LEHNINGER PRINCIPLES OF BIOCHEMISTRY - VOLUME 3. INFORMATION PATHWAYS - 2017

PART III. INFORMATION PATHWAYS

24. GENES AND CHROMOSOMES

24.2. DNA Supercoiling

Cellular DNA, as we have seen, is exceptionally compact and therefore possesses a high degree of structural Organization. The packaging mechanism serves not only to store DNA efficiently, but also to ensure access to the information it contains. Before examining The Role of this mechanism in cellular processes such as Replication and METABOLISM/31.html">Transcription, we must discuss an important structural feature of DNA: supercoiling.

Supercoiling is the twisting of an already existing helix. A familiar example of this Structure is a coiled telephone cord. One or more supercoils frequently form in the line between the handset and the base (Fig. 24-10). A DNA molecule is wound into a double helix in which the two DNA strands wrap around a common axis. Further twisting of this axis around itself (Fig. 24-11) results in supercoiled DNA. As we will see later, supercoiling is typically the result of structural strain. When DNA is not twisted about its axis, it is said to be relaxed.

Class="center">Figure 24-10. Supercoiling. An ordinary telephone cord is twisted into a helix like DNA, and can itself form additional supercoil loops. This analogy is also apt because it was the telephone cord that helped Jerome Vinograd and his colleagues realize that many properties of small circular DNA molecules can be explained by supercoiling. They first discovered The phenomenon of DNA Supercoiling in 1965 in samples of small Viral DNA molecules.

Figure 24-11. DNA supercoiling. When the DNA double helix is twisted around its axis, a new helix (a superhelix) is formed. Supertwisted DNA is commonly referred to as a supercoil.

It is logical to assume that packaging DNA generates various types of supercoiling. Less obvious is the fact that DNA replication and Transcription also affect and depend upon supercoiling. Both processes involve the Separation of DNA strands, which promotes The formation of additional helical turns (as shown in Fig. 24-12).

Figure 24-12. Strand separation induces supercoiling. Twist two rubber bands into a right-handed double helix as shown in the top diagram. Have someone hold one end and try to separate the strands from the other end. You will observe supercoiling.

The fact that tightly packed cellular DNA twists around itself into a superhelical structure seems logical and even trivial, were it not for one observation: many cellular circular DNA molecules remain highly supercoiled even after they are extracted and purified, free of Proteins and other cellular components. This implies that supercoiling is an inherent property of The quaternary structure of DNA. It is characteristic of all cellular DNA molecules and is strictly regulated by every Cell.

Supercoils are characterized by several measurable parameters; these data have provided a deeper understanding of DNA Structure and function. These studies rely heavily on concepts from topology, a branch of mathematics that investigates The properties of an object under continuous deformation. In the case of DNA, continuous deformation includes conformational changes caused by thermal motion and interactions with proteins or other molecules, whereas discontinuous deformation involves breaks in the DNA strands. For circular DNA molecules, topological properties are those that do not change during the deformation of the DNA strands as long as no breaks occur. Topological properties change only As a result of the breakage and subsequent rejoining of one or both DNA strands.

We now consider the fundamental properties and physical basis of supercoiling.

Most Cellular DNAs Are Underwound

To understand The Nature of supercoiling, we first focus on the properties of small circular DNA molecules, such as Plasmids and small Introduction/6.html">DNA-containing Viruses. If such DNA molecules have no breaks in either strand, they are called closed-circular DNA molecules. If the shape of a closed-circular DNA closely approximates the B-form of DNA (the Watson-Crick structure; see Fig. 8-13 in Vol. 1) with one turn of The Double Helix per 10.5 bp, it is relaxed rather than supercoiled (Fig. 24-13). Supercoiling occurs when There is a certain amount of structural strain. Purified closed-Circular DNA is rarely relaxed, regardless of its biological origin. Furthermore, DNA molecules isolated from a specific cellular source possess a characteristic degree of supercoiling. Consequently, DNA is strained to such an extent that superturns arise, and this state is regulated by The Cell.

Figure 24-13 Relaxed and supercoiled plasmid DNA. The far-left electron micrograph shows a relaxed molecule; the degree of supercoiling increases from left to right.

At virtually any given moment, strains in the DNA double helix arise from its partial unwinding. In other words, the DNA contains fewer helical turns than does the B-form. The consequences of partial unwinding are summarized in Fig. 24-14. An 84 bp segment of circular DNA in the relaxed form can contain eight turns of the double helix, or one turn per 10.5 bp. If one of these turns is removed, there are 84 bp / 7 = 12.0 bp per turn, which is greater than the 10.5 bp characteristic of B-DNA (Fig. 24-14b). This deviation from the most stable form of DNA creates thermodynamic strain within the molecule, resulting in an energetically unfavorable DNA conformation. Part of this strain can be relieved by twisting the DNA around its own axis to form a superhelix (Fig. 24-14c; some of the strain in the 84 bp region may simply be distributed across the unwound structure of a larger DNA fragment). In principle, the strain can also be compensated for by the separation of the two DNA strands over a span of about 10 bp (Fig. 24-14d). In isolated closed-circular DNA, the strain caused by partial unwinding is typically accommodated more by supercoiling than by strand separation, because twisting the DNA axis generally requires less energy than breaking the Hydrogen Bonds between Base Pairs. However, it should be noted that following partial unwinding in vivo, the DNA strands separate more easily, which facilitates the readout of the information they contain.

Figure 24-14. Consequences of DNA unwinding. (a) A segment of a closed-circular DNA molecule 84 bp in length in a relaxed form with eight helical turns. (b) Removal of a single turn introduces structural strain. (c) Strain is typically alleviated by supercoiling. (d) Partial unwinding of DNA somewhat facilitates strand separation. It is shown that, theoretically, unwinding by one turn can facilitate strand separation over a region of about 10 bp. However, strand separation over such a short region is normally hindered by hydrogen bonds between base pairs, and the effect becomes significant only for longer DNA segments and more extensive unwinding.

Each cell actively unwinds its DNA through enzymatic processes (described below), and the resulting strained state serves to store energy. Cells maintain DNA in a partially unwound state to facilitate its compact packaging. DNA unwinding is also essential for the function of Enzymes involved in DNA metabolism, which must separate the two DNA strands to carry out their tasks.

DNA can exist in such an unwound state only if it is closed into a circle or bound and stabilized by proteins in such a way that the strands cannot freely rotate relative to one another. If one of the strands in an isolated, protein-free circular DNA is broken, free rotation occurs spontaneously at that site, and the unwound DNA transitions into a relaxed state. In a closed-circular DNA molecule, the number of helical turns cannot be altered without at least a temporary break in one of the DNA strands. For this reason, the number of helical turns in a DNA molecule is a characteristic measure of supercoiling.

The degree of DNA twisting is determined by a topological parameter known as the linking number.

Topology offers several concepts that help us discuss DNA supercoiling, particularly THE CONCEPT OF the linking number. The linking number is a topological property of double-stranded DNA because it remains unchanged when the DNA is bent or deformed as long as both strands stay intact. The linking number is denoted as Lr—from the English term linking number (Fig. 24-15).

Fig. 24-15 Linking number (Lk). Here, as usual, each blue ribbon represents one of the strands of double-stranded DNA. For the molecule in Fig. a, Lk = 1; for the molecule in Fig. b, Lk = 6. One of the strands in Fig. b is shown untwisted to more clearly illustrate the boundary of an imaginary surface (pale blue). The number of intersections of this surface by the spiral strand corresponds to the linking number.

Let us consider The process of separating the strands of a double-stranded circular DNA molecule. If the two strands are linked as shown in Fig. 24-15a, their tight connection can be called a topological bond. Even if all Hydrogen bonds and stacking interactions between bases are broken, thereby disrupting the physical contact between the strands, they remain topologically linked. Imagine that one of the circular strands bounds a certain surface (such as The surface of a soap film inside a ring before a soap bubble is blown from it). The linking number can be defined as the number of times the second strand intersects this surface. In the molecule of Fig. 24-15a, Lk = 1; in the molecule of Fig. 24-15b, Lk = 6. For a closed circular DNA, the linking number is always expressed as an integer. By convention, the linking number is positive (+) for a right-handed helix and negative (-) for a left-handed helix. In DNA, a negative linking number does not occur.

Let us apply these considerations to a closed circular DNA molecule consisting of 2,100 bp (Fig. 24-16a). When the molecule is relaxed, finding the linking number is straightforward: it is The ratio of the total number of base pairs to the number of base pairs per turn of the helix (approximately 10.5 bp); consequently, in this case, Lk = 200. For a circular DNA molecule to be characterized by a definite linking number, neither of its strands must contain breaks. If even a single strand has a break, the helix can be completely separated into two strands. In this case, topological linkages are absent, and Lk cannot be defined (Fig. 24-16b).

We can now describe DNA twisting in terms of Changes in the linking number. The reference point is taken as the linking number of relaxed DNA, Lk₀. For the molecule in Fig. 24-16a, Lk₀ = 200; if two turns are removed from the molecule, Lk = 198.

∆Lk = Lk - Lk₀ (24-1)

∆Lk = 198 - 200 = -2

Fig. 24-16. Linking number illustrated using closed circular DNA molecules. A 2,100 bp circular DNA is shown in three forms: (a) relaxed, Lk = 200; (b) relaxed with a single-stranded break (nick), where Lk cannot be defined; (c) partially unwound by two turns, Lk = 198. The partially unwound molecule typically exists in a supercoiled state, but partial unwinding can also facilitate the separation of DNA strands.

It is usually convenient to express The change in the linking number through a parameter independent of the DNA molecule's length. This parameter is called the superhelical density (σ), or the specific linking difference; the superhelical density is equal to the ratio of the change in the number of helical turns to the number of turns in relaxed DNA:

σ = ∆Lk/Lk₀ (24-2)

In the example of Fig. 24-16c, σ = -0.01, which means that 1% (2 out of 200) of the helical turns in the DNA (in the B-form) have been removed. Typically, the degree of cellular DNA unwinding is 5–7%, i.e., σ ranges from -0.05 to -0.07. A negative value indicates that the change in the linking number is associated with DNA unwinding. Thus, supercoiling caused by partial unwinding is negative supercoiling. Conversely, under certain circumstances, DNA can be overwound, which is expressed by a positive superhelical value. Note that when DNA is partially uncoiled (negative supercoiling), the part of the DNA helix wound around its axis is the mirror image of an overwound DNA helix (positive supercoiling) (Fig. 24-1). Supercoiling is not a random process; the nature of supercoiling is largely described by the torsional strain that arises in DNA when the linking number is decreased or increased relative to B-form DNA.

Fig. 24-17. Negative and positive supercoiling. For the relaxed DNA molecule shown in Fig. 24-16a, partial unwinding or overwinding by two helical turns (Lk = 198 or 202) results in negative or positive supercoiling, respectively. Note that the axis of the DNA helix coils in opposite directions in both cases.

The linking number can change by ±1 when One DNA strand is broken, one of the ends is rotated 360° around the second strand, and the broken ends are rejoined. Such a change does not affect the number of nucleotide pairs or the number of atoms in the circular DNA molecule. Two forms of circular DNA that differ only in such a topological property as the linking number are called topoisomers.

Example 24-1 Determining Superhelical Density

What is the superhelical density of a closed circular DNA molecule of 4,200 bp with a linking number Lk = 374? What is the superhelical density of a DNA of the same length with Lk = 412? Are these molecules positively or negatively supercoiled?

Solution. Let us find Lk₀. To do this, we divide the length of the circular DNA (in bp) by 10.5 bp/turn: (4,200 bp) / (10.5 bp/turn) = 400. Now, using formula 24-1, we can find ∆Lk: ∆Lk = Lk - Lk₀ = 374 - 400 = -26. Substituting The values of ∆Lk and Lk₀ into formula 24-2: σ = ∆Lk/Lk₀ = -26/400 = -0.065.

Since the supercoiling is negative, the DNA molecule carries negative supercoils.

If Lk = 412 for the same DNA molecule, ∆Lk = 412 - 400 = 12, consequently, σ = 12/400 = 0.03. The supercoiling is positive, and the DNA molecule carries positive supercoils.

The linking number has two structural components: twist (Tw) and writhe (Wr) (Fig. 24-18). They are much more difficult to describe than the linking number, but twist can be viewed as a measure of the coiling (number of turns) of the helical axis, and writhe as a measure of the local bending or spatial intertwining of adjacent base pairs (number of supercoils). When the linking number changes, part of the resulting stress is typically compensated by twisting (supercoiling) and bending, which is expressed by the equation:

Lk = Tw + Wr

Fig. 24-18. Twist and writhe modeled with a rubber cord. The pink rubber cord represents the axis of a relaxed DNA molecule. The tension generated by twisting the rubber (as in partial DNA unwinding) manifests as twist or writhe. Topological changes in the linking number are usually accompanied by changes in both twist and writhe.

The parameters Tw and Wr are not necessarily integers. Twisting and writhing have a geometric rather than topological nature, as they change upon deformation of a closed circular DNA molecule.

Partial unwinding of DNA promotes supercoiling, simplifies strand separation to a certain extent, and facilitates A number of structural changes within the molecule. While the physiological significance of these specific changes is modest, they help illustrate the consequences of partial unwinding. Recall that the cruciform structure (see Fig. 8-19 in Vol. 1) typically contains several unpaired bases, and it is precisely the insufficient twisting of DNA that helps maintain strand separation (Fig. 24-19). Partial unwinding of the right-handed DNA helix also facilitates the formation of short stretches of left-handed Z-DNA in regions where The nucleotide sequence permits this conformation (Chapter 8).

Fig. 24-19. Facilitation of cruciform structure formation by partial DNA unwinding. In principle, cruciform structures can form at palindromic sequences (see Fig. 8-19 in Vol. 1), but they rarely arise in relaxed DNA because linear DNA accommodates more base pairs than cruciform structures do. Partial DNA unwinding facilitates strand separation and the formation of a cruciform structure at the corresponding sequence regions.

Topoisomerases Catalyze Changes in the Linking Number of DNA

DNA supercoiling is a tightly regulated process that affects various aspects of DNA metabolism. Every cell contains enzymes whose sole function is the partial unwinding and/or relaxation of DNA. Enzymes that increase or decrease the degree of DNA partial unwinding are called topoisomerases; they alter the linking number of DNA. These enzymes play a particularly crucial role in DNA replication and packaging. There are two classes of topoisomerases. Type I topoisomerases transiently break one of the two DNA strands, pass the unbroken strand through the break, and reseal the broken ends; they change Lk in increments of 1. Type II topoisomerases break both DNA strands and change Lk in increments of 2.

Fig. 24-20. Detection of topoisomers. In this experiment, all DNA molecules contain the same number of base pairs but differ in their degree of supercoiling. Because supercoiled DNA molecules are more compact than relaxed molecules, they move faster during gel Electrophoresis. The gels shown separate topoisomers with similar superhelix density values (molecules move from top to bottom). In lane 1, highly supercoiled DNA migrates as a single band, although different topoisomers may be present. Lanes 2 and 3 show the results of treating supercoiled DNA with type I topoisomerase; the DNA in lane 3 was exposed to the enzyme longer than the DNA in lane 2. When the superhelix density decreases to a point where the gel can resolve individual topoisomers, discrete bands become visible. Each band bracketed to the right of lane 3 contains circular DNA molecules with the same linking number; for two adjacent bands, the linking numbers differ by one.

Fig. 24-21. Reaction mechanism. Bacterial type I topoisomerases alter the linking number. The reaction sequence of type I topoisomerase is shown. The enzyme can exist in closed and open Conformations. (a) The DNA molecule binds to the enzyme in the closed conformation, and one DNA strand is cleaved. (b) The enzyme transitions to the open conformation, and the second DNA strand passes through the break in the first strand. (c) The enzyme returns to the closed conformation, and the DNA strand is rejoined.

The action of these Enzymes can be demonstrated using agarose gel electrophoresis (Fig. 24-20). A set of identical plasmids with the same linking number migrates in the gel as discrete bands. This method makes it possible to resolve topoisomers whose Lr values differ by as little as 1 and to detect changes in the linking number caused by topoisomerases.

E. coli cells possess at least four different topoisomerases (I through IV). Type I topoisomerases (topoisomerases I and III) generally relax DNA by removing negative supercoils (increasing Lr). The mechanism by which bacterial type I topoisomerases alter the linking number is illustrated in Fig. 24-21. The bacterial type II enzyme (type II topoisomerase, or DNA gyrase) can introduce negative supercoils (decreasing Lr). This enzyme utilizes ATP energy to drive the reaction. To change the DNA linking number, type II topoisomerases break both strands of the DNA molecule and pass another duplex through the resulting gap. The degree of bacterial DNA supercoiling is maintained by regulating the combined activities of topoisomerases I and II.

Eukaryotic cells also possess type I and type II topoisomerases. Topoisomerases I and III are type I enzymes, whereas vertebrates have a single type II enzyme that exists in two isoforms, IIα and IIβ. Most type II topoisomerases, including archaeal DNA gyrase, are similar and belong to a single family—type IIA. In addition, archaea possess unusual type IV topoisomerases, which form the type IIB family. Eukaryotic type II topoisomerases cannot partially unwind DNA (form negative supercoils), but they can relax both positive and negative supercoils (Fig. 24-22).

Fig. 24-22. Proposed mechanism of linking number alteration by eukaryotic type IIA topoisomerases. ① The multi-subunit enzyme binds one DNA molecule (blue bars). The opened cavities above and below the bound DNA are designated the N- and C-gates. ② A second segment of the same DNA molecule (red bars) binds at the N-gate and becomes trapped ③. Both strands of the first DNA segment are cleaved (the reaction mechanism is analogous to that shown in Fig. 24-20b), and ④ the second DNA segment passes through the break. ⑤ The cleaved DNA is resealed, and the second DNA segment is released through the G-gate. During this cycle, two ATP molecules are bound and hydrolyzed; one ATP molecule is presumably hydrolyzed during complex formation at step ④. Further details of ATP Hydrolysis in this reaction remain to be established.

As we will see in subsequent chapters, topoisomerases play a pivotal role in DNA metabolism, making them important targets for drugs designed to combat bacterial infections and malignancies (Box 24-1).

Compact Packaging of DNA Requires a Special Form of Supercoiling

Supercoiled DNA molecules share several common features. In negatively supercoiled molecules, supercoils are right-handed (Fig. 24-17) and tend to form thin, long loops with numerous branches rather than compact structures (Fig. 24-23). At the superhelix density characteristic of the cell, the length of supercoiled regions, including branches, accounts for approximately 40% of the total DNA length. This type of supercoiling is termed plectonemic (from the Greek plektos, twisted, and *nema*, thread). This term can describe any structure with threads twisted in some simple and regular manner, and it adequately explains the overall structure of supercoiled DNA in solution.

Fig. 24-23. Plectonemic supercoiling. (a) Electron micrograph of plectonemic supercoiled plasmid DNA; (b) interpretation of the observed pattern, with purple lines indicating the axes of the supercoils (note the branching of the helix); (c) a simplified representation of this structure.

Plectonemic supercoiling—the form in which DNA is typically isolated under laboratory conditions—is not compact enough for packaging DNA within a cell. Partially unwound DNA can exist in another superhelical conformation known as the solenoidal form (Fig. 24-24). Unlike the extended right-handed plectonemic superhelix, the solenoidal form features tight left-handed loops. This structure resembles a garden hose neatly wound onto a spool. Although plectonemic and solenoidal superhelices are fundamentally different in structure, both forms of negative supercoiling can arise in the exact same region of partially unwound DNA and are interconvertible. The plectonemic form is more stable in solution, whereas the solenoidal form is stabilized by proteins and is found within Chromatin. DNA in this conformation is much more compact (Fig. 24-24b). Solenoidal supercoiling is the mechanism by which partial unwinding contributes to the compact packaging of DNA.

Fig. 24-24. Plectonemic and solenoidal forms of supercoiling. (a) In the plectonemic superhelix, DNA forms extended right-handed turns. In the solenoidal negative superhelix, the molecule resembles a tube composed of tight left-handed turns. Both forms can interconvert, although the solenoidal form generally does not form unless DNA is bound to specific proteins. (b) The plectonemic (top) and solenoidal forms of the same DNA molecule differ in size. DNA in the solenoidal form is significantly more compact.

Box 24-1. MEDICINE. Treatment of Diseases by Inhibition of Topoisomerases

The topology of cellular DNA is directly linked to its function. Without the aid of topoisomerases, cells cannot replicate or package their DNA, nor can they express genes, ultimately leading to cell death. Consequently, topoisomerase inhibitors represent crucial pharmaceutical agents in the fight against infectious pathogens and malignant cells.

Two classes of bacterial topoisomerase inhibitors are employed as Antibiotics. Coumarins, including novobiocin and coumermycin A1, are natural products isolated from Streptomyces species. They inhibit the binding of ATP by bacterial type II topoisomerases, namely DNA gyrase and topoisomerase IV. Although these antibiotics are rarely used today to treat human infectious diseases, the search for effective clinical candidates within this class remains ongoing.

Quinolone antibiotics, which also inhibit bacterial DNA gyrase and topoisomerase IV, entered medical practice in 1962 with the introduction of nalidixic acid. Although this compound has limited efficacy and is no longer used clinically in the United States, extensive research into this class of substances led to The Development of fluoroquinolones, such as ciprofloxacin (Cipro). Quinolones act by inhibiting The final stage of the topoisomerase reaction, which involves sealing breaks in the DNA strands. Ciprofloxacin is a broad-spectrum antibiotic. It is one of the few drugs effective against anthrax pathogens and is regarded as a potent countermeasure against biological weapons. Quinolones act exclusively on bacterial topoisomerases; inhibition of eukaryotic enzymes requires concentrations several orders of magnitude higher than the therapeutic dose.

Some of the most potent chemotherapeutic agents used in Cancer treatment are human topoisomerase inhibitors. Because tumor cells typically exhibit elevated concentrations of topoisomerases, drugs that inhibit these enzymes are significantly more toxic to tumors than to healthy Tissues. Both type I and type II topoisomerase inhibitors are utilized as antitumor drugs.

Camptothecin, isolated from an ornamental Chinese plant and first tested clinically in the 1970s, inhibits eukaryotic type I topoisomerase. Clinical trials revealed limited efficacy, despite promising results in preclinical mouse models. Later, in the 1990s, two effective derivatives of this compound were developed—irinotecan and topotecan—used for the treatment of colorectal and Ovarian cancer, respectively. Additional related drugs may be released in the near future. All medications in this group function by binding to the topoisomerase-DNA complex while the DNA is in a cleaved state, thereby inhibiting ligation.

A variety of antitumor agents target human type II topoisomerases, including doxorubicin (adriamycin), etoposide (etopophos), and ellipticine. Doxorubicin, a member of the anthracycline class, is effective against several types of cancer. Most drugs in this group stabilize the covalent bonds within the Cleavage complex formed by topoisomerase and broken DNA.

Generally, all of these antitumor drugs increase The amount of DNA damage in rapidly proliferating tumor tissues. Unfortunately, however, healthy tissues can also be affected, leading to systemic toxicity and adverse side effects that must be managed during Chemotherapy. As antitumor therapy becomes more effective and patient prognoses improve, The Emergence of secondary, treatment-related malignancies has come to the forefront. Topoisomerases will undoubtedly remain a major focus of intensive research in the search for novel anti-cancer drugs.

Summary of Section 24.2 DNA Supercoiling

■ Most cellular DNA is supercoiled. Partial unwinding reduces the total number of DNA helical turns compared to the relaxed B-form. To maintain this underwound state, the DNA must be circular or protein-bound. Partial unwinding is quantified by a topological parameter known as the linking number, Lk.

■ Partial unwinding is measured by the superhelical density σ (the specific linking difference), expressed as (Lk − Lk₀) / Lk₀. For cellular DNA, σ typically ranges from −0.05 to −0.07, meaning that the DNA lacks 5% to 7% of its helical turns. DNA underwinding facilitates strand separation by enzymes involved in DNA metabolism.

■ DNA molecules that differ only in their linking number are called topoisomers. Enzymes that facilitate the partial unwinding or relaxation of DNA are called topoisomerases; they catalyze changes in the linking number. Type I and type II topoisomerases alter Lk by increments of one or two units per catalytic cycle, respectively.