Beyond the static DNA model of Watson and Crick

DNA supercoiling, the coiling of the Watson-Crick double helix about itself, affects every aspect of how DNA is read, how it is replicated and transcribed, and its multiplicity of interactions with biological molecules. Despite its importance, much about supercoiled DNA (positively supercoiled DNA, in particular) remains unknown. We utilized electron cryo-tomography to investigate the 3D structures of individual and highly pure DNA minicircles, 336 bp in length, with specific and defined degrees of positive or negative supercoiling. Minicircles in each supercoiling state (topoisomer) adopt a wide distribution of 3D conformations unique to that specific supercoiling level. Probing for disruptions to base pairing revealed that localized distortions from the DNA double helix, evident as exposed DNA bases, allow the DNA to adopt conformations that would otherwise be energetically unfavorable. Counterintuitively, we also detected exposed bases in positively supercoiled minicircles beyond a sharp supercoiling threshold, probably as a consequence of extreme bending strain in the highly writhed minicircles. Our data support the "cooperative kinking model" of Lionberger, Stasiak, and colleagues, in which sharp bending at one location on the supercoiled minicircle induces kinking at a DNA site diametrically opposite. Modeling these DNA bending sites, we simulated how they modify the supercoiling-dependent 3D shapes of the minicircles. These experiments revealed unexpected DNA sequence- and supercoiling-dependent structural alterations in DNA and are a step toward creating gene therapy vectors with specific shapes for use in treating human diseases.