Effect of ethidium binding and superhelix density on the supercoiling free energy and torsion and bending constants of p30δ DNA

Effects of Na+and Mg2+on the Structures of Supercoiled DNAs: Comparison of Simulations with Experiments

“…Agreement with the measured D 0 values at different superhelix densities was much better, when a relatively small torsional rigidity (C = 1.36 × 10 −19 dyne cm) was employed instead of the substantially larger and more popular value, C = 3.0 × 10 −19 dyne cm. In addition, simulations of p30d DNA (4752 bp) using P 0 = 500 Å , d = 50 Å , and C = 2.0 × 10 −19 dyne cm, which is nearly identical to the independently measured value for that same DNA, yielded supercoiling free energies in good agreement with the experimental data in 0.1 M NaCl over the full range of superhelix density from s = 0 to −0.05 Clendenning et al, 1994). When the higher torsional rigidity (C = 3.0 × 10 −19 dyne cm) was used, the agreement with experiment was rather poor.…”

“…Samples with various values of the linking difference were prepared by incubating the native supercoiled DNA with topoisomerase I from chicken erythrocytes at 37°C in 100/10/1 STE buffer (pH 7.8), in the presence of various amounts of ethidium, as described earlier Clendenning et al, 1994). Reactions were halted after 30 minutes by addition of phenol.…”

“…brightest band) in each sample at 21°C in 100/10/1 STE buffer was determined by the band counting method of Keller (1975), as described Clendenning et al, 1994).…”

Effect of polyethylene glycol on the supercoiling free energy of DNA

“…Both experiments 24 and simulations 23 show that Eq. (2) holds over the range of superhelix density ( ϵ ⌬l/ l 0 ) from 0 to native (ϳ Ϫ0.05) in 0.1M NaCl.…”

“…24,26 In addition, a quantitative image of the distribution of fluorescence intensity throughout the gel was obtained by using a UVP Gel Documentation System and UVP Image Store 7500 software (UVP, Inc.). The integrated fluorescence intensity associated with each band was determined after background subtraction with the aid of an Image Quant program, Version 3.3, from Molecular Dynamics.…”