Biochemistry: The Chemical Reactions of Living Cells, Volume 1 - D. Metzler 1980

The Molecules We Are Made Of
Nucleic Acids
Circular and supercoiled structures; intercalation

DNA molecules can exist not only as open, linear molecules; their ends are frequently covalently joined to one another. For instance, the E. coli chromosome consists of a single closed circle. Circular DNA molecules are frequently found in Cell/35.html">Mitochondria and also in certain Viruses [80].

Like Proteins, The Structure of DNA can be significantly distorted by the Introduction of extra supercoils (superhelices). To achieve this effect, a torsional moment must be applied to one end of the chain. For example, if we take a slightly twisted, freely hanging rubber band and twist it tighter (as is done when preparing model airplanes for flight), positive supercoiling occurs. A similar situation—The formation of positive (or negative) supercoils (tertiary coiling)—can also occur in DNA. Supercoils are frequently found in circular DNA molecules. When a normal double-helical complex (duplex) is "twisted," the total winding number a of one strand relative to the other equals the number of turns in the Secondary structure ß, which corresponds to the unstrained helical duplex (i.e., the Watson-Crick structure), plus the number of supercoils т:

Class="center">a = τ + ß.       (2-14)

The value of ß is always positive, but τ can also be negative—in this case, the formation of a fully formed secondary structure (the Watson-Crick helix) is accompanied by the appearance of a certain number of left-handed supercoils [81].

The superhelix density (degree of supercoiling) of a DNA molecule is usually expressed by the value σ, which is equal to the number of supercoils per 10 Base Pairs [80, 82]. For naturally occurring circular DNA molecules, σ is most often negative; its most typical value is —0.05 (~5 supercoils per 1000 base pairs). The presence of supercoils in circular DNA molecules can be easily established because the sedimentation constant of DNA changes accordingly [81]. Thus, supercoiled native polyoma virus DNA sediments quite rapidly. After introducing a break into one of the strands of The Double Helix by brief enzyme Treatment, a "relaxed" form of the molecule is generated, which sediments more slowly. Supercoiling affects the viscosity of DNA solutions as well as the electrophoretic mobility of the molecules (Fig. 2-27,A) [82a]. In some cases, the presence of supercoils can be observed using an Electron microscope.

FIG. 2-27. A. Electrophoresis of SV 40 virus DNA preparation (Supplement 4-C), representing a mixture of molecules with varying numbers of supercoils. Native DNA molecules (electrophoretogram 1) migrate rapidly toward the anode as a series of bands, each differing from its neighbor in the number of supercoils in the corresponding DNA by 1. The average number of supercoils is 25. Incubation of DNA in the presence of a "relaxation" enzyme (Chap. 15, Sec. D.1) causes a sequential decrease in the number of supercoils: the enzyme introduces single-strand breaks and immediately reseals the strand. Incubation of DNA with this enzyme at 0°C for 1, 3, 6, 10, and 30 min (electrophoretograms 2–6) gradually converts the DNA into a form with an average number of supercoils equal to zero [82]. Electrophoresis was performed in a mixture of 0.5% agarose gel and 1.9% polyacrylamide gel; plate dimensions—17X18X0.3 cm. To visualize the bands, electrophoretograms were stained with the fluorescent intercalating dye ethidium bromide. (Kindly provided by W. Keller.)

What causes the formation of supercoils in natural DNAs? A key to this question is provided by studies of intercalation [83–86]—the insertion of flat aromatic rings between DNA base pairs. Many drugs (particularly Antibiotics), Dyes, and other substances possess The ability to undergo such intercalation. These include daunomycin, proflavin, ethidium bromide, and hycanthone (Fig. 2-27,B). Hycanthone, one of the most widely used drugs in the world, is used in the treatment of Schistosomiasis (Chap. 1, Sec. D.1). Because intercalating compounds possess mutagenic activity, The Use of such drugs entails a certain risk.

Intercalation is often used to assess the degree of negative supercoiling in DNA molecules. By adding increasing amounts of an intercalating agent, one monitors Changes in the sedimentation constant (or other properties) of the DNA. As the degree of intercalation increases, the secondary structure turns of the DNA unwind [ß in equation (2-14)]. Each intercalating ring causes the helix to unwind by ~26°. Since for a covalently closed duplex the value of a [equation (2-14)] is constant, a decrease in ß with increasing intercalation leads to an increase in τ. When intercalation reaches a level at which τ increases to 0, a minimum sedimentation rate will be observed. Further addition of the intercalating agent will result in positive supercoiling.

The "replicative form" of virus —X174 DNA (Supplement 4-C)—a small circular molecule of ~5000 base pairs—was treated with proflavin [82]. The binding of 0.06 moles of proflavin per mole of NUCLEOTIDES reduced τ to zero. From this, the estimate ¹σ = 0.055 was obtained; this means that at 25°C, pH 6.8, and an Ionic strength of ~0.2, the molecule contains —26 supercoils. Superhelix density is strongly influenced by Temperature, pH, and ionic composition. In general, σ becomes less negative by ~3.3X10^-4 per 1° increase in temperature [86c]. For example, for —X174 virus DNA at an ionic strength of 0.2, σ was found to be —0.059 at 15°C and —0.040 (—19 supercoils) at 75°C.

FIG. 2-27. B. Structural formulas of several intercalating compounds (see also the STRUCTURE OF THE actinomycin-DNA complex (Supplement 15-B).

¹ The estimate given here is based on the premise that intercalation of a single proflavin molecule produces an unwinding angle of ~16°; for comparison, ethidium bromide leads to an unwinding angle of 26°. In earlier literature, the unwinding angle for ethidium bromide was taken to be 12°, and the resulting superhelix density was lower than those cited nowadays [84, 86a, b].

Recent studies have shown that electrophoretic Separation of both natural and artificially produced supercoiled DNA molecules frequently reveals approximately 10 forms, each differing from the other by a single superhelical turn (Fig. 2-27,A). The relative Abundance of these topologically isomeric forms roughly corresponds to a Gaussian distribution. It is believed that these isomers arise As a result of Thermal Fluctuations in the degree of supercoiling at the moment the DNA chain is enzymatically closed into a circle.

Is the intercalation of planar molecules into nucleic acid chains coupled with any biochemical function? Apparently, yes. For example, in proteins that interact with Nucleic Acids, the aromatic rings of amino acid side chains can insert between the base planes in the DNA helical structure much like bookmarks inside a book [85, 86]. Changes in superhelix density caused by intercalation or alterations in the ionic environment may play a role in ensuring the proper sequence in the interaction of DNA with intracellular enzyme systems.