Supercoiled DNA: Structure

Abstract

Supercoiling is introduced into DNA molecules when the double helix is twisted around its own axis in three‐dimensional space. Experimental techniques that are sensitive to molecular shape can be used to analyse the topological states of DNA, but the approaches used most successfully are high‐speed centrifugation, high‐resolution microscopy and gel electrophoresis. Generally, DNA molecules are negatively supercoiled inside cells, although the level of supercoiling is not equal throughout the genome and many supercoils may be constrained by bound proteins. Supercoiling increases the free energy of DNA and influences DNA metabolism by promoting or hindering specific enzymatic processes. DNA topoisomerases are the main enzymes that regulate DNA topology and several different types of enzymes are present in all cells.

Key Concepts

  • Double‐stranded DNA helices can wind in three‐dimensional space to form further helices of higher order, forming supercoiled DNA.
  • Since the early 1960s, the importance of DNA supercoiling to cellular processes has been apparent, with its most obvious consequence being that it aids compaction of large DNA molecules into the relatively small volume of cells.
  • The extent of supercoiling in a DNA molecule is influenced by environmental conditions, such as ionic strength and temperature; since supercoiling of DNA influences the biological pathways in which it is involved, the level of DNA supercoiling inside cells is tightly regulated.
  • Supercoiling provides a significant amount of free energy to DNA molecules and, inside cells, this can be used to drive structural transitions and other metabolic processes that would normally be thermodynamically unfavourable, such as opening of the DNA helix during replication and transcription.
  • Mathematical and modelling studies have provided insight for quantitative analyses of DNA supercoiling, leading to definitions for twist, which describes how the individual strands of DNA coil around its axis, and writhe, which describes how the helix axis coils in three‐dimensional space.
  • DNA inside cells contains supercoils of two types: interwound supercoiling occurs when circular DNA winds around its own axis and toroidal supercoiling occurs when the DNA helix forms a series of spirals around an imaginary ring.
  • Any technique that is sensitive to molecular shape will be useful for experimental analysis of supercoiled DNA, but the large size of the molecules means few techniques have been used successfully; those that have been widely used include high‐speed centrifugation, high‐resolution microscopy (e.g. electron microscopy (EM) and scanning‐force microscopy) and agarose gel electrophoresis.
  • A wide variety of proteins that bind to DNA alter the local geometry of its helix and influence DNA topology; an important characterised example of this effect is the winding of DNA around the eukaryotic histone octamer to form the nucleosome.
  • A fundamental feature of closed domains of DNA, such as a circular molecule, is that the two strands of DNA are topologically linked and strand separation can be achieved only by breakage of one of the strands; the main enzymes that regulate DNA topology are DNA topoisomerases and they usually act to remove or introduce negative supercoils or they may remove both positive and negative supercoils.
  • Cellular processes that move macromolecular assemblies along DNA may generate localised DNA supercoiling since as the large protein complex moves along the DNA, its rotation around the DNA may be inhibited.

Keywords: DNA ; linking number; supercoiling; topology; twist; writhe

Figure 1. Relationship of linking number, twist and writhe of closed circular forms of DNA. Closed DNA circles can be made by formation of covalent 5′–3′ phosphodiester bonds on each strand of a linear molecule. For a linear molecule with 36 helical turns, the linking number of this unconstrained state ( 0) is 36. Closure into an unconstrained planar circle, as shown on the left side of the figure, produces a molecule with twist ( ) = 36 and writhe ( ) = 0. If the number of helical turns is altered before closure, the DNA molecule adopts a supercoiled conformation. On the right side of the figure, four helical turns are removed from the molecule, reducing the linking number ( ) to 32. For simplicity, the figure shows all unwinding partitioned as , although such changes are usually partitioned between and . Unwinding of helical turns produces negatively supercoiled DNA (or −Δ ) as shown, whereas the inclusion of additional turns produces positively supercoiled DNA. For DNA with −Δ in the interwound form, the superhelical turns are right‐handed. Note that separation of DNA strands removes negative supercoils (equivalent to the addition of positive supercoils).
Figure 2. Measurement of linking number by gel electrophoresis. (a) Schematic illustration of a DNA sample separated by electrophoresis through an agarose gel with and without an intercalator. DNA isolated from bacterial cells contains molecules with different topology: some have their backbones unbroken and are negatively supercoiled (SC), some have one strand broken and are referred to as ‘nicked’ (N) and some have both strands broken to produce a linear molecule (L). Note that the supercoiled DNA consists of a Gaussian distribution of different topoisomers. Upon addition of intercalator, the migration of intact molecules is altered, but that of nicked and linear molecules is not changed. (b) Enzymatic relaxation of plasmid DNA in the presence of varying concentrations of intercalator produces samples containing topoisomers at different levels of supercoiling. Utilisation of multiple gels with different concentrations of intercalator allows measurement of Δ . For each sample, average superhelical density ( ) is shown above the lane. Note that in each gel, samples can have positively or negatively supercoiled topoisomers. The inclusion of intercalator in the running buffer alters the electrophoretic mobility of all topoisomers equivalently. Superhelical density can be measured for experimental samples (‘native’) by comparison with those of known . (c) Two‐dimensional agarose gel electrophoresis of topoisomers ranging from high negative to moderate positive . A DNA sample is loaded in a single well in a large agarose gel and electrophoresis is performed under specific conditions (usually without intercalator) in direction D1. After soaking of the gel in buffer containing intercalator, electrophoresis is continued in direction D2 (90° to D1). The gel shown contained 20 µg mL−1 chloroquine during the second electrophoresis, resulting in all topoisomers having positive . Deviation of topoisomers from a smooth curve indicates that structural transitions in the DNA molecules reduced their negative during the first direction of electrophoresis. Spots marked ‘N’ and ‘L’ indicate the position of migration of ‘nicked’ and ‘linear’ DNA molecules, respectively.
Figure 3. Representation of knots and catenanes. (a) Topological knots may be formed in closed circles of DNA. The simplest knot that can be formed is called a trefoil because there are three lobes to the structure when it is laid flat. Two isomers of the trefoil knot are shown. Many other more complex knots may be formed within cccDNA molecules. (b) Catenanes are formed when two circular DNA molecules are interlocked. Catenanes containing complex, multiple links and involving many DNA circles have been observed in naturally occurring DNA. The arrows indicate that the polarity of a knot or catenane is influenced by the directionality of the sequence in the DNA molecule.
Figure 4. Twin domains of supercoiling are generated during transcription. (a) The shaded cylinders flank a closed domain of DNA containing eight helical turns. (b) To accommodate the transcriptional complex, some unwinding of the DNA helix occurs producing slight overwinding of the remaining DNA within each closed domain. (c) During transcription elongation, rotation of the large transcriptional complex around the DNA is hindered and positive and negative supercoiling are generated ahead and behind the polymerase, respectively. In this diagram, positive and negative supercoiling is represented by the presence of the same number of helical turns over a shorter and longer distance of DNA, respectively. Several biological mechanisms exist to remove these supercoils.
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Further Reading

Calladine CR , Drew HR , Luisi B and Travers AA (2004) Understanding DNA: The Molecule and How it Works, 3rd edn edn. Academic Press: London .

Cozzarelli NR and Wang JC (eds) (1990) DNA Topology and its Biological Effects. Cold Spring Harbor, NY: Cold Spring Harbor Press .

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Travers A (1993) DNA‐Protein Interactions. UK: Chapman & Hall .

Vologodskii A (1992) Topology and Physics of Circular DNA. Boca Raton, USA: CRC Press .

Wang JC (1994) Appendix I: an introduction to DNA supercoiling and DNA topoisomerase‐catalyzed linking number changes of supercoiled DNA. In: Liu LF (ed) DNA Topoisomerases: Topoisomerase‐targeting Drugs, pp. 257–270. San Diego: Academic Press .

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Bowater, Richard P(Jul 2015) Supercoiled DNA: Structure. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0001040.pub3]