Scientific Understanding of Consciousness
Supercoils of DNA
Science 5 October 2012: Vol. 338 no. 6103 pp. 94-97
Dynamics of DNA Supercoils
M. T. J. van Loenhout, et.al.
Delft University of Technology, Department of Bionanoscience, Kavli Institute of Nanoscience, Lorentzweg 1, 2628CJ Delft, Netherlands.
DNA in cells exhibits a supercoiled state in which the double helix is additionally twisted to form extended intertwined loops called plectonemes. Although supercoiling is vital to many cellular processes, its dynamics remain elusive. In this work, we directly visualize the dynamics of individual plectonemes. We observe that multiple plectonemes can be present and that their number depends on applied stretching force and ionic strength. Plectonemes moved along DNA by diffusion or, unexpectedly, by a fast hopping process that facilitated very rapid (<20 milliseconds) long-range plectoneme displacement by nucleating a new plectoneme at a distant position. These observations directly reveal the dynamics of plectonemes and identify a mode of movement that allows long-distance reorganization of the conformation of the genome on a millisecond time scale.
Supercoiling and changes in the supercoiling state are ubiquitous in cellular DNA and affect virtually all genomic processes. Proteins moving along the helical path of the DNA, for example, generate torsional stress, which produces twist (the over- or underwinding of the DNA double helix around its axis) and writhe (the coiling of the duplex axis around itself). Supercoiling affects the cell because it alters the conformation of the genome on two basic levels. First, supercoiling can induce local changes in the DNA structure, such as a locally destabilized or deformed duplex, which subsequently affect transcription or trigger protein binding. Second, supercoiling can induce global changes in the conformation of the genome, which bring distant DNA sequences together, thereby facilitating DNA compaction and site-specific recombination. Genomic DNA is organized in topological domains of 10 to 100 kilobases (kb) that isolate topological changes from neighboring regions. Within these regions, torsion can rapidly transmit, allowing for long-range communication between distant genomic locations. To understand the cellular processes that are affected by supercoiling, it is essential to comprehend its dynamics.
To visualize the dynamics of plectonemes directly along a single DNA molecule, we developed a magnetic tweezers apparatus that pulls a fluorescently labeled DNA molecule sideways and visualizes it along its length using epi-fluorescence. We took all measurements on 21-kb DNA molecules, which are similar in length to the topological domains observed in genomic DNA. We observed individual plectonemes in supercoiled DNA molecules in images acquired with 20-ms time resolution. We observed that multiple plectonemes were present that appeared and disappeared and moved along the DNA.
The number of plectonemes present in a DNA molecule will be set by the free-energy balance between the change in enthalpy and the change in entropy upon the formation of an additional plectoneme. Entropy will favor the presence of multiple plectonemes, as they can occupy multiple positions along the molecule and distribute the writhe between them. The energy cost required to bend the DNA in the plectoneme will favor a single plectoneme. The structure of a plectoneme can be simplified to consist of an intertwined section and an end loop. The formation of an end loop is energetically more costly than extending the intertwined region, making it unfavorable to form multiple plectonemes. Surprisingly, the data showed that multiple plectonemes were present in DNA molecules.
In the dynamics of plectonemes, we observed two different types of motion: (i) diffusive motion, in which a plectoneme randomly moved along a DNA molecule, and (ii) hopping, in which a plectoneme suddenly shrank or disappeared while simultaneously a new plectoneme nucleated at a different location.
The observed dynamics of DNA supercoils reveal how plectonemes change the DNA conformation. We found that multiple plectonemes are present in a supercoiled DNA molecule under applied force, with a typical density of one plectoneme per 10 kb. Diffusion of plectonemes was strongly dependent on applied stretching force, suggesting that it is hindered by local inhomogeneities in the DNA mechanical properties. In contrast, hopping of plectonemes results in a fast, long-range rearrangement of the DNA conformation, which may explain the fast search times for site juxtaposition of two distant DNA regions. Hopping can also aid to recruit a plectoneme to a DNA sequence that exhibits inherent curvature or to a site of protein-induced DNA bending. Such a mechanism will allow for changes in the conformation of the genome at the millisecond timescale, thereby triggering protein binding or influencing gene expression.
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