《生物化学原理》（英文版）INFORMATION PATHWAYS

8885d_c24_920-9472/11/041:36 PM Page931mac76mac76:385 24.2 DNA Supercoiling dNA double helix(coill Axis Cail- DNA FIGURE 24-10 Supercoils. A typical phone cord is coiled like a DNA helix, and the coiled cord can itself coil in a supercoil. The illustra- tion is especially appropriate because an examination of phone cords many properties of small circular DNAs can be explained by super- coiling. They first detected DNA supercoiling, in small circular viral DNAs. in 1965 FIGURE 24-11 Supercoiling of DNA. When the axis of the DNA dou- ble helix is coiled on itself, it forms a new helix(superhelix). The DNA superhelix is usually called a superc require a separation of dNA strands process com- plicated by the helical interwinding of the strands (as demonstrated in Fig. 24-12) That DNA would bend on itself and become super- coiled in tightly packaged cellular DNA would seem log- cal, then, and perhaps even trivial, were it not for one additional fact: many circular DNA molecules remain highly supercoiled even after they are extracted and pu rified, freed from protein and other cellular components This indicates that supercoiling is an intrinsic property of DNA tertiary structure. It occurs in all cellular DNAs and is highly regulated by each cell. A number of measurable properties of supercoiling have been established, and the study of supercoiling has provided many insights into DNA structure and func tion. This work has drawn heavily on concepts derived from a branch of mathematics called topology, the udy of the properties of an object that do not change under continuous deformations For DNA. continuous deformations include conformational changes due to FIGURE 24-12 Supercoiling induced by separating the strands of a thermal motion or an interaction with proteins or other helical structure. Twist two linear strands of rubber band into a right molecules; discontinuous deformations involve DNA handed double helix as shown. Fix one end by having a friend hold strand breakage. For circular DNA molecules, a topolo- to it, then pull apart the two strands at the other end. Th al property is one that is unaffected by deformations strain will produce supercoiling

require a separation of DNA strands—a process com￾plicated by the helical interwinding of the strands (as demonstrated in Fig. 24–12). That DNA would bend on itself and become super￾coiled in tightly packaged cellular DNA would seem log￾ical, then, and perhaps even trivial, were it not for one additional fact: many circular DNA molecules remain highly supercoiled even after they are extracted and pu￾rified, freed from protein and other cellular components. This indicates that supercoiling is an intrinsic property of DNA tertiary structure. It occurs in all cellular DNAs and is highly regulated by each cell. A number of measurable properties of supercoiling have been established, and the study of supercoiling has provided many insights into DNA structure and func￾tion. This work has drawn heavily on concepts derived from a branch of mathematics called topology, the study of the properties of an object that do not change under continuous deformations. For DNA, continuous deformations include conformational changes due to thermal motion or an interaction with proteins or other molecules; discontinuous deformations involve DNA strand breakage. For circular DNA molecules, a topolo￾gical property is one that is unaffected by deformations FIGURE 24–10 Supercoils. A typical phone cord is coiled like a DNA helix, and the coiled cord can itself coil in a supercoil. The illustra￾tion is especially appropriate because an examination of phone cords helped lead Jerome Vinograd and his colleagues to the insight that many properties of small circular DNAs can be explained by super￾coiling. They first detected DNA supercoiling, in small circular viral DNAs, in 1965. DNA double helix (coil) DNA supercoil Axis FIGURE 24–11 Supercoiling of DNA. When the axis of the DNA dou￾ble helix is coiled on itself, it forms a new helix (superhelix). The DNA superhelix is usually called a supercoil. FIGURE 24–12 Supercoiling induced by separating the strands of a helical structure. Twist two linear strands of rubber band into a right￾handed double helix as shown. Fix one end by having a friend hold onto it, then pull apart the two strands at the other end. The resulting strain will produce supercoiling. 24.2 DNA Supercoiling 931 8885d_c24_920-947 2/11/04 1:36 PM Page 931 mac76 mac76:385_reb:

8885dc24920-9472/11/041:36 PM Page932mac76mac76:385 932 Chapter 24 Genes and Chromosomes FIGURE 24-13 Relaxed and supercoiled plasmid DNAs. The molecule in the leftmost electron micrograph is relaxed; the degree of supercoiling increases from left to right of the dna strands as long as no breaks are introduced. ture of the larger DNA molecule). In principle, the strain Topological properties are changed only by breakage could also be accommodated by separating the two dNA and rejoining of the backbone of one or both Dna strands over a distance of about 10 bp (Fig. 24-14d)In strands isolated closed-circular DNA, strain introduced by un- We now examine the fundamental properties and derwinding is generally accommodated by supercoiling physical basis of supercoiling rather than strand separation, because coiling the axis of the dNa usually requires less energy than breaking Most cellular dna is underwound the hydrogen bonds that stabilize paired bases. Note, To understand supercoiling we must first focus on the however, that the underwinding of dNa in vivo makes small viral DNAs. When these DNAs have no brea g properties of small circular DNAs such as plasmids either strand, they are referred to as closed-circular DNAS. If the dna of a closed-circular molecule con (a) Relaxed (8 turns) forms closely to the B-form structure(the Watson-Crick structure: see Fig. 8-15), with one turn of the double 2000 helix per 10.5 bp, the DNA is relaxed rather than su- (b)Strained (7 turns) is subject to some form of structural strain Purified MM closed-circular dna is rarely relaxed, regardless of its biological origin. Furthermore, DNAs derived from a given cellular source have a characteristic degree of su- percoiling. DNA structure is therefore strained in aman- ner that is regulated by the cell to induce the super- In almost every instance, the strain is a result of un- derwinding of the dna double helix in the closed cir- (c)Supercoil le. In other words, the dNa has fewer helical turns than would be expected for the B-form structure. The effects of underwinding are summarized in Figure 24-14. An 84 bp segment of a circular dna in the re- (d) Strand separation laxed state would contain eight double-helical turns one for every 10.5 bp. If one of these tums were re- within a closed-circular molecule, 84 bp long, in its relaxed form with helical tums. (b)Removal of one turn induces structural strain. rather than the 10.5 found in B-DNA (Fig. 24-14b). This (@) The strain is generally accommodated by formation of a supercoil is a deviation from the most stable DNA form, and the (d) DNA underwinding also makes the separation of strands some- molecule is thermodynamically strained as a result. Gen- hat easier. In principle, each turn of underwinding should facilitate erally, much of this strain would be accommodated by rand separation over about 10 bp, as shown. However, the hydrogen. coiling the axis of the DNA on itself to form a supercoil bonded base pairs would generally preclude strand separation over (ig. 24-14c; some of the strain in this 84 bp segment such a short distance, and the effect becomes important only for longer would simply become dispersed in the untwisted struc- DNAs and higher levels of DNA underwinding

of the DNA strands as long as no breaks are introduced. Topological properties are changed only by breakage and rejoining of the backbone of one or both DNA strands. We now examine the fundamental properties and physical basis of supercoiling. Most Cellular DNA Is Underwound To understand supercoiling we must first focus on the properties of small circular DNAs such as plasmids and small viral DNAs. When these DNAs have no breaks in either strand, they are referred to as closed-circular DNAs. If the DNA of a closed-circular molecule con￾forms closely to the B-form structure (the Watson-Crick structure; see Fig. 8–15), with one turn of the double helix per 10.5 bp, the DNA is relaxed rather than su￾percoiled (Fig. 24–13). Supercoiling results when DNA is subject to some form of structural strain. Purified closed-circular DNA is rarely relaxed, regardless of its biological origin. Furthermore, DNAs derived from a given cellular source have a characteristic degree of su￾percoiling. DNA structure is therefore strained in a man￾ner that is regulated by the cell to induce the super￾coiling. In almost every instance, the strain is a result of un￾derwinding of the DNA double helix in the closed cir￾cle. In other words, the DNA has fewer helical turns than would be expected for the B-form structure. The effects of underwinding are summarized in Figure 24–14. An 84 bp segment of a circular DNA in the re￾laxed state would contain eight double-helical turns, or one for every 10.5 bp. If one of these turns were re￾moved, there would be (84 bp)/7  12.0 bp per turn, rather than the 10.5 found in B-DNA (Fig. 24–14b). This is a deviation from the most stable DNA form, and the molecule is thermodynamically strained as a result. Gen￾erally, much of this strain would be accommodated by coiling the axis of the DNA on itself to form a supercoil (Fig. 24–14c; some of the strain in this 84 bp segment would simply become dispersed in the untwisted struc￾ture of the larger DNA molecule). In principle, the strain could also be accommodated by separating the two DNA strands over a distance of about 10 bp (Fig. 24–14d). In isolated closed-circular DNA, strain introduced by un￾derwinding is generally accommodated by supercoiling rather than strand separation, because coiling the axis of the DNA usually requires less energy than breaking the hydrogen bonds that stabilize paired bases. Note, however, that the underwinding of DNA in vivo makes 932 Chapter 24 Genes and Chromosomes 0.2 m FIGURE 24–13 Relaxed and supercoiled plasmid DNAs. The molecule in the leftmost electron micrograph is relaxed; the degree of supercoiling increases from left to right. (a) Relaxed (8 turns) (d) Strand separation (b) Strained (7 turns) (c) Supercoil FIGURE 24–14 Effects of DNA underwinding. (a) A segment of DNA within a closed-circular molecule, 84 bp long, in its relaxed form with eight helical turns. (b) Removal of one turn induces structural strain. (c) The strain is generally accommodated by formation of a supercoil. (d) DNA underwinding also makes the separation of strands some￾what easier. In principle, each turn of underwinding should facilitate strand separation over about 10 bp, as shown. However, the hydrogen￾bonded base pairs would generally preclude strand separation over such a short distance, and the effect becomes important only for longer DNAs and higher levels of DNA underwinding. 8885d_c24_920-947 2/11/04 1:36 PM Page 932 mac76 mac76:385_reb:

8885d_c24_920-9472/11/041:36 PM Page933mac76mac76:385 24.2 DNA Supercoiling it easier to separate DNA strands, giving access to the information they contain. Every cell actively underwinds its DNA with the aid of enzymatic processes (described below), and the resulting strained state represents a form of stored en- ergy. Cells maintain dNa in an underwound state to fa- cilitate its compaction by coiling. The underwinding of DNA is also important to enzymes of DNA metabolism (a)Lk=1 that must bring about strand separation as part of their function The underwound state can be maintained only if the DNA is a closed circle or if it is bound and stabilized by proteins so that the strands are not free to rotate about each other if there is a break in one strand of an iso- lated, protein-free circular DNA, free rotation at that point will cause the underwound dNa to revert spon- taneously to the relaxed state. In a closed-circular dNA molecule. however the number of helical turns cannot be changed without at least transiently breaking one of the dna strands The number of helical turns in a dna (b)Lk= 6 molecule therefore provides a precise description of FIGURE 24-15 Linking number, Lk. Here, as usual, each blue ribbon supercoiling represents one strand of a double-stranded DNA molecule. For the DNA Underwinding Is Defined by Topological molecule in(a), Lk= 1. For the molecule in( b), Lk=6. One of the strands in(b) is kept untwisted for illustrative purposes, to define Linking Number the border of an imaginary surface (shaded blue). The The field of topology provides a number of ideas that times the twisting strand penetrates this surface provides a rigorous are useful to this discussion, particularly the concept of definition of linking number linking number. Linking number is a topological prop- erty of double-stranded dNA, because it does not vary is relaxed, the linking number is simply the number of when the dna is bent or deformed, as long as both dna base pairs divided by the number of base pairs per turn, strands remain intact. Linking number (Lk) is illustrated which is close to 10.5; so in this case, Lk= 200. For a in Figure 24-15 circular DNA molecule to have a topological property Let's begin by visualizing the separation of the two such as linking number, neither strand may contain a strands of a double-stranded circular dNA. If the two break. If there is a break in either strand. the strands strands are linked as shown in Figure 24-15a, they are can, in principle, be unraveled and separated com- effectively joined by what can be described as a pletely. In this case, no topological bond exists and lk topological bond. Even if all hydrogen bonds and base- is undefined(Fig. 24-16b) stacking interactions were abolished such that the We can now describe dNa underwinding in terms strands were not in physical contact, this topological of changes in the linking number. The linking number bond would still link the two strands. Visualize one of in relaxed DNA, Lko, is used as a reference. For the mol- the circular strands as the boundary of a surface(such ecule shown in Figure 24-16a, Lko= 200; if two turns as a soap film spanning the space framed by a circular are removed from this molecule, Lk= 198. The change wire before you blow a soap bubble). The linking num- can be described by the equation ber can be defined as the number of times the second △Lk=Lk-Lko=198-200=-2 strand pierces this surface. For the molecule in Figure 24-15a, Lk= l; for that in Figure 24-15b, Lk=6. The It is often convenient to express the change in linking linking number for a closed-circular DNA is always an number in terms of a quantity that is independent of the integer. By convention, if the links between two dNA length of the DNA molecule. This quantity, called the strands are arranged so that the strands are interwound specifie linking difference (o), or superhelical a right-handed helix, the linking number is defined density, is a measure of the number of turns removed as positive (+) for strands interwound in a left-handed relative to the number present in relaxed dNA the linking number is negative(-). Negative link- numbers are, for all practical purposes, not countered in dnA. We can now extend these ideas to a closed-circular In the example in Figure 24-16c, 0=-0.01, which NA with 2, 100 bp(ig. 24-16a) When the molecule means that 1%(2 of 200)of the helical turns present

it easier to separate DNA strands, giving access to the information they contain. Every cell actively underwinds its DNA with the aid of enzymatic processes (described below), and the resulting strained state represents a form of stored en￾ergy. Cells maintain DNA in an underwound state to fa￾cilitate its compaction by coiling. The underwinding of DNA is also important to enzymes of DNA metabolism that must bring about strand separation as part of their function. The underwound state can be maintained only if the DNA is a closed circle or if it is bound and stabilized by proteins so that the strands are not free to rotate about each other. If there is a break in one strand of an iso￾lated, protein-free circular DNA, free rotation at that point will cause the underwound DNA to revert spon￾taneously to the relaxed state. In a closed-circular DNA molecule, however, the number of helical turns cannot be changed without at least transiently breaking one of the DNA strands. The number of helical turns in a DNA molecule therefore provides a precise description of supercoiling. DNA Underwinding Is Defined by Topological Linking Number The field of topology provides a number of ideas that are useful to this discussion, particularly the concept of linking number. Linking number is a topological prop￾erty of double-stranded DNA, because it does not vary when the DNA is bent or deformed, as long as both DNA strands remain intact. Linking number (Lk) is illustrated in Figure 24–15. Let’s begin by visualizing the separation of the two strands of a double-stranded circular DNA. If the two strands are linked as shown in Figure 24–15a, they are effectively joined by what can be described as a topological bond. Even if all hydrogen bonds and base￾stacking interactions were abolished such that the strands were not in physical contact, this topological bond would still link the two strands. Visualize one of the circular strands as the boundary of a surface (such as a soap film spanning the space framed by a circular wire before you blow a soap bubble). The linking num￾ber can be defined as the number of times the second strand pierces this surface. For the molecule in Figure 24–15a, Lk  1; for that in Figure 24–15b, Lk  6. The linking number for a closed-circular DNA is always an integer. By convention, if the links between two DNA strands are arranged so that the strands are interwound in a right-handed helix, the linking number is defined as positive (); for strands interwound in a left-handed helix, the linking number is negative (). Negative link￾ing numbers are, for all practical purposes, not en￾countered in DNA. We can now extend these ideas to a closed-circular DNA with 2,100 bp (Fig. 24–16a). When the molecule is relaxed, the linking number is simply the number of base pairs divided by the number of base pairs per turn, which is close to 10.5; so in this case, Lk  200. For a circular DNA molecule to have a topological property such as linking number, neither strand may contain a break. If there is a break in either strand, the strands can, in principle, be unraveled and separated com￾pletely. In this case, no topological bond exists and Lk is undefined (Fig. 24–16b). We can now describe DNA underwinding in terms of changes in the linking number. The linking number in relaxed DNA, Lk0, is used as a reference. For the mol￾ecule shown in Figure 24–16a, Lk0  200; if two turns are removed from this molecule, Lk  198. The change can be described by the equation Lk  Lk  Lk0  198  200  2 It is often convenient to express the change in linking number in terms of a quantity that is independent of the length of the DNA molecule. This quantity, called the specific linking difference (), or superhelical density, is a measure of the number of turns removed relative to the number present in relaxed DNA:    L L k k 0 In the example in Figure 24–16c,   0.01, which means that 1% (2 of 200) of the helical turns present 24.2 DNA Supercoiling 933 (b) Lk = 6 (a) Lk = 1 FIGURE 24–15 Linking number, Lk. Here, as usual, each blue ribbon represents one strand of a double-stranded DNA molecule. For the molecule in (a), Lk  1. For the molecule in (b), Lk  6. One of the strands in (b) is kept untwisted for illustrative purposes, to define the border of an imaginary surface (shaded blue). The number of times the twisting strand penetrates this surface provides a rigorous definition of linking number. 8885d_c24_920-947 2/11/04 1:36 PM Page 933 mac76 mac76:385_reb:

8885dc24920-9472/11/041:36 PM Page934mac76mac76:385 934 Chapter 24 Genes and Chromosomes Relaxed DNA △Lk= △Lk=+2 strand △Dk=-2 (a)Lk=200= FIGURE 24-17 Negative and positive supercoils. For the relaxed DNA (c)Lh=198 molecule of Figure 24-16a, underwinding or overwinding by two helical turns(Lk=198 or 202)will produce negative or positive su. FIGURE 24-16 Linking number applied to closed-circular DNA mol- percoiling respectively. Note that the DNA axis twists in opposite ecules A2, 100 bp circular DNA is shown in three forms: (a)relaxed, directions in the two cases Lk= 200,(b)relaxed with a nick(break) in one strand, Lk undefined and(c) underwound by two turns, Lk=198. The underwound mole- cule generally exists as a supercoiled molecule, but underwinding also boring base pairs. When the linking number changes facilitates the separation of DNA strands some of the resulting strain is usually compensated for by writhe(supercoiling) and some by changes in twist in the DNa (in its B form) have been removed. The de- giving rise to the equation gree of underwinding in cellular dnas generally falls in Lk= Tw+ wr the range of 5% to 7%; that is, o=-005 to-0.07. The negative sign indicates that the change in linking num and Wr need not be integers. Twist and writhe are ber is due to underwinding of the DNA. The supercoil- geometric rather than topological properties, because g induced by underwinding is therefore defined as they may be changed by deformation of a closed-circular negative supercoiling. Conversely, under some condi- DNA molecule. tions DNA can be overwound, resulting in positive su- In addition to causing supercoiling and making percoiling. Note that the twisting path taken by the axis strand separation somewhat easier, the underwinding of of the dna helix when the dna is underwound (nega- tive supercoiling) is the mirror image of that taken when the dna is overwound (positive supercoiling)(Fig. 24-17). Supercoiling is not a random process; the path WVAVAVAVAVAVAVAV of the supercoiling is largely prescribed by the torsional strain imparted to the dNa by decreasing or increasing Straight ribbon(relaxed DNA the linking number relative to B-DNA. one DNa strand, rotating one of the ends 360 about the unbroken strand, and rejoining the broken ends. This change has no effect on the number of base pairs or the number of atoms in the circular dna molecule. two Large writhe, small change in twist forms of a circular DNa that differ only in a topological property such as linking number are referred to as topoisomers. Zero writhe large change in twist nking number can be broken de FIGURE 24-18 Ribbon model for illustrating twist and writhe. the tural components called writhe (r) and twist (Tw) pink ribbon represents the axis of a relaxed DNA molecule. Strain (Fig. 24-18). These are more difficult to describe than introduced by twisting the ribbon(underwinding the DNA) can be linking number, but writhe may be thought of as a meas- manifested as writhe or twist. Changes in linking number are usually ure of the coiling of the helix axis and twist as deter- accompanied by changes in both writhe and twist

in the DNA (in its B form) have been removed. The de￾gree of underwinding in cellular DNAs generally falls in the range of 5% to 7%; that is,   0.05 to 0.07. The negative sign indicates that the change in linking num￾ber is due to underwinding of the DNA. The supercoil￾ing induced by underwinding is therefore defined as negative supercoiling. Conversely, under some condi￾tions DNA can be overwound, resulting in positive su￾percoiling. Note that the twisting path taken by the axis of the DNA helix when the DNA is underwound (nega￾tive supercoiling) is the mirror image of that taken when the DNA is overwound (positive supercoiling) (Fig. 24–17). Supercoiling is not a random process; the path of the supercoiling is largely prescribed by the torsional strain imparted to the DNA by decreasing or increasing the linking number relative to B-DNA. Linking number can be changed by 1 by breaking one DNA strand, rotating one of the ends 360 about the unbroken strand, and rejoining the broken ends. This change has no effect on the number of base pairs or the number of atoms in the circular DNA molecule. Two forms of a circular DNA that differ only in a topological property such as linking number are referred to as topoisomers. Linking number can be broken down into two struc￾tural components called writhe (Wr) and twist (Tw) (Fig. 24–18). These are more difficult to describe than linking number, but writhe may be thought of as a meas￾ure of the coiling of the helix axis and twist as deter￾mining the local twisting or spatial relationship of neigh￾boring base pairs. When the linking number changes, some of the resulting strain is usually compensated for by writhe (supercoiling) and some by changes in twist, giving rise to the equation Lk  Tw  Wr Tw and Wr need not be integers. Twist and writhe are geometric rather than topological properties, because they may be changed by deformation of a closed-circular DNA molecule. In addition to causing supercoiling and making strand separation somewhat easier, the underwinding of 934 Chapter 24 Genes and Chromosomes Relaxed DNA Lk  200 ∆Lk  2 ∆Lk  2 Negative supercoils Lk  198 Positive supercoils Lk  202 FIGURE 24–17 Negative and positive supercoils. For the relaxed DNA molecule of Figure 24–16a, underwinding or overwinding by two helical turns (Lk  198 or 202) will produce negative or positive su￾percoiling, respectively. Note that the DNA axis twists in opposite directions in the two cases. Straight ribbon (relaxed DNA) Zero writhe, large change in twist Large writhe, small change in twist FIGURE 24–18 Ribbon model for illustrating twist and writhe. The pink ribbon represents the axis of a relaxed DNA molecule. Strain introduced by twisting the ribbon (underwinding the DNA) can be manifested as writhe or twist. Changes in linking number are usually accompanied by changes in both writhe and twist. (a) Lk  200  Lk0 (b) Lk undefined (c) Lk = 198 strand break ∆Lk  2 Nick FIGURE 24–16 Linking number applied to closed-circular DNA mol￾ecules. A 2,100 bp circular DNA is shown in three forms: (a) relaxed, Lk  200; (b) relaxed with a nick (break) in one strand, Lk undefined; and (c) underwound by two turns, Lk  198. The underwound mole￾cule generally exists as a supercoiled molecule, but underwinding also facilitates the separation of DNA strands. 8885d_c24_920-947 2/11/04 1:36 PM Page 934 mac76 mac76:385_reb:

88524-920-9472/11041:36age935mac76ma76:385 24.2 DNA Supercoiling DNA facilitates a number of structural changes in the portant role in processes such as replication and dna molecule. These are of less physiological importance but packaging. There are two classes of topoisomerases help illustrate the effects of underwinding. Recall that Type I topoisomerases act by transiently breaking one a cruciform(see Fig 8-21) generally contains a few un- of the two DNA strands, passing the unbroken strand paired bases; DNA underwinding helps to maintain the through the break, and rejoining the broken ends; they required strand separation(Fig. 24-19). Underwinding change Lk in increments of 1. Type Il topoisomerases of a right-handed dna helix also facilitates the forma- break both dna strands and change Lk in increments tion of short stretches of left-handed Z-DNA in regions of 2 where the base sequence is consistent with the z form The effects of these enzymes can be demonstrated (Chapter 8) using agarose gel electrophoresis(Fig. 24-20). A pop- ulation of identical plasmid DNAs with the same linking Topoisomerases Catalyze Changes in the Linking number migrates as a discrete band during electro- Number of dna phoresis. Topoisomers with Lk values differing by as little as l can be separated by this method, so change DNA supercoiling is a precisely regulated process that in linking number induced by topoisomerases are read- influences many aspects of DNA metabolism. Every cell ily detected. has enzymes with the sole function of underwinding and/or relaxing DNA. The enzymes that increase or de- 3 crease the extent of DNa underwinding are topoiso merases; the property of dNa that they change is the linking number. These enzymes play an especially im- Relaxed Relaxed dNA Underwound dNA FIGURE 24-20 Visualization of topoisomers. In this experiment, all DNA molecules have the same number of base pairs but exhibit some range in the degree of supercoiling. Because supercoiled DNA mole- nles are more compact than relaxed molecules, they migrate more rapidly during gel electrophoresis. The gels shown here separate t somers(moving from top to bottom)over a limited range of superbe cal density. In lane 1, highly supercoiled DNA migrates in a single Cruciform dna band, even though different topoisomers are probably present. Lanes 2 and 3 illustrate the effect of treating the supercoiled DNA with FIGURE 24-19 Promotion of cruciform structures by DNA under- type I topoisomerase; the DNA in lane 3 was treated for a longer time winding. In principle, cruciforms can form at palindromic sequences than that in lane 2. As the superhelical density of the DNA is reduced (see Fig. 8-21), but they seldom occur in relaxed DNA because the to the point where it corresponds to the range in which the gel can linear DNA accommodates more paired bases than does the cruci. resolve individual topoisomers, distinct bands appear. Individual bands form structure. Underwinding of the DNA facilitates the partial strand in the region indicated by the bracket next to lane 3 each contain separation needed to promote cruciform formation at appropriate DNA circles with the same linking number; the linking number anges by 1 from one band to the next

DNA facilitates a number of structural changes in the molecule. These are of less physiological importance but help illustrate the effects of underwinding. Recall that a cruciform (see Fig. 8–21) generally contains a few un￾paired bases; DNA underwinding helps to maintain the required strand separation (Fig. 24–19). Underwinding of a right-handed DNA helix also facilitates the forma￾tion of short stretches of left-handed Z-DNA in regions where the base sequence is consistent with the Z form (Chapter 8). Topoisomerases Catalyze Changes in the Linking Number of DNA DNA supercoiling is a precisely regulated process that influences many aspects of DNA metabolism. Every cell has enzymes with the sole function of underwinding and/or relaxing DNA. The enzymes that increase or de￾crease the extent of DNA underwinding are topoiso￾merases; the property of DNA that they change is the linking number. These enzymes play an especially im- 24.2 DNA Supercoiling 935 Relaxed DNA Underwound DNA Cruciform DNA FIGURE 24–19 Promotion of cruciform structures by DNA under￾winding. In principle, cruciforms can form at palindromic sequences (see Fig. 8–21), but they seldom occur in relaxed DNA because the linear DNA accommodates more paired bases than does the cruci￾form structure. Underwinding of the DNA facilitates the partial strand separation needed to promote cruciform formation at appropriate sequences. Relaxed DNA Highly supercoiled DNA 12 3 Decreasing Lk FIGURE 24–20 Visualization of topoisomers. In this experiment, all DNA molecules have the same number of base pairs but exhibit some range in the degree of supercoiling. Because supercoiled DNA mole￾cules are more compact than relaxed molecules, they migrate more rapidly during gel electrophoresis. The gels shown here separate topoi￾somers (moving from top to bottom) over a limited range of superhe￾lical density. In lane 1, highly supercoiled DNA migrates in a single band, even though different topoisomers are probably present. Lanes 2 and 3 illustrate the effect of treating the supercoiled DNA with a type I topoisomerase; the DNA in lane 3 was treated for a longer time than that in lane 2. As the superhelical density of the DNA is reduced to the point where it corresponds to the range in which the gel can resolve individual topoisomers, distinct bands appear. Individual bands in the region indicated by the bracket next to lane 3 each contain DNA circles with the same linking number; the linking number changes by 1 from one band to the next. portant role in processes such as replication and DNA packaging. There are two classes of topoisomerases. Type I topoisomerases act by transiently breaking one of the two DNA strands, passing the unbroken strand through the break, and rejoining the broken ends; they change Lk in increments of 1. Type II topoisomerases break both DNA strands and change Lk in increments of 2. The effects of these enzymes can be demonstrated using agarose gel electrophoresis (Fig. 24–20). A pop￾ulation of identical plasmid DNAs with the same linking number migrates as a discrete band during electro￾phoresis. Topoisomers with Lk values differing by as little as 1 can be separated by this method, so changes in linking number induced by topoisomerases are read￾ily detected. 8885d_c24_920-947 2/11/04 1:36 PM Page 935 mac76 mac76:385_reb: