《生物化学原理》（英文版）INFORMATION PATHWAYS

8885d_c24_920-9472/11/041:36 PM Page936mac76mac76:385 O-P=0 3′5 After DNA binds(step ) an active-site Tyr attacks a phosphodiester bond on one DNA strand in step 2, cleaving it, creating a covalent and liberating the 3-hydroxyl group of the adjacent nucleotide conformation 3.5 Bar 0-P=0 0 Release, or begin new cyle With the enzyme in the closed conformation, the liberated 3-hydroxyl group attacks the 5' P-Tyr protein-DNA linkage in step@to MECHANISM FIGURE 24-21 Bacterial type I topoisomerases alter DNA strand is cleaved. (b) The enzyme changes to its open confor aking number. A proposed reaction sequence for the bacterial topoi- mation, and the other DNA strand moves through the break in the first is illustrated. The enzyme has closed and open conforma. strand. (c) In the closed conformation, the DNA strand is religated tions. (a)a DNA molecule binds to the closed conformation and one

5 Tyr Closed conformation 3 3 5 Open conformation 53 3 35 After DNA binds (step 1 ), an active-site Tyr attacks a phosphodiester bond on one DNA strand in step 2 , cleaving it, creating a covalent 5- P –Tyr protein-DNA linkage, and liberating the 3-hydroxyl group of the adjacent nucleotide. In step 3 the enzyme switches to its open conformation, and the unbroken DNA strand passes through the break in the first strand. With the enzyme in the closed conformation, the liberated 3-hydroxyl group attacks the 5- P –Tyr protein-DNA linkage in step 4 to religate the cleaved DNA strand. Release, or begin new cyle : 1 2 3 (a) (b) (c) O O O –O P O O O OH CH2 CH2 O H Tyr O O O –O P O O CH2 CH2 O H Tyr O –O O P O CH2 CH2 Tyr O : 4 5 O– OH O O P H+ O O O CH2 CH2 Tyr 53 3 35 5 3 3 5 Base Base Base O O Base O Base O Base O Base O O O Base MECHANISM FIGURE 24–21 Bacterial type I topoisomerases alter linking number. A proposed reaction sequence for the bacterial topoi￾somerase I is illustrated. The enzyme has closed and open conforma￾tions. (a) A DNA molecule binds to the closed conformation and one DNA strand is cleaved. (b) The enzyme changes to its open confor￾mation, and the other DNA strand moves through the break in the first strand. (c) In the closed conformation, the DNA strand is religated. 8885d_c24_920-947 2/11/04 1:36 PM Page 936 mac76 mac76:385_reb:

8885dc24920-9472/11/041:36 PM Page937mac76mac76:385 24.2 DNA Supercoiling E. coli has at least four different individual topo- Isomerases through I). Those of type I(topoiso- erases I and in) generally relax dNa by removing negative supercoils (increasing Lk). The way in which bacterial type I topoisomerases change linking number is illustrated in Figure 24-21. A bacterial type ll enzyme called either topoisomerase I or DNa gyrase, can in- ③ number, type lI topoisomerases cleave both strands of C-gate a dna molecule and pass another duplex through the break. The degree of supercoiling of bacterial DNA is maintained by regulation of the net activity of topoiso- erases i and II Eukaryotic cells also have type I and type I topo- isomerases. The type Enzymes are topoisomerases I and Ill; the type I enzymes are topoisomerases lla and lB The eukaryotic type Il topoisomerases cannot un 爸一 wind DNA (introduce negative supercoils), but they can relax both positive and negative supercoils. We consider e probable origin of negative supercoils in eukaryotic cells in our discussion of chromatin in section 24 3. The FIGURE 24-22 Proposed mechanism for the alteration of linking process catalyzed by eukaryotic type II topoisomerases number by eukaryotic type llA topoisomerases. O The multisubunit is illustrated in Figure 24-22 enzyme binds one DNA molecule(blue). Gated cavities above and below the bound DNA are called the N-gate and the C-gate. 2A DNA Compaction Requires a Special Form second segment of the same DNA molecule(red) is bound at the N of Supercoiling gate and 3 trapped. Both strands of the first DNA are now cleaved chemistry is similar to that in Fig. 24-20b), and (4 the second Supercoiled DNA molecules are uniform in a number of DNA segment is passed through the break. The broken DNA is re- respects. The supercoils are right-handed in a negatively ligated, and the second DNA segment is released through the C-gate supercoiled DNA molecule(Fig. 24-17), and they tend Two ATPs are bound and hydrolyzed during this cycle; it is likely that to be extended and narrow rather than compacted, of- one is hydrolyzed in the step leading to the complex in step .Ad- ten with multiple branches(Fig. 24-23). At the super- ditional details of the ATP hydrolysis component of the reaction re. helical densities normally encountered in cells, the main to be worked out. length of the supercoil axis, including branches, is about 40% of the length of the DNA. This type of supercoiling is referred to as plectonemic(from the greek plektos Plectonemic supercoiling, the form observed in " twisted, and nema, " thread). This term can be ap- isolated DNAs in the laboratory, does not produce suf- plied to any structure with strands intertwined in some ficient compaction to package DNa in the cell. A sec- simple and regular way, and it is a good description of ond form of supercoiling, solenoidal (Fig. 24-24),can the general structure of supercoiled DNA in solution. be adopted by an underwound DNA. Instead of the Branch FIGURE 24-23 Plectonemic supercoiling (a) Electron micrograph of plectonemically supercoiled plasmid DNA and (b)ar interpretation of the observed structure The purple lines show the axis of the supercoil; note the branching of the supercoil.(c)An idealized representation

E. coli has at least four different individual topo￾isomerases (I through IV). Those of type I (topoiso￾merases I and III) generally relax DNA by removing negative supercoils (increasing Lk). The way in which bacterial type I topoisomerases change linking number is illustrated in Figure 24–21. A bacterial type II enzyme, called either topoisomerase II or DNA gyrase, can in￾troduce negative supercoils (decrease Lk). It uses the energy of ATP to accomplish this. To alter DNA linking number, type II topoisomerases cleave both strands of a DNA molecule and pass another duplex through the break. The degree of supercoiling of bacterial DNA is maintained by regulation of the net activity of topoiso￾merases I and II. Eukaryotic cells also have type I and type II topo￾isomerases. The type I enzymes are topoisomerases I and III; the type II enzymes are topoisomerases II and II
. The eukaryotic type II topoisomerases cannot under￾wind DNA (introduce negative supercoils), but they can relax both positive and negative supercoils. We consider one probable origin of negative supercoils in eukaryotic cells in our discussion of chromatin in Section 24.3. The process catalyzed by eukaryotic type II topoisomerases is illustrated in Figure 24–22. DNA Compaction Requires a Special Form of Supercoiling Supercoiled DNA molecules are uniform in a number of respects. The supercoils are right-handed in a negatively supercoiled DNA molecule (Fig. 24–17), and they tend to be extended and narrow rather than compacted, of￾ten with multiple branches (Fig. 24–23). At the super￾helical densities normally encountered in cells, the length of the supercoil axis, including branches, is about 40% of the length of the DNA. This type of supercoiling is referred to as plectonemic (from the Greek plektos, “twisted,” and nema, “thread”). This term can be ap￾plied to any structure with strands intertwined in some simple and regular way, and it is a good description of the general structure of supercoiled DNA in solution. 24.2 DNA Supercoiling 937 3 5 4 2 1 N-gate C-gate FIGURE 24–22 Proposed mechanism for the alteration of linking number by eukaryotic type IIA topoisomerases. 1 The multisubunit enzyme binds one DNA molecule (blue). Gated cavities above and below the bound DNA are called the N-gate and the C-gate. 2 A second segment of the same DNA molecule (red) is bound at the N￾gate and 3 trapped. Both strands of the first DNA are now cleaved (the chemistry is similar to that in Fig. 24–20b), and 4 the second DNA segment is passed through the break. 5 The broken DNA is re￾ligated, and the second DNA segment is released through the C-gate. Two ATPs are bound and hydrolyzed during this cycle; it is likely that one is hydrolyzed in the step leading to the complex in step 4 . Ad￾ditional details of the ATP hydrolysis component of the reaction re￾main to be worked out. Plectonemic supercoiling, the form observed in isolated DNAs in the laboratory, does not produce suf￾ficient compaction to package DNA in the cell. A sec￾ond form of supercoiling, solenoidal (Fig. 24–24), can be adopted by an underwound DNA. Instead of the (a) (c) Branch points Supercoil axis (b) FIGURE 24–23 Plectonemic supercoiling. (a) Electron micrograph of plectonemically supercoiled plasmid DNA and (b) an interpretation of the observed structure. The purple lines show the axis of the supercoil; note the branching of the supercoil. (c) An idealized representation of this structure. 8885d_c24_920-947 2/11/04 1:36 PM Page 937 mac76 mac76:385_reb:

8885dc24920-9472/11/041:36 PM Page938mac76mac76:385 938 Chapter 24 Genes and Chromosomes density), which is(Lk- Lko /Lko. For cellular DNAS, o is typically -0.05 to-0.07, which neans that approximately 5% to 7% of the elical turns in the dna have been removed DNA underwinding facilitates strand separation of dna metabolism. Plectonemic I DNAs that differ only in linking number are called topoisomers. Enzymes that underwind and/or relax DNA, the topoisomerases, catalyze Solenoidal changes in linking number. The two classes of topoisomerases, type I and type ll, change Lk in increments of I or 2, respectively, per O catalytic event FIGURE 24-24 Plectonemic and solenoidal supercoiling. (a)Plec- 24. 3 The Structure of chromosomes tonemic supercoiling takes the form of extended right-handed coils. The term"chromosome"is used to refer to a nucleic Solenoidal negative supercoiling takes the form of tight left-handed acid molecule that is the repository of genetic informa- tums about an imaginary tubelike structure. The two forms are read- tion in a virus, a bacterium, a eukaryotic cell, or an or- served unless certain proteins are bound to the DNA. b) Plectonemic in the nuclei of dye-stained eukaryotic cells, as visual to scale. Solenoidal supercoiling provides a much greater dey ized using a light microscope Chromatin Consists of dna and proteins The eukaryotic cell cycle(see Fig. 12-41) produces re markable changes in the structure of chromosomes(Fig. extended right-handed supercoils characteristic of the 24-25). In nondividing eukaryotic cells (in GO) and plectonemic form, solenoidal supercoiling involves tight those in interphase (Gl, S, and G2), the chromosomal material, chromatin, is amorphous and appears to be garden hose neatly wrapped on a reel. Although their randomly dispersed in certain parts of the nucleus. In structures are dramatically different, plectonemic and the S phase of interphase the DNA in this amorphous solenoidal supercoiling are two forms of negative super- state replicates, each chromosome producing two sister coiling that can be taken up by the same segment of chromosomes(called sister chromatids )that remain as- underwound DNA. The two forms are readily intercon- sociated with each other after replication is complete vertible. Although the plectonemic form is more stable The chromosomes become much more condensed dur- in solution, the solenoidal form can be stabilized by ing prophase of mitosis, taking the form of a species- protein binding and is the form found in chromatin. It specific number of well-defined pairs of sister chro- provides a much greater degree of compaction(Fig. matids(Fig. 24-5) Chromatin consists of fibers containing protein and which underwinding contributes to DNA compaction. DNA in approximately equal masses, along with a small amount of RNA. The DNA in the chromatin is very SUMMARY 24.2 DNA Supercoiling tightly associated with proteins called histones, which package and order the dNa into structural units called Most cellular DNAs are supercoiled. Under- nucleosomes(Fig. 24-26). Also found in chromatin are many nonhistone proteins, some of which help maintain winding decreases the total number of helical chromosome structure, others that regulate the turns in the dna relative to the relaxed, B form. pression of specific genes(Chapter 28). Beginning with To maintain an underwound state. DNA must nucleosomes, eukaryotic chromosomal DNA is packaged be either a closed circle or bound to protein. to a succession of higher-order structures that ulti- Underwinding is quantified by a topological nately yield the compact chromosome seen with the light microscope. We now turn to a description of this I Underwinding is measured in terms of specific structure in eukaryotes and compare it with the pack- linking difference, o(also called superhelical aging of dNa in bacterial cells

extended right-handed supercoils characteristic of the plectonemic form, solenoidal supercoiling involves tight left-handed turns, similar to the shape taken up by a garden hose neatly wrapped on a reel. Although their structures are dramatically different, plectonemic and solenoidal supercoiling are two forms of negative super￾coiling that can be taken up by the same segment of underwound DNA. The two forms are readily intercon￾vertible. Although the plectonemic form is more stable in solution, the solenoidal form can be stabilized by protein binding and is the form found in chromatin. It provides a much greater degree of compaction (Fig. 24–24b). Solenoidal supercoiling is the mechanism by which underwinding contributes to DNA compaction. SUMMARY 24.2 DNA Supercoiling ■ Most cellular DNAs are supercoiled. Under￾winding decreases the total number of helical turns in the DNA relative to the relaxed, B form. To maintain an underwound state, DNA must be either a closed circle or bound to protein. Underwinding is quantified by a topological parameter called linking number, Lk. ■ Underwinding is measured in terms of specific linking difference,  (also called superhelical density), which is (Lk  Lk0)/Lk0. For cellular DNAs,  is typically 0.05 to 0.07, which means that approximately 5% to 7% of the helical turns in the DNA have been removed. DNA underwinding facilitates strand separation by enzymes of DNA metabolism. ■ DNAs that differ only in linking number are called topoisomers. Enzymes that underwind and/or relax DNA, the topoisomerases, catalyze changes in linking number. The two classes of topoisomerases, type I and type II, change Lk in increments of 1 or 2, respectively, per catalytic event. 24.3 The Structure of Chromosomes The term “chromosome” is used to refer to a nucleic acid molecule that is the repository of genetic informa￾tion in a virus, a bacterium, a eukaryotic cell, or an or￾ganelle. It also refers to the densely colored bodies seen in the nuclei of dye-stained eukaryotic cells, as visual￾ized using a light microscope. Chromatin Consists of DNA and Proteins The eukaryotic cell cycle (see Fig. 12–41) produces re￾markable changes in the structure of chromosomes (Fig. 24–25). In nondividing eukaryotic cells (in G0) and those in interphase (G1, S, and G2), the chromosomal material, chromatin, is amorphous and appears to be randomly dispersed in certain parts of the nucleus. In the S phase of interphase the DNA in this amorphous state replicates, each chromosome producing two sister chromosomes (called sister chromatids) that remain as￾sociated with each other after replication is complete. The chromosomes become much more condensed dur￾ing prophase of mitosis, taking the form of a species￾specific number of well-defined pairs of sister chro￾matids (Fig. 24–5). Chromatin consists of fibers containing protein and DNA in approximately equal masses, along with a small amount of RNA. The DNA in the chromatin is very tightly associated with proteins called histones, which package and order the DNA into structural units called nucleosomes (Fig. 24–26). Also found in chromatin are many nonhistone proteins, some of which help maintain chromosome structure, others that regulate the ex￾pression of specific genes (Chapter 28). Beginning with nucleosomes, eukaryotic chromosomal DNA is packaged into a succession of higher-order structures that ulti￾mately yield the compact chromosome seen with the light microscope. We now turn to a description of this structure in eukaryotes and compare it with the pack￾aging of DNA in bacterial cells. 938 Chapter 24 Genes and Chromosomes (a) (b) Plectonemic Solenoidal FIGURE 24–24 Plectonemic and solenoidal supercoiling. (a) Plec￾tonemic supercoiling takes the form of extended right-handed coils. Solenoidal negative supercoiling takes the form of tight left-handed turns about an imaginary tubelike structure. The two forms are read￾ily interconverted, although the solenoidal form is generally not ob￾served unless certain proteins are bound to the DNA. (b) Plectonemic (top) and solenoidal supercoiling of the same DNA molecule, drawn to scale. Solenoidal supercoiling provides a much greater degree of compaction. 8885d_c24_920-947 2/11/04 1:36 PM Page 938 mac76 mac76:385_reb:

8885dc24920-9472/11/041:36 PM Page939mac76mac76:385 24.3 The structure of chromosomes 939 FIGURE 24-25 Changes in chromosome structure during the eukaryotic cell cycle. Cellular DNA is uncondensed throughout interphase. The interphase period can be Cohesin subdivided (see Fig 12-41)into the G1(gap) phase; the S (synthesis) phase, when the DNA is replicated; and the Duplex phase, in which the replicated chromosomes cohere to one another. The DNA undergoes condensation in the prophase Replication occurs proteins involved in cohesion and condensation(discussed from multiple rigins of replication; later in the chapter). The architecture of the cohesin- daughter chromatids condensin-DNA complex is not yet established, and the are linked by cohesins attractions shown here are figurative, simply suggesting their role in condensation of the chromosome. During completed metaphase, the condensed chromosomes line up along a Interphase o。o plane halfway between the spindle poles. One chromosome Mitosis that extend between the spindle and the centromere. The sister chromatids separate at anaphase, each drawn toward the spindle pole to which it is connected. After cell division is complete, the chromosomes decondense and the cycle 《》关 ins anew Cohesins Histone core Linker dna Histones Are Small, Basic Proteins Found in the chromatin of all eukaryotic cells, histones have molecular weights between 11,000 and 21,000 and are very rich in the basic amino acids arginine and ly sine(together these make up about one- fourth of the amino acid residues). All eukaryotic cells have five m jor classes of histones, differing in molecular weight amino acid composition Table 24-3). The H3 histones are nearly identical in amino acid sequence in all eukaryotes, as are the H4 histones, suggesting strict conservation of their functions. For example, only 2 of 102 amino acid residues differ between the ha histone molecules of peas and cows, and only 8 differ between the H4 histones of humans and yeast. Histones H1, H2A and H2B show less sequence similarity among eukary otic species Each type of histone has variant forms, because cer- tain amino acid side chains are enzymatically modified by methylation, ADP-ribosylation, phosphorylation, gly- cosylation, or acetylation. Such modifications affect the net electric charge, shape, and other properties of FIGURE 24-26 Nucleosomes Regularly spaced nucleosomes consist histones, as well as the structural and functional prop- of histone complexes bound to DNA. (a) Schematic illustration and erties of the chromatin, and they play a role in the reg (b)electron micrograph ulation of transcription(Chapter 28)

Histones Are Small, Basic Proteins Found in the chromatin of all eukaryotic cells, histones have molecular weights between 11,000 and 21,000 and are very rich in the basic amino acids arginine and ly￾sine (together these make up about one-fourth of the amino acid residues). All eukaryotic cells have five ma￾jor classes of histones, differing in molecular weight and amino acid composition (Table 24–3). The H3 histones are nearly identical in amino acid sequence in all eukaryotes, as are the H4 histones, suggesting strict conservation of their functions. For example, only 2 of 102 amino acid residues differ between the H4 histone molecules of peas and cows, and only 8 differ between the H4 histones of humans and yeast. Histones H1, H2A, and H2B show less sequence similarity among eukary￾otic species. Each type of histone has variant forms, because cer￾tain amino acid side chains are enzymatically modified by methylation, ADP-ribosylation, phosphorylation, gly￾cosylation, or acetylation. Such modifications affect the net electric charge, shape, and other properties of histones, as well as the structural and functional prop￾erties of the chromatin, and they play a role in the reg￾ulation of transcription (Chapter 28). 24.3 The Structure of Chromosomes 939 Interphase Mitosis Metaphase Anaphase Prophase Spindle pole G1 G2 condensation replication and cohesion Condensins Cohesins Replication completed Cohesin Duplex DNA S Replication occurs from multiple origins of replication; daughter chromatids are linked by cohesins alignment separation FIGURE 24–25 Changes in chromosome structure during the eukaryotic cell cycle. Cellular DNA is uncondensed throughout interphase. The interphase period can be subdivided (see Fig. 12–41) into the G1 (gap) phase; the S (synthesis) phase, when the DNA is replicated; and the G2 phase, in which the replicated chromosomes cohere to one another. The DNA undergoes condensation in the prophase of mitosis. Cohesins (green) and condensins (red) are proteins involved in cohesion and condensation (discussed later in the chapter). The architecture of the cohesin￾condensin-DNA complex is not yet established, and the interactions shown here are figurative, simply suggesting their role in condensation of the chromosome. During metaphase, the condensed chromosomes line up along a plane halfway between the spindle poles. One chromosome of each pair is linked to each spindle pole via microtubules that extend between the spindle and the centromere. The sister chromatids separate at anaphase, each drawn toward the spindle pole to which it is connected. After cell division is complete, the chromosomes decondense and the cycle begins anew. Histone core of nucleosome Linker DNA of nucleosome (a) (b) 50 nm FIGURE 24–26 Nucleosomes. Regularly spaced nucleosomes consist of histone complexes bound to DNA. (a) Schematic illustration and (b) electron micrograph. 8885d_c24_920-947 2/11/04 1:36 PM Page 939 mac76 mac76:385_reb:

885024-920-947211041:36age940nac76ma76:385律 940 Chapter 24 Genes and Chromosomes H2B Nucleosomes Are the Fundamental Organizational Units of chromatin The eukaryotic chromosome depicted in Figure 24-5 represents the compaction of a DNA molecule about 10 um long into a cell nucleus that is typically 5 to 10 um in diameter. This compaction involves several levels of highly organized folding Subjection of chromo- somes to treatments that partially unfold them reveals a structure in which the dna is bound tightly to beads H2A of protein, often regularly spaced(Fig. 24-26). The beads in this"beads-on-a-string"arrangement are com- plexes of histones and DNA. The bead plus the con- necting DNA that leads to the next bead form the nu- cleosome, the fundamental unit of organization upon which the higher-order packing of chromatin is built. The bead of each nucleosome contains eight histone molecules: two copies each of H2A, H2B, H3, and H4 The spacing of the nucleosome beads provides a re- peating unit typically of about 200 bp, of which 146 bp e bound tightly around the eight-part histone core and the remainder serve as linker dna between nucleosome beads. Histone hi binds to the linker dna. brief treat- ment of chromatin with enzymes that digest dNA causes preferential degradation of the linker DNA, releasing his- tone particles containing 146 bp of bound DNA that have been protected from digestion. Researchers have crys- tallized nucleosome cores obtained in this way, and x-ray diffraction analysis reveals a particle made up of the eight histone molecules with the DNA wrapped round it in the form of a left-handed solenoidal super col (Fig. 24-2 A close inspection of this structure reveals why eu- karyotic DNA is underwound even though eukaryotic cells lack enzymes that underwind DNA Recall that the solenoidal wrapping of dNa in nucleosomes is but one form of supercoiling that can be taken up by under- wound (negatively supercoiled) DNA. The tight wrap- ping of DNA around the histone core requires the re- moval of about one helical turn in the dna. when the protein core of a nucleosome binds in vitro to a relaxed closed-circular dna, the binding introduces a negative supercoil. Because this binding process does not break the dna or change the linking number, the formation of a negative solenoidal supercoil must be accompanied by a compensatory positive supercoil in the unbound re- gion of the dNA (Fig. 24-28). As mentioned earlier, eu- karyotic topoisomerases can relax positive supercoils. FIGURE 24-27 DNA wrapped around a nucleosome core. (a)Space. Relaxing the unbound positive supercoil leaves the neg- filling representation of the nucleasome protein core, with different tive supercoil fixed(through its binding to the nucle- colors for the different histones(PDB ID 1AO)(b) Top and(c)side osome histone core)and results in an overall decrease views of the crystal structure of a nucleosome with 146 bp of bound in linking number. Indeed, topoisomerases have proved DNA. The protein is depicted as a gray surface contour, with the bound necessary for assembling chromatin from purified his- DNA in blue. The DNA binds in a left-handed solenoidal supercoil tones and closed-circular dna in vitro that circumnavigates the histone complex 1.8 times. A schematic draw. Another factor that affects the binding of DNA to ing is included in (c) for comparison with other figures depicting histones in nucleosome cores is the sequence of the

Nucleosomes Are the Fundamental Organizational Units of Chromatin The eukaryotic chromosome depicted in Figure 24–5 represents the compaction of a DNA molecule about 105 m long into a cell nucleus that is typically 5 to 10 m in diameter. This compaction involves several levels of highly organized folding. Subjection of chromo￾somes to treatments that partially unfold them reveals a structure in which the DNA is bound tightly to beads of protein, often regularly spaced (Fig. 24–26). The beads in this “beads-on-a-string” arrangement are com￾plexes of histones and DNA. The bead plus the con￾necting DNA that leads to the next bead form the nu￾cleosome, the fundamental unit of organization upon which the higher-order packing of chromatin is built. The bead of each nucleosome contains eight histone molecules: two copies each of H2A, H2B, H3, and H4. The spacing of the nucleosome beads provides a re￾peating unit typically of about 200 bp, of which 146 bp are bound tightly around the eight-part histone core and the remainder serve as linker DNA between nucleosome beads. Histone H1 binds to the linker DNA. Brief treat￾ment of chromatin with enzymes that digest DNA causes preferential degradation of the linker DNA, releasing his￾tone particles containing 146 bp of bound DNA that have been protected from digestion. Researchers have crys￾tallized nucleosome cores obtained in this way, and x-ray diffraction analysis reveals a particle made up of the eight histone molecules with the DNA wrapped around it in the form of a left-handed solenoidal super￾coil (Fig. 24–27). A close inspection of this structure reveals why eu￾karyotic DNA is underwound even though eukaryotic cells lack enzymes that underwind DNA. Recall that the solenoidal wrapping of DNA in nucleosomes is but one form of supercoiling that can be taken up by under￾wound (negatively supercoiled) DNA. The tight wrap￾ping of DNA around the histone core requires the re￾moval of about one helical turn in the DNA. When the protein core of a nucleosome binds in vitro to a relaxed, closed-circular DNA, the binding introduces a negative supercoil. Because this binding process does not break the DNA or change the linking number, the formation of a negative solenoidal supercoil must be accompanied by a compensatory positive supercoil in the unbound re￾gion of the DNA (Fig. 24–28). As mentioned earlier, eu￾karyotic topoisomerases can relax positive supercoils. Relaxing the unbound positive supercoil leaves the neg￾ative supercoil fixed (through its binding to the nucle￾osome histone core) and results in an overall decrease in linking number. Indeed, topoisomerases have proved necessary for assembling chromatin from purified his￾tones and closed-circular DNA in vitro. Another factor that affects the binding of DNA to histones in nucleosome cores is the sequence of the 940 Chapter 24 Genes and Chromosomes H2B H4 H2A H2A H3 H4 H3 H2B (a) (b) (c) FIGURE 24–27 DNA wrapped around a nucleosome core. (a) Space￾filling representation of the nucleosome protein core, with different colors for the different histones (PDB ID 1AOI). (b) Top and (c) side views of the crystal structure of a nucleosome with 146 bp of bound DNA. The protein is depicted as a gray surface contour, with the bound DNA in blue. The DNA binds in a left-handed solenoidal supercoil that circumnavigates the histone complex 1.8 times. A schematic draw￾ing is included in (c) for comparison with other figures depicting nucleosomes. 8885d_c24_920-947 2/11/04 1:36 PM Page 940 mac76 mac76:385_reb: