DNA Topology: Supercoiling and Linking

Janet E Lindsley, University of Utah, Salt Lake City, Utah, USA

The topology of DNA has a major impact on its structure and reactivity. Two different aspects of DNA topology are the linkage of two single strands comprising the DNA double helix which can cause DNA supercoiling and the linkage of double helixes in the form of knots and catenanes.

Secondary article
Article Contents
. Definition of a Topological Property . DNA Supercoiling . The Solution Structure of Supercoiled DNA . Linking Number, Twist and Writhe . Knots and Catenanes . Kinetoplast DNA

Definition of a Topological Property

A topological property is one that remains unchanged, even when the object of interest is distorted, so long as no surfaces are torn. In other words, two entities are topologically equivalent if one can be smoothly deformed into the other. An example of two topologically equivalent objects is a doughnut and a tea cup (Figure 1a). Clearly, these items have dierent shapes, or geometries, but they have the same topology. A doughnut shape made of a malleable substance can be smoothly deformed into a tea cup, and vice versa, without breaking connections or forming new connections within the substance; the hole in the doughnut is retained as the hole in the tea cup handle, and no new holes are introduced. Therefore, a topological property is a property that is invariant unless integrity of the entity is broken.

(a)

(b)

(c)
Figure 1 Topology basics. (a) A doughnut and a tea cup have the same topology, as evidenced by the fact that one can be freely deformed into the other, while clearly having different geometries. (b) The DNA strands of a double helix become topologically linked when the 5 end of each strand is ligated to the 3 end of the same strand; the linking number equals the initial number of helical turns. In this illustration, the linking number between the blue and red strands is three. (c) The topology or linking number of the strands does not change, no matter how distorted, as long as neither strand is broken.

Topology has traditionally been a eld of mathematics that deals with the fundamental properties of abstract spaces. However, shortly after the double-helical structure of DNA was elucidated, the usefulness of applying topology theory to understanding DNA structures was appreciated. In the 1960s, Vinograd et al. discovered that dierent topological forms of the same circular, double stranded DNA viruses exist (Vinograd et al., 1965). These dierent topological isomers, or topoisomers, are the result of the strands of DNA being linked to each other dierent numbers of times. To think about the linkage of DNA strands, imagine the two sugarphosphate backbones as two strands of thread that wind around each other in the right-handed sense. If one end of each strand is tied to the opposite end of the same strand, making two closed circles, then the two strands will be topologically linked to each other a number of times equal to the initial number of helical turns (Figure 1b). Once the ends of the threads, or a DNA segment, are attached, the linkage between the strands will not change, no matter how they are distorted, so long as the attachment of the strands is not broken (Figure 1c). Therefore, the linkage between strands is a topologic property. It also becomes clear that in order for the strands to be topologically linked, the beginning and end of the threads, or strands of a DNA segment, must be xed relative to each other. This is the case for circular DNA, where the ends of the segment are covalently attached through continuous 5 3 phosphodiester bonds. This is also the case when the ends of a DNA segment are held xed together, as is found for chromosomal loops in cells. No matter how much a DNA molecule is distorted by twisting, denaturation, binding by proteins or small molecules, short of breaking the backbone or disrupting the structure that holds the ends together, its topology remains unchanged because the number of links between the two strands does not change. The linking, or topology, of DNA strands is biologically important because it inuences essentially every reaction in which DNA participates, including the binding of transcriptional regulatory factors, the initiation of DNA replication, the segregation of replicated chromosomes to daughter cells, the chemical reactivity and compaction of
1

ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net

DNA Topology: Supercoiling and Linking

DNA. One of the most common and well-known consequences of DNA topology is DNA supercoiling.

DNA Supercoiling
Supercoiling literally means the coiling of a coil or helix. Most complementary DNA sequences exist as a B-form double helix, where each strand wraps around the other and a common helical axis in the right-handed sense. In addition, for much of the DNA within cells, the helical axis coils upon itself, giving rise to the higher order structure called supercoiling. This can be most easily seen in circular, covalently closed viral and plasmid DNAs that remain supercoiled upon purication from cells, as long as the DNA backbone is not broken. Additionally, the DNA comprising linear chromosomes and held in topologically constrained domains or loops is also supercoiled within cells. Supercoiling is the physical consequence of the topologic linking of the two strands of DNA diering from the structurally most stable linking. When the two strands are topologically linked fewer times than would be most stable, the helical axis coils about itself in the right-handed sense, forming negatively supercoiled DNA (Figure 2). When the two strands are linked more times than is most stable, the DNA positively supercoils with the helical axis coiling about itself in the left-handed sense. Most of the DNA in mesophilic cells is negatively supercoiled. At least the plasmid DNA, and possibly the chromosomal DNA, in some hyperthermophilic archea is positively supercoiled, and the level of positive supercoiling increases as the growth temperature increases (Lopez-Garcia and Forterre, 1997). Additionally, when a protein such as a polymerase tracks along right-handed double-helical DNA, if the protein does not rotate around the helix as fast as it tracks forward, then transient domains of positive and negative supercoiling will form ahead of and behind the tracking protein. This is especially prominent in prokaryotes, where transcription and translation are coupled and rotation of the RNA polymerase/nascent RNA/ribosome/nascent polypeptide complex around the

DNA helix is slow. This transient, transcription-driven supercoiling may have important consequences for gene regulation and DNA conformational transitions (Liu and Wang, 1987). Supercoiled DNA is a high-energy, strained state for DNA and the removal of supercoils is an energetically favourable process. Many biological processes take advantage of the free energy available from supercoiled DNA. For example, it requires less energy to denature a region of negatively supercoiled DNA than relaxed DNA. Therefore, proteins and small molecules that preferentially bind single-strand DNA have a higher anity for negatively supercoiled DNA than for relaxed DNA. Transcription and DNA replication, two processes that require at least transient separation of the DNA strands, generally require that the DNA substrate be negatively supercoiled.

The Solution Structure of Supercoiled DNA

There are two general forms of supercoiling: interwound (or plectonemic) and toroidal (or solenoidal) (Figure 3). Puried supercoiled DNA in solution forms interwound supercoils right-handed for negatively supercoiled and left-handed for positively supercoiled. A combination of experimental and theoretical studies indicate that in a solution of moderate ionic strength, puried negatively supercoiled DNA adopts a structure that, when projected on a at surface, looks similar to that shown in Figure 3a (Cozzarelli et al., 1990). One result of this structure is that two distant DNA sequences are predicted to be  100 times more likely to be juxtaposed in a supercoiled DNA molecule than in the equivalent relaxed molecule (Vologodskii and Cozzarelli, 1996). The structures tend to be branched, and at the branch points, three dierent sequences are brought in close proximity. This increased juxtaposition frequency means that proteins that bind distal sequences, but that must interact to form a

Break strands

Reseal ends

Untwist ends

Figure 2 Reducing the number of times two strands are linked creates negatively supercoiled DNA. If the strands of a relaxed DNA circle are broken and untwisted several times before being resealed, the resulting closed circle will negatively supercoil.

ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net

DNA Topology: Supercoiling and Linking

(a)

(b)

Figure 3 Interwound (a) and toroid structures (b) of negatively supercoiled DNA. The DNA double helix is shown as a single line for simplicity in these illustrations. For each model, the DNA length is 3 kilobase pairs and the specific linking difference is 2 0.06. (a) Purified, negatively supercoiled DNA is predicted to have a noncollapsed, branched structure. (b) The same DNA shown in (a), however the DNA is packaged as nucleosomes stacked end-to-end with no linker DNA. See Cozzarelli et al. (1990) for a detailed description.

functional complex, will have a higher probability of doing so on plectonemically supercoiled DNA. In contrast to the structure of puried supercoiled DNA, DNA that is wrapped around histones, forming nucleosomes, exhibits toroidal supercoils (Figure 3b). The handedness of toroidal supercoils is opposite that of interwound supercoils; negatively supercoiled DNA forms left-handed toroidal supercoils and positively supercoiled DNA forms right-handed toroidal supercoils. Figure 3 shows two models of the same length circle of DNA, supercoiled to approximately the same extent, with either interwound or toroid supercoils. From this illustration it is clear that toroidal supercoils are an ecient way to condense DNA for packaging into a restricted size nucleus. It therefore makes sense that eukaryotes, with generally large genomes, use toroidal supercoils, in the form of nucleosomes, as the rst level of compaction of long DNA molecules into chromosomes that must t into the nucleus.

Linking Number, Twist and Writhe

Supercoiled DNA is the structural consequence of the topologic linking of DNA strands. The relationship between the topology and structure, or geometry, of DNA is described by the terms linking number, twist and writhe. The concept of linking number for the two strands of a DNA double helix was introduced above (Figure 1b). If the ends of a linear double-stranded DNA molecule are joined together to form a covalently closed circle, then the number of times the two strands are topologically linked together approximates the number of double helical turns in the original linear molecule. This number is the linking number or Lk (for a strict mathematical denition of linking number, see Cozzarelli et al., 1990). A real linking number must be an integer. When the ends of the linear DNA are joined under minimal torsional stress and

standard reaction conditions (20378C, 0.2 mol L 2 1 NaCl), the standard linking number will be the closest integer to the number of base pairs (N) divided by the number of base pairs per turn (h); under standard conditions, h  10.5. For example, the standard linking number for the covalently closed circular plasmid pBR322 (4361 base pairs) is 415 (the closest integer to 4361/10.5). The value Lk8 represents the hypothetically most relaxed linking number, which exactly equals N/h and is often not an integer. For pBR322, Lk8 5 415.3. It is important to realize that unlike the true linking number, Lk, Lk8 is not a topological property. The value for Lk8 varies if the conditions specifying the relaxed state (such as temperature or binding of a small molecule or protein) are varied. Supercoiling, more formally called writhing, of the DNA results when the real Lk diers signicantly from Lk8. If Lk is smaller than Lk8 (if Lk 2 Lk8 5 0, giving a negative value for the linking dierence), then the DNA will supercoil negatively. If Lk 2 Lk8 4 0, then the DNA will supercoil positively. If Lk  Lk8, then the DNA is relaxed. The DNA within most cells is negatively supercoiled because the true linking number is less than the theoretically most relaxed linking number. The specic linking dierence, or superhelical density (s) is dened as: (Lk 2 Lk8)/Lk8. Since the specic linking dierence for DNA isolated from Escherichia coli is approximately 2 0.06, the most abundant topoisomer of pBR322 isolated from this organism has Lk  390 (since s 5 2 0.06 = (390 415.3)/415.3). While Lk is a topologic property that does not change unless the integrity of the DNA backbone is transiently broken, it is also the sum of two geometric properties, twist (Tw) and writhe (Wr), such that Lk = Tw 1 Wr. Tw is essentially the number of times either strand of DNA coils around the helical axis, while Wr is the number of times the helical axis coils upon itself, or supercoils (for more mathematically rigorous denitions, see Cozzarelli et al., 1990). When the ends of linear DNA are ligated closed under minimal torsional stress to form a covalently closed circle, such that Lk  Lk8, then Lk  Tw and Wr  0, i.e. the DNA is relaxed and not supercoiled. When Lk6Lk8, it has been empirically found that the linking dierence (Lk 2 Lk8) is distributed  75% as writhe and  25% as twist, as long as the DNA molecule is larger than  3 kilobase pairs (kb). This means that for pBR322 isolated from E. coli with an Lk of  390, and Lk 2 Lk8 of  2 25, the average Wr will be  2 19 and the average Tw will be  409. For small DNA circles, writhing becomes energetically unfavourable, and the linking dierence is primarily seen as a change in twist. Since Lk 5 Tw 1 Wr, as long as Lk does not change, any change in conditions that alters Tw will result in a compensatory change in Wr, and vice versa. For example, as the temperature is increased, the DNA helix untwists slightly and the number of base pairs per turn (h) increases. This results in a decrease in Tw (since N/h decreases as h
3

ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net

DNA Topology: Supercoiling and Linking

increases) and an increase in Wr. Therefore, if the temperature of a relaxed covalently closed DNA circle is increased, then the DNA will begin to positively supercoil. Likewise, the binding of an intercalating molecule, such as the uorescent dye ethidium bromide, increases the number of base pairs per turn, decreases Tw and therefore increases Wr. Since the geometry or writhing of a DNA molecule aects how it migrates in an agarose gel, covalently closed DNA circles migrate dierently in the presence and absence of ethidium bromide. Depending on the concentration of ethidium present and the linking dierence (Lk 2 Lk8) of the original topoisomers, the increase in Wr due to the ethidium binding can either increase or decrease the mobility of the DNA compared to a gel run without the intercalator. This relationship between Lk, Tw and Wr also indicates that if the phosphodiester backbone of the DNA is broken, and the DNA is rearranged such that upon religation Lk has changed, then there must be a corresponding change in either Tw or Wr, or both. This is formally stated as DLk 5 DTw 1 DWr. As mentioned above, supercoiled DNA, or DNA for which Lk6Lk8, is a high-energy state of the DNA. The Gibbs free energy, DG, available from DNA supercoiling is directly proportional to the specic linking dierence squared (s2), as long as the DNA circle or loop in question is 4 2 kb in size. For smaller DNA circles, the free energy of supercoiling increases as the length of the DNA decreases (Horowitz and Wang, 1984). It is this available free energy that makes the supercoiling of DNA so biologically important. This free energy can cause local changes in DNA structure such as denaturation, cruciform extrusion, and formation of Z- and H-form DNA. These structural changes usually occur at specic nucleotide sequences as the result of negative supercoiling, or supercoiling in conjunction with specic protein binding. Negative supercoiling makes it easier for proteins to denature double-strand DNA (essential for replication and transcription initiation) and wrap DNA in a lefthanded coil, as in nucleosomes.

two parental strands must be reduced exactly to zero for the two resulting daughter DNA molecules to be fully synthesized and separated. Enzymes called topoisomerases unlink the DNA both ahead of and behind the replication fork. At the end of replication, often several links remain, and the daughter DNA molecules are interlinked or catenated. Type II DNA topoisomerases, which function by transporting a double-stranded segment of DNA through a transient break in a separate double-strand DNA segment, are required to unlink the catenated daughter DNA molecules. Essentially the same phenomenon is thought to occur in eukaryotic cells where the long linear DNA is organized into loops of  30100 kb. These loops are thought to be attached to the nuclear matrix or scaold through proteinDNA interactions. Each loop is a topologically isolated domain. After DNA replication, the loops of daughter chromosomes are often catenated, and must be unlinked by a type II DNA topoisomerase prior to chromosome segregation. DNA catenanes represent a unique way to link two separate DNA sequences; the topologic bond that holds together singly catenated DNA circles is maintained independent of direct interaction between the circles. This property makes DNA catenanes very useful for certain types of biochemical studies. For example, a protein bound at one DNA sequence can inuence the activity of another bound at a distal sequence by several possible mechanisms. After binding to its cognate site, the rst protein could track or slide along the DNA until it encounters the second protein. Alternatively, the two proteins could interact by looping out the DNA between them. These two possibilities can be clearly distinguished by using catenated DNA rings, where one protein-binding site is on each ring. Proteins that interact by the rst mechanism will not show activation using the catenanes, but those that interact via the second mechanism will. This method has been used to study how enhancer sequences interact with promoter sequences to activate transcription from a distance (Wedel et al., 1990).

Knots and Catenanes

Another important aspect of the topology of DNA is its ability to form knots and interlinked DNA circles or catenanes. Although DNA knots are not commonly seen in nature, studying how they are formed and resolved in vitro by various site-specic recombination enzymes has been useful in determining the mechanisms of these enzymes (Wasserman and Cozzarelli, 1986). Catenanes, from the Latin catena for chain, are biologically important DNA structures, particularly in relation to the replication of the circular genomes of bacteria, plasmids and viruses. When circular DNA is replicated, the linking number between the
4

Kinetoplast DNA
The topology of the mitochondrial or kinetoplast DNA (kDNA) of trypanosomes and related protozoan parasites is highly unusual. The DNA in these organelles exists as a network of catenated circles. The best-studied kDNA is from Crithidia fasciculata and is composed of  25 large circles (37 kb) and 5000 mini-circles (2.5 kb). Each of the circles is relaxed (i.e. not supercoiled) and singly linked to about three other circles, creating a network that somewhat resembles the chain mail used in medieval armour. Although it is unclear why this organelle has evolved such an unusual structure for its DNA, it is clear that this structure eectively solves the problem of chromosome

ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net

DNA Topology: Supercoiling and Linking

compaction. While nuclear DNA is compacted about 104fold, at least partially because of nucleosome-bound toroidal supercoils, kDNA is compacted to approximately the same extent by mini-circle catenation (Chen et al., 1995).

References
Chen J, Rauch CA, White JH, Englund PT and Cozzarelli NR (1995) The topology of the kinetoplast DNA network. Cell 80: 6169. Cozzarelli NR, Boles TC and White JH (1990) A primer on the topology and geometry of DNA supercoiling. In: Cozzarelli NR and Wang JC (eds) DNA Topology and Its Biological Eects, pp. 139184. Cold Spring Harbor, NY: Cold Spring Harbor Press. Horowitz DS and Wang JC (1984) Torsional rigidity of DNA and length dependence of the free energy of DNA supercoiling. Journal of Molecular Biology 173: 7591. Liu LF and Wang JC (1987) Supercoiling of the DNA template during transcription. Proceedings of the National Academy of Sciences of the USA 84: 70247027. Lopez-Garcia P and Forterre P (1997) DNA topology in hyperthermophilic archaea: reference states and their variation with growth phase, growth temperature, and temperature stresses. Molecular Microbiology 23: 12671279.

Vinograd J, Lebowitz R, Radlo R, Watson R and Laipis P (1965) The twisted circular form of polyoma viral DNA. Proceedings of the National Academy of Sciences of the USA 53: 11041111. Vologodskii A and Cozzarelli NR (1996) Eect of supercoiling on the juxtaposition and relative orientation of DNA sites. Biophysics Journal 70: 25482556. Wasserman SA and Cozzarelli NR (1986) Biochemical topology: applications to DNA recombination and replication. Science 232: 951960. Wedel A, Weiss DS, Popham D, Droge P and Kustu S (1990) A bacterial enhancer functions to tether a transcriptional activator near a promoter. Science 248: 486490.

Further Reading
Bates AD and Maxwell A (1993) DNA Topology. Oxford: IRL Press. Bauer WR, Crick FH and White JH (1980) Supercoiled DNA. Scientic American 243: 100113. Cozzarelli NR and Wang JC (eds) (1990) DNA Topology and Its Biological Eects. Cold Spring Harbor, NY: Cold Spring Harbor 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: Topoisomerasetargeting Drugs, pp. 257270. San Diego: Academic Press.

ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net