Abstract
The characterization of knots formed in duplex DNA has proved useful to infer biophysical properties and the spatial trajectory of DNA, both in free solution and across its macromolecular interactions. Since knotting, like supercoiling, makes DNA molecules more compact, DNA knot probability and knot complexity can be assessed by the electrophoretic velocity of nicked DNA circles. However, the chirality of the DNA knots has to be determined by visualizing the sign of their DNA crossings by means of electron microscopy. This procedure, which requires purifying the knotted DNA molecules and coating them with protein, is semi-quantitative and it is impracticable in biological samples that contain little amount of knotted DNA forms. Here, we took advantage of an earlier observation that the two chiral forms of a trefoil knot acquire slightly different electrophoretic velocity when the DNA is supercoiled. We introduced a second gel dimension to reveal these chiral forms in DNA mixtures that are largely unknotted. The result is a high-resolution 2D-gel electrophoresis procedure that quantitatively discerns the fractions of positive- and negative-noded trefoil knots formed in vitro and in vivo systems. This development in DNA knot analysis may uncover valuable information toward disclosing the architecture of DNA ensembles.
Highlights
The analysis of knots found in circular molecules of DNA provides precious information because knots are topological invariants that footprint the left- and right-handed turns of DNA in the tridimensional space
After prolonged gel electrophoresis, the linking number (Lk) topoisomers of supercoiled DNA molecules containing a trefoil can split into doublets that correspond to the two chiral forms of the trefoil knot (Figure 2B)
The second feature is that Lk topoisomers of negatively supercoiled DNA molecules that are unknotted can be separated from those Lk topoisomers that contain a knot by running a 2D gel in the presence of a DNA intercalator [5]
Summary
The analysis of knots found in circular molecules of DNA provides precious information because knots are topological invariants that footprint the left- and right-handed turns of DNA in the tridimensional space. DNA knots can form during the topological closure of linear DNA molecules [1]. They are formed by recombinases that rearrange intramolecular DNA sequences [2] and by type-2 topoisomerases that pass intramolecular segments of duplex DNA through each other [3,4,5]. The detection and topological characterization of these knots is not exempt of technical limitations, which often preclude the extraction of their valuable information
Sign-in/Register to view highlights & summary 
Published Version (
Free)
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call