radius fell to between 60 and 70nm from 90nm indicating a large structural change, as did alterations in the P(θ) function. In experiments with the intercalating dye proflavine from values of bound proflavine of 0–0.06mol of dye/mol equiv. of nucleotide which correspond to values of σ from −0.025 to −0.0004 (−12 to 0 superhelical turns) a similar transition was found when the superhelix density was changed by the same amount, and the molecule was shown to go through a further structural change as the unwinding of the duplex proceeded. At σ=−0.018 (−9 superhelical turns) the structure was compatible with a toroid, and at σ+−0.0004 it was compatible with a circle but at no point in the sequence of structure transitions was the structure compatible with the conventional straight interwound model normally visualized as the shape of supercoiled DNA.