DNA supercoiling-induced shapes alter minicircle hydrodynamic properties

Abstract

DNA in cells is organized in negatively supercoiled loops. The resulting torsional and bending strain allows DNA to adopt a surprisingly wide variety of 3-D shapes. This interplay between negative supercoiling, looping, and shape influences how DNA is stored, replicated, transcribed, repaired, and likely every other aspect of DNA activity. To understand the consequences of negative supercoiling and curvature on the hydrodynamic properties of DNA, we submitted 336 bp and 672 bp DNA minicircles to analytical ultracentrifugation (AUC). We found that the diffusion coefficient, sedimentation coefficient, and the DNA hydrodynamic radius strongly depended on circularity, loop length, and degree of negative supercoiling. Because AUC cannot ascertain shape beyond degree of non-globularity, we applied linear elasticity theory to predict DNA shapes, and combined these with hydrodynamic calculations to interpret the AUC data, with reasonable agreement between theory and experiment. These complementary approaches, together with earlier electron cryotomography data, provide a framework for understanding and predicting the effects of supercoiling on the shape and hydrodynamic properties of DNA.

DNA-based assay for calorimetric determination of protein concentrations in pure or mixed solutions

“…Samples were centrifuged at 24,000, 32,000, and 40,000 rpm, with a minimum equilibration time of 30 hours per speed. Buoyant molecular weights were determined using Heteroanalysis and PSV values were calculated using the Svedberg equation [37,38]. All experiments were conducted in standard NaCl buffer, with a density of 1.005584 g/ml.…”