Researchers have observed the dynamics of individual supercoiled DNA molecules

Biological context
A double-stranded DNA molecule forms a double-helix with one helical turn every 10.5 basepairs. However, if torsion is applied to it, the molecule can become 'supercoiled': the pitch of the double-helix is modified (twist) and/or plectonemic structures appear along DNA (writhe). In living cells, the degree of DNA supercoiling influences many important biological functions and needs to be carefully regulated. This regulation results from a complex balance between processes such as replication and transcription which generate torsion along DNA (figure 1), and the action of specialized enzymes called topoisomerases which are able to modify the topology of DNA molecules via a mechanism of transient DNA strand breakage and religation.

DNA dynamics are an important aspect of the supercoiling regulation process. The single-molecule experiments performed at Delft University made it possible to follow directly the motion of individual supercoiled DNA molecules in real time, yielding quantitative information about these dynamics.

A new experimental approach combining magnetic tweezers with optical trapping
These experiments were mainly based on the 'magnetic tweezers' technique, which relies on the attachment of the two ends of DNA molecules respectively to a surface and to a magnetic bead, on which forces can be exerted through a pair of permanent magnets. This technique allows one to apply torsion to DNA molecules by rotating the magnets and, therefore, the supercoiling state of the DNA molecules can be precisely controlled.

In the new experiments performed in Delft, magnetic tweezers were supplemented with an optical trap, allowing for a sudden perturbation of a DNA molecule initially at equilibrium (figure 2a). Typically, the extension of the DNA molecule under study, initially imposed by the external force created on the bead by the magnets , was reduced by optically trapping the bead and moving the trap position towards the surface. Subsequently shutting off the laser trap led to motion of the magnetic bead back to its equilibrium position under the magnetic force, a process during which a part of the plectonemes initially present along DNA were removed. Alternatively, dynamic studies were also performed using an enzyme which creates a single-stranded break on a specific site of the DNA molecules (figure 2b). The creation of such a break leads to the complete enzymatic relaxation of the torsional constraint, and therefore to an increase of the extension of the DNA molecules, as supercoils are relaxed.

The results support fast dynamics of supercoiled DNA
The dynamics of supercoiled DNA observed both in combined tweezers and enzymatic experiments could be described in a very satisfying way with a simple theoretical model, whose main assumption is a fast internal dynamics of DNA. Surprisingly, the relaxation of plectonemic structures that occured during these experiments did not significantly slow down the dynamics, and the dynamics were dominated by DNA stretching.

This study, the first to address the dynamics of supercoiled DNA at the single-molecule level, provides a solid baseline for the dynamics of supercoiled DNA in the absence of proteins. In the future, Delft researchers are interested in extending this work to situations closer to those occurring in living cells, in which various proteins are bound to DNA. It will be interesting to determine whether the presence of such proteins significantly modifies DNA dynamics.

Reference: "Fast dynamics of supercoiled DNA revealed by single-molecule experiments", Aurélien Crut, Daniel A. Koster, Chris H. Wiggins, Ralf Seidel and Nynke H. Dekker, Proc. Natl. Acad. Sci. (USA), 17 July 2007. 

For more information, please contact Dr. Aurélien Crut (telephone: +31 (0)15 278 35 52) or Dr. Nynke Dekker (telephone: +31 (0)15 278 32 19).