Internal dynamics of supercoiled DNA molecules

Abstract

The intramolecular diffusive motion within supercoiled DNA molecules is of central importance for a wide array of gene regulation processes. It has recently been shown, using fluorescence correlation spectroscopy, that plasmid DNA exhibits unexpected acceleration of its internal diffusive motion upon supercoiling to intermediate density. Here, we present an independent study that shows a similar acceleration for fully supercoiled plasmid DNA. We have developed a method that allows fluorescent labeling of a 200-bp region, as well as efficient supercoiling by Escherichia coli gyrase. Compared to plain circular or linear DNA, the submicrosecond motion within the supercoiled molecules appears faster by up to an order of magnitude. The mean-square displacement as a function of time reveals an additional intermediate regime with a lowered scaling exponent compared to that of circular DNA. Although this unexpected behavior is not fully understood, it could be explained by conformational constraints of the DNA strand within the supercoiled topology in combination with an increased apparent persistence length.

Figure 1

Schematic representation of the sample preparation.

Figure 2

(a) Normalized correlation function, G(t), of the isolated labeled fragment S. (b) Mean-square displacement of the labeled fragment S. The data follows a power law t¹ (solid line), indicating a point-source diffusive behavior.

Figure 3

(a) Normalized correlation functions, G(t), of linear DNA (black) and open circular plasmid DNA (gray). (b) Mean-square displacement of the data in a for linear DNA (black) and open circular DNA (gray). Straight lines indicate fits to the different regimes for linear DNA data for times >50 ms, MSD ∼t¹, and for linear and open circular DNA data at times <1 ms: MSD t^{0.59 ± 0.065} and t^{0.52 ± 0.05}, respectively.

Figure 4

(a) Normalized correlation functions, G(t), of open circular DNA (black, upper curve) and supercoiled plasmid DNA (gray, lower curve). (b) Mean-square displacement of the data in a. Straight lines indicate fits to the different regimes. Fits to the OC data are as in Fig. 3b. The data for SC DNA is fit in the regime below 0.2 ms (t^{0.54 ± 0.06}) and in the intermediate regime between 0.3 ms and 3 ms (t^{0.43 ± 0.05}).