Supercoiling-dependent DNA binding: quantitative modeling and applications to bulk and single-molecule experiments

Abstract

DNA stores our genetic information and is ubiquitous in applications, where it interacts with binding partners ranging from small molecules to large macromolecular complexes. Binding is modulated by mechanical strains in the molecule and can change local DNA structure. Frequently, DNA occurs in closed topological forms where topology and supercoiling add a global constraint to the interplay of binding-induced deformations and strain-modulated binding. Here, we present a quantitative model with a straight-forward numerical implementation of how the global constraints introduced by DNA topology modulate binding. We focus on fluorescent intercalators, which unwind DNA and enable direct quantification via fluorescence detection. Our model correctly describes bulk experiments using plasmids with different starting topologies, different intercalators, and over a broad range of intercalator and DNA concentrations. We demonstrate and quantitatively model supercoiling-dependent binding in a single-molecule assay, where we directly observe the different intercalator densities going from supercoiled to nicked DNA. The single-molecule assay provides direct access to binding kinetics and DNA supercoil dynamics. Our model has broad implications for the detection and quantification of DNA, including the use of psoralen for UV-induced DNA crosslinking to quantify torsional tension in vivo, and for the modulation of DNA binding in cellular contexts.

DNA supercoiling enhances DNA condensation by ParB proteins

“…We exploited this feature to directly introduce supercoiling of the desired handedness, either positive or negative supercoiling, to the DNA molecule. We achieved this by changing the concentrations of the intercalator dye SYTOX Orange (SxO) that is also used for fluorescent visualization of the DNA [40][41][42] (Fig. 1B, see Methods for details).…”

“…1E; Movie S1). The effect of supercoiling on ParB binding and condensation was monitored for both supercoiled and torsionally unconstrained DNA molecules in the same field of view 42 (Fig. S1).…”