EFFECT OF BENDING STRAIN ON THE TORSION ELASTIC-CONSTANT OF DNA

The torsion constants of both circular and linear forms of the same 18 1 bp DNA were investigated by time-resolved fluorescence polarization anisotropy (FTA) of intercalated ethidium. The ratio of intrinsic ethi dium binding constants of the circular and linear species was determin ed 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 circul arization, the torsion constant increased by a factor of 1.42, the int rinsic binding constant for ethidium increased by about fourfold, and the CD spectrum underwent a significant change. These effects are attr ibuted to an altered secondary structure induced by the bending strain . Quantitative agreement between torsion constants obtained from the p resent FPA studies and previous topoisomer distribution measurements o n circular DNAs containing 205 to 217 bp removes a long-standing appar ent discrepancy between those two methods. After storage at 4 degrees 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 bind ing constant ratio lie close to the corresponding values obtained prev iously 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 D NAs with less than 250 bp are specific to the altered secondary struct ure(s) therein, and are not applicable to linear and much larger circu lar DNAs with lower mean bending strains. (C) 1996 Academic Press Limi ted