Effect of Bending Strain on the Torsion Elastic Constant of DNA

The torsion constants of both circular and linear forms of the same 181 bp DNA were investigated by time-resolved fluorescence polarization anisotropy (FPA) of intercalated ethidium. The ratio of intrinsic ethidium binding constants of the circular and linear species was determined from the relative fluorescence intensities of intercalated and non-intercalated dye in each case. Possible changes in secondary structure were also probed by circular dichroism (CD) spectroscopy. Upon circularization, the torsion constant increased by a factor of 1.42, the intrinsic binding constant for ethidium increased by about fourfold, and the CD spectrum underwent a significant change. These effects are attributed to an altered secondary structure induced by the bending strain. Quantitative agreement between torsion constants obtained from the present FPA studies and previous topoisomer distribution measurements on circular DNAs containing 205 to 217 bp removes a long-standing apparent discrepancy between those two methods. After storage at 4°C for eight months, the torsion constant of the circular DNA increased by about 1.25-fold, whereas that of the linear DNA remained unchanged. For these aged circles, both the torsion constant and intrinsic binding constant ratio lie close to the corresponding values obtained previously for a 247 bp DNA by analyzing topoisomer distributions created in the presence of various amounts of ethidium. The available evidence strongly implies that torsion constants measured for small circular DNAs with less than 250 bp are specific to the altered secondary structure(s) therein, and are not applicable to linear and much larger circular DNAs with lower mean bending strains.