Effect of ethidium on the torsion constants of linear and supercoiled DNAs.

The torsion elastic constants (alpha) of linear pBR322 (4363 bp) and pUC8 (2717 bp) DNAs and supercoiled pBR322 and pJMSII (4375 bp) DNAs are measured in 0.1 M NaCl as a function of added ethidium/base-pair (EB/BP) ratio by studying the fluorescence polarization anisotropy (FPA) of the intercalated ethidium. The time-resolved FPA is measured by using a picosecond dye laser for excitation and time-correlated single photon counting detection. Previously developed theory for the emission anisotropy is generalized to incorporate rotations of the transition dipole due to excitation transfer. The excitation transfers are simulated by a Monte Carlo procedure (Genest et al., Biophys. Chem. 1 (1974) 266-278) and the consequent rotations of the transition dipole are superposed on the Brownian rotations. After accounting for excitation transfer, the torsion constants of the linear DNAs are found to be essentially independent of intercalated ethidium up to a binding ratio r = 0.10 dye/bp. Dynamic light scattering measurements on linear pUC8 DNA confirm that the torsion constant is independent of binding ratio up to r = 0.20 dye/bp. If alpha d denotes the torsion constant between ethidium and a base-pair, and alpha 0 that between two base-pairs, then our data imply that alpha d/alpha 0 lies in the range 0.65 to 1.64 with a most probable value of 1.0. The torsion constants of supercoiled DNAs decrease substantially with increasing binding ratio even after accounting for excitation transfer. At the binding ratio r* = 0.064, where the superhelix density vanishes and superhelical strain is completely relaxed, the torsion constant of the supercoiled pBR322 DNA/dye complex lies below that of the corresponding linear DNA/dye complex by about 30%. This contradicts the conventional view according to which linear, nicked circular, and supercoiled DNA/dye complexes with r = r* should coexist with the same concentration of free dye, display the same distribution of bound dye, and exhibit identical secondary structures, twisting and bending rigidities, and FPA dynamics. These and other observations imply the existence of metastable secondary structure in freshly relaxed supercoiled DNAs. A tentative explanation is presented for these and other unexpected observations on supercoiled DNAs.