A Direct Observation of Highly Bent and Twisted DNA at the Single Molecule Level

Many DNA-binding proteins interact with twisted or bent DNA. To characterize the activity of these proteins as a function of the torsional and bending stresses, we must first understand how these mechanical stresses affect the DNA tertiary structure (topology). To experimentally define this relationship on scales that are biologically relevant to DNA-binding proteins requires DNA molecules which stably maintain high degrees of stress and deformation on a length scale appreciably below the persistence length. DNA minicircles of ∼100bp in size offer a unique opportunity to achieve our required specifications. We have prepared circular DNA constructs (100bp, 106bp, and 108bp) sustaining comparable magnitudes of bending stress and varying degrees of torsional stress (which arises when linear DNA molecules of a non-integral number of helical turns are circularized). Using cryo-electron microscopy (cryo-EM) combined with 3-D image reconstruction, we have been able to quantitatively characterize the structural details at the molecular level of the topological effects of torsional stress within these minicircle constructs. We have observed the three species of minicircles under conditions of both weak and mild electrostatic repulsion, and measured the observed distributions of curvature (indicative of kink formation) and writhe (reflective of torsional stress). Despite the significant torsional stress sustained within the most highly stressed construct, all three are roughly planar, though the writhe and curvature distributions do depart significantly from theoretically predicted values. We are attempting to resolve the discrepancies between theoretical expectations and our observed experimental data using Brownian dynamics simulations of DNA minicircles sustaining varying degrees of torsional stress. We expect that this work will begin to define the behavior of highly stressed DNA at biologically relevant scales, and will broaden our understanding of how sub-persistence length DNA responds to mechanical stress.