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

PART III. INFORMATION PATHWAYS

24. GENES AND CHROMOSOMES

24.3. Chromosome Structure

Chromosomes are nucleic acid molecules that serve as the repositories of Genetic information in Viruses, Bacteria, Prokaryotic Cells, or eukaryotic Organelles. The term also refers to the intensely stained bodies visible under a Light Microscope after staining the nuclei of Eukaryotic cells.

Chromatin Consists of DNA and Proteins

During The Introduction/5.html">Eukaryotic Cell cycle (see Fig. 12-43 in Vol. 1), Chromosome Structure undergoes remarkable changes (Fig. 24-25). In non-dividing eukaryotic cells (G0 stage) and during the interphase stages of division (G1, S, and G2), the chromosomal material, chromatin, is amorphous and presumably distributed randomly throughout the nuclear interior. During the S phase of interphase, this amorphous DNA is replicated, with each chromosome forming two sister chromosomes (termed sister chromatids) that remain tethered to each other after Replication is complete. Chromosomes become much more condensed during prophase of mitosis and assume characteristic shapes that depend on the Organism species (Fig. 24-5).

Class="center">Figure 24-25. Changes in chromosome structure during the eukaryotic Cell Cycle. The relative durations of the phases are depicted here merely for convenience. The duration of each phase varies among different cell types and under different growth conditions (for unicellular organisms) or metabolic states (for multicellular ones). The mitotic phase is typically the shortest. As shown schematically in the diagram, cellular DNA is decondensed during interphase. Interphase (see Fig. 12-43 in Vol. 1) can be subdivided into the G1 phase (from the German Lücke, meaning gap), the S phase (synthesis) when METABOLISM/36.html">DNA replication occurs, and the G2 phase during which the replicated chromosomes (chromatids) align with one another. Mitosis comprises four distinct phases. DNA Condensation occurs during prophase. During metaphase, the condensed chromosomes align in pairs at the center of the mitotic spindle. Each chromosome in every pair is connected to the mitotic spindle—oriented toward a cell pole—via microtubules extending between the spindle and the centromere. Sister chromatids separate in anaphase, with each moving toward the pole to which it is tethered by the spindle. Upon completion of Cell Division, the chromosomes decondense, and the cycle begins anew.

Chromatin is composed of fibers containing approximately equal proportions (by mass) of protein and DNA, along with a small amount of RNA. The DNA molecule in chromatin is very tightly bound to Histones, which package and organize the DNA into structural units called nucleosomes (Fig. 24-26). Chromatin also contains numerous non-histone proteins—some help maintain chromosome structure, while others regulate the expression of specific genes (Chapter 28). Starting from nucleosomes, eukaryotic DNA forms increasingly higher-order structures that ultimately assemble into the compact chromosome visible under a light microscope. Let us examine The structure of eukaryotic chromosomes and compare it with the packaging of DNA in bacterial cells.

Figure 24-26. Nucleosomes. Regularly spaced nucleosomes consist of histone complexes bound to DNA. (a) Schematic representation; (b) electron micrograph.

Histones are small Basic Proteins

Histones, with molecular weights ranging from 11,000 to 21,000, are found in all eukaryotic cells; these proteins are distinguished by a high content of the basic Amino Acids Arginine and Lysine (which together account for a quarter of all amino acid residues). All Eukaryotic cells contain five Major Classes of histones, which differ in molecular weight and Amino Acid Composition (Table 24-4). Histone H3 has an almost identical Amino Acid Sequence across all eukaryotic cells, as does histone H4, indicating a high degree of functional conservation. For example, the H4 histone molecules of peas and cows differ by only two amino acid residues out of 102; human and Yeast H4 histones differ by eight residues. The sequences of histones H1, H2A, and H2B are less conserved among different eukaryotic species.

Table 24-4. Classes of Histones and Their Properties

* The sizes of these histones vary slightly among different species. The data shown refer to bovine histones.

Histones of each class undergo enzymatic modifications—such as methylation, ADP-ribosylation, phosphorylation, glycosylation, or Acetylation. Such modifications affect the net electrical charge, conformation, and Other properties of histones, as well as the Structural and functional properties of chromatin, and are crucial for the Regulation of Transcription (Chapter 28).

Additionally, some eukaryotic histones exist as multiple variants, particularly histones H2A and H3, which are discussed in more detail below. These histones and their modifications play specialized roles in DNA metabolism.

Nucleosomes Are the Fundamental Structural Units of Chromatin

Eukaryotic chromosomes, depicted in Figure 24-5, represent a compact packaging of a DNA molecule approximately 10⁵ µm long within a Cell Nucleus whose diameter is typically 5 to 10 µm. This remarkable compaction is achieved through a multilevel organizational hierarchy. When this folding is partially disrupted, chromosomes reveal a structure in which DNA is tightly bound to protein globules (beads), often separated by regular intervals (Fig. 24-27). The beads in these "beads-on-a-string" structures are histones complexed with DNA. Each bead, along with the DNA thread connecting it to the next bead, constitutes a nucleosome—the fundamental structural unit upon which further chromatin folding occurs. The globule of each nucleosome contains eight histone molecules: two copies each of H2A, H2B, H3, and H4. The DNA segments between the beads represent repeating units, typically 200 bp in length, of which 146 bp are tightly wrapped around the octameric histone core, while the remaining DNA links the globules together. Histone H1 interacts with the linker DNA. Mild Treatment of chromatin with DNA-cleaving Enzymes primarily degrades the linker DNA, resulting in the release of histone particles containing 146 bp of bound DNA that was protected from Digestion. Researchers have crystallized these nucleosome core particles and, using X-ray crystallography, established that the DNA is wound around the eight histone molecules in a left-handed solenoidal superhelix (Fig. 24-27).

Figure 24-27. DNA wraps around the nucleosome core. (a) Space-filling model of the proteins in a nucleosome core particle from the African clawed frog Xenopus laevis; different histones are shown in different colors (PDB ID 1A0I). (b and c) Top and side views of the crystal STRUCTURE OF THE nucleosome (surface contour shown in gray) with the wrapped 146 bp bound DNA (blue). The DNA molecule is wound in a left-handed solenoidal superhelix, making 1.8 turns around the histone complex. A schematic representation of the nucleosome is provided for clarity ((c), bottom right).

Detailed study of this structure explained why eukaryotic DNA undergoes partial unwinding even in the absence of specialized unwinding enzymes in eukaryotic cells. Recall that the solenoidal coiling of DNA in nucleosomes is one of two superhelical forms that partially unwound (negatively supercoiled) DNA can adopt. Tightly wrapping DNA around the histone core requires the removal of approximately one turn from the DNA. When the histone core binds in vitro to relaxed closed-circular DNA, negative supercoiling is induced. Because this binding process neither breaks the DNA nor changes its linking number, The formation of a negative solenoidal superhelix must be compensated by the formation of a positive supercoil in the unbound DNA segment (Fig. 24-28). As mentioned previously, eukaryotic topoisomerases can relax positive supercoils. Relaxation of the unbound, positively supercoiled DNA locks in the negatively supercoiled DNA (upon its binding to the histone core) and is reflected in an overall decrease in the linking number. Topoisomerases have been shown to be essential for the assembly of chromatin from purified histones and closed-circular DNA in vitro.

Figure 24-28. Chromatin assembly. (a) Relaxed closed-circular DNA. (b) Binding of the histone core and formation of a nucleosome introduces one negative supercoil. If neither strand is nicked, a positive supercoil must form elsewhere in the DNA (ΔLk = 0). (c) Relaxation of this positive supercoil by a topoisomerase leaves one negative supercoil (ΔLk = -1).

Another factor influencing the binding of DNA to histones in nucleosomes is the DNA sequence itself. The histone core does not bind to just any DNA, but rather to specific regions. The exact principle governing this Selection is not yet fully understood, but in some cases it is likely determined by the number of A = T Base Pairs in the segment of the DNA helix that contacts the histones (Fig. 24-29). Narrowing of the DNA helix's minor groove at these points facilitates tight packing of the DNA around the histone core; a combination of two or three A = T nucleotide pairs increases the likelihood of this narrowing. Nucleosomes show a particularly strong preference for sequences where AA, AT, or TT dinucleotides occur at intervals of 10 bp, and this exact property is exhibited by up to 50% of the sequences that bind to histones in vivo.

Fig. 24-29. Nucleosome positioning that optimizes The Use of A = T nucleotide pairs at the contact sites between the histone core and the minor groove of the DNA helix.

Proper positioning of certain nucleosomal particles on DNA requires additional proteins. In some organisms, specific proteins bind to particular DNA sequences, facilitating the formation of a nucleosomal particle nearby. During replication or other processes that require the temporary displacement of nucleosomes, the nucleosomes remain bound to the DNA. This binding occurs in a stepwise manner: first, a tetramer formed by two H3 histones and two H4 histones binds, followed by the binding of H2A-H2B dimers. The reattachment of nucleosomes to chromosomes following DNA replication involves the RCAF protein complex (replication-coupling assembly factor). The RCAF complex consists of acetylated histones H3 and H4, a tripartite protein called chromatin assembly factor 1 (CAF1), and the ASF1 protein (antisilencing factor 1). The Mechanism of nucleosome binding is not yet fully understood, although it is known that elements of the RCAF complex interact directly with Components of the replication machinery. When nucleosomes need to be reassembled following DNA Repair or other processes, RCAF is replaced by another specialized protein complex that mediates the binding. In some cases, histone chaperone factors allow the replacement of canonical histones with variant histones. The correct positioning of these histone variants is critically important; for instance, the absence of a single histone variant is lethal to mouse embryos at early Stages of development (Box 24-2). Precise positioning of nucleosomal particles can also play a key role in the expression of certain eukaryotic genes (Chap. 28).

Nucleosomes form higher-order structures

Wrapping around the nucleosome core reduces the length of DNA approximately sevenfold. However, overall compaction within a chromosome shortens the DNA by more than 10,000-fold, indicating the existence of higher levels of DNA Organization. Very mild chromosome isolation techniques reveal that nucleosome cores are organized into structures known as 30-nm fibers (Fig. 24-30). This packaging requires one molecule of histone H1 per nucleosome. Nevertheless, stretches of this folded structure are interrupted by segments of DNA bound to specific non-histone proteins. Furthermore, the presence of 30-nm fibers depends on the transcriptional activity of the DNA region; transcriptionally active regions are less condensed and contain very little, if any, histone H1.

Fig. 24-30. The 30-nm fiber represents a higher level of nucleosome organization. (a) Schematic model of the proposed fiber structure; (b) electron micrograph.

The 30-nm fiber constitutes the second level of chromatin organization, providing a 100-fold reduction in DNA length. Higher levels of DNA organization are still under investigation, but specific DNA regions are likely anchored to a chromosomal scaffold (Fig. 24-31). Scaffold-associated regions are separated by DNA loops ranging from 20 to 100 kbp in size. The DNA within such a loop may contain a set of related genes. For example, in Drosophila, the complete set of histone-encoding genes is clustered within loops attached to the chromosome scaffold (Fig. 24-32). The scaffold itself contains several proteins, notably high concentrations of histone H1 (positioned on the inner side of the fiber) and topoisomerase II. The presence of topoisomerase II further highlights The Link Between local DNA unwinding and Chromatin Structure. Topoisomerase II is so essential for maintaining chromatin structure that inhibitors of this enzyme can rapidly destroy dividing cells. Several chemotherapeutic agents act as topoisomerase II inhibitors—they allow the enzyme to initiate strand breaks but prevent it from resealing them.

Fig. 24-31. Partially unwound human chromosome, showing numerous DNA loops attached to the chromosomal scaffold.

Fig. 24-32. Chromosomal DNA loops attached to the chromosomal scaffold. The DNA molecules within the loops are packaged into 30-nm fibers, making these loops the next level of chromosome organization. Loops frequently contain groups of functionally related genes. Complete sets of histone genes, as shown in this diagram, form clusters in loops of this type. Unlike most genes, histone genes are present in large copy numbers in many Eukaryotic Genomes.

Box 24-2. MEDICINE. Epigenetics, Nucleosome Structure, and Histone Variants

Information that is transmitted from one generation to the next (to daughter cells during cell division or from parents to offspring) but is not encoded in the DNA sequence itself is termed epigenetic. Much of this information is stored in the form of covalent histone modifications and/or the incorporation of histone variants into chromosomes.

Regions of chromatin where active Gene Expression (transcription) takes place are generally found in a decondensed state known as euchromatin. In these regions, histones H3 and H2A are frequently replaced by the histone variants H3.3 and H2AZ, respectively (Fig. 1). The complexes that mediate DNA binding to nucleosomes containing histone variants are similar to those responsible for binding canonical histone nucleosomes. The binding of nucleosomes containing histone H3.3 is carried out by a complex in which chromatin assembly factor 1 (CAF1) is replaced by the HIRA protein (named after the yeast HIR class of proteins, histone repressor). Both CAF1 and HIRA can be regarded as histone chaperones that assist in ensuring proper nucleosome assembly and positioning. Histone H3.3 differs from H3 by only four amino acid residues, yet these residues play a critical role in histone binding.

Fig. 1. Several variants of histones H3, H2A, and H2B are known. Canonical histones and several well-characterized variants are shown. Highlighted regions indicate sites of Lys/Arg residue methylation and Ser residue phosphorylation. The HFD (histone-fold domain) is a structural domain present in all canonical histones.

Like histone H3.3, the histone variant H2AZ is associated with a specific binding complex and is typically localized to actively transcribed chromatin regions. The incorporation of H2AZ stabilizes the nucleosome octamer but impedes certain cooperative interactions between nucleosomes that are required to form a compact chromosome structure. This results in a more open chromatin conformation, which facilitates GENE EXPRESSION IN the H2AZ-containing domains. The gene encoding H2AZ is essential for mammalian development; in Drosophila, the absence of H2AZ prevents the organism from developing past the larval stage.

Another H2A variant, H2AX, is implicated in DNA repair and genetic recombination. In mice, the absence of H2AX leads to genomic instability and male sterility. Small amounts of H2AX appear to be distributed throughout The Genome. Upon the formation of a DNA double-strand break, nearby H2AX molecules are phosphorylated at the Ser¹³⁹ residue located at the C-terminus of the protein. Experimentally blocking this phosphorylation inhibits the formation of Protein Complexes required for DNA repair.

A histone H3 variant known as CENPA is associated with repetitive DNA sequences within centromeres. Centromeric chromatin contains the histone chaperones CAF1 and HIRA, both of which appear to participate in the assembly of CENPA-containing nucleosomes. Knockout of the CENPA gene in mice is lethal.

Genomics approaches can be applied to study the Functions and genomic distribution of histone variants. One particularly useful technique is chromatin immunoprecipitation (ChIP). The method involves precipitating nucleosomes containing a specific histone variant via binding to variant-specific Antibodies. Although nucleosomes can be studied independently of DNA, the associated DNA is typically preserved during the analysis, making it possible to map the exact nucleosome binding sites. The recovered DNA can be labeled and used as a probe in microarray assays (see Fig. 9-22, Vol. 1) to generate a high-resolution map of the genomic sequences bound by each specific nucleosome. This approach is widely known as ChIP-on-chip (Fig. 2).

Fig. 2. Chromatin immunoprecipitation on a microarray (ChIP-on-chip) allows the identification of genomic sequences bound by a specific histone variant. (a) A histone variant tagged with an epitope (a protein or chemical structure recognized by an antibody; see Chaps. 5 and 9, Vol. 1) is introduced into a specific cell type, where it is incorporated into nucleosomes. (In some cases, such tags are unnecessary because antibodies are available that bind directly to modified residues of the endogenous histone of interest.) Chromatin is then isolated from the cells and digested with micrococcal nuclease. Nucleosome-bound DNA is protected from digestion, whereas linker DNA is cleaved, releasing DNA segments corresponding to one or two nucleosomes. Upon The addition of antibodies, nucleosomes containing the tagged histone variant are selectively precipitated. The DNA associated with these nucleosomes is extracted from the precipitate, labeled, and used as a probe for microarray Hybridization against the genome sequences of that cell type. (b) Microarray hybridization reveals the DNA sequences associated with nucleosomes containing a specific histone variant. In this example (top panel), hybridization data are shown for DNA associated with histone H3.3 across a small region of the Drosophila genome. Annotated genes in this genomic region are shown in the bottom panel (broad bars). DNA segments bound by histone H3.3 localize near actively transcribed genes (red bars).

Histone variants, as well as many covalent histone modifications, help define chromatin function. They serve as epigenetic marks that facilitate or repress specific functions, such as chromosome segregation, transcription, and DNA repair. These modifications persist through cell division and Meiosis, thereby becoming part of the heritable information passed from generation to generation in all eukaryotic organisms.

Evidence suggests an even more complex Organization of Eukaryotic chromosomes, with each successive level significantly increasing packaging compaction. One model illustrating how DNA achieves this compact structure is shown in Fig. 24-33. Higher-order chromatin structures likely vary among different chromosomes, across different Regions of the same chromosome, and at different Stages of the cell cycle. No single model can adequately capture all of these variations.

Fig. 24-33. Compact packing of DNA in A eukaryotic chromosome. The model illustrates the hierarchical levels of eukaryotic DNA organization that account for chromosome compaction. Within The Cell, higher-order structures (beyond the 30-nm fiber level) are likely less uniform than depicted in this diagram.

Nevertheless, the principle is clear: the remarkable compaction of eukaryotic chromosomal DNA is achieved through successive, coiled-upon-coiled folding...

The Structure of Condensed Chromosomes is Maintained by SMC Proteins

Alongside histones and topoisomerases, a third major class of chromatin proteins exists: the so-called SMC proteins (structural maintenance of chromosomes). The Primary Structure of an SMC protein consists of five distinct domains (Fig. 24-34a). The N- and C-terminal globular domains (at the N- and C-termini), each contributing a portion of the ATP-Hydrolysis Active Site, are separated by two long α-helical segments (see Fig. 4-10, Vol. 1) that are interrupted by a flexible hinge domain. Proteins of this family typically form a V-shaped dimer by associating through their hinge domains (Fig. 24-34b, c). One N- and one C-domain come together to form a functional ATP-hydrolysis center at each free end of the V-shaped complex.

Fig. 24-34. Structure of SMC proteins. (a) The five domains of the SMC primary structure, with N and C denoting the N- and C-terminal sequences, respectively. (b) Each polypeptide folds such that the two helical domains wrap around each other, bringing the N- and C-domains together to form a complete ATP-binding site. Two such Polypeptides associate via their hinge regions to form a V-shaped dimer. (c) Electron micrograph of SMC proteins from Bacillus subtilis. (d) Cohesins consist of SMC1–SMC3 protein pairs, whereas condensins consist of SMC2–SMC4 pairs. These eukaryotic SMC proteins associate with a kleisin subunit and other regulatory proteins (not shown). (e) ATP hydrolysis may drive the opening and closing of the ATPase domain of the SMC dimer, which remains associated with kleisin and other proteins (not shown).

Proteins of the SMC family are found in all organisms, from bacteria to humans. Eukaryotes possess two primary types of SMC proteins—cohesins and condensins—both of which associate with various regulatory and accessory proteins (Fig. 24-34d). Cohesins play an essential role in tethering sister chromatids together immediately following DNA Replication and holding them in this state during chromosome condensation as the cell enters metaphase. This linkage is crucial for accurate chromosome segregation during cell division. Cohesins, together with a third protein type known as kleisin, appear to form a ring structure around replicated chromosomes that holds them together until chromosome segregation begins in anaphase. This ring can expand and contract in response to ATP hydrolysis (Fig. 24-34e). Condensins are vital for chromosome condensation as the cell enters mitosis. In vitro, condensins bind to DNA in a manner that introduces positive supercoils—forcing the DNA into a supercoiled state, in stark contrast to the partial unwinding caused by nucleosome binding. While it remains unclear exactly how this facilitates compact chromatin folding, the wrapping of DNA around condensins likely plays an important role in DNA compaction. Together, cohesins and condensins mediate many of the dynamic structural changes chromosomes undergo throughout the eukaryotic cell cycle (Fig. 24-35).

Fig. 24-35. The roles of cohesins and condensins in the eukaryotic cell cycle. Cohesins are loaded onto chromosomes during G1 phase and hold sister chromatids together through replication. Upon entry into mitosis, condensins bind to the chromatids to maintain them in a condensed state. During anaphase, cohesin linkages are cleaved by separase activity. Following chromatid Separation, condensins dissociate, allowing daughter chromosomes to decondense back to their interphase state.

Bacterial DNA is also complexly organized

Let us now briefly discuss the structure of bacterial chromosomes. Bacterial DNA is packaged into a structure called the nucleoid; the nucleoid can occupy a significant portion of the cell volume (Fig. 24-36). It has been established that DNA is attached at one or more points to the inner surface of The Plasma Membrane. Much less is known about the structure of the nucleoid than about eukaryotic chromatin, but the details of its complex organization are gradually coming to light. In E. coli cells, a distinctive scaffolding system organizes the circular chromosome into roughly 500 loop domains, each averaging about 10,000 bp (Fig. 24-37), as described above for chromatin. These domains are topologically constrained; for example, if a DNA strand break occurs in one domain, relaxation is confined to that specific domain. The domains do not have fixed endpoints. Instead, their boundaries constantly shift along the DNA sequence during replication. Bacterial DNA apparently lacks a structure comparable to eukaryotic nucleosomes. E. coli cells contain many histone-like proteins (the best-studied being the two-subunit HU protein, M_r 19,000), but these proteins associate and dissociate within a matter of minutes, and no regular, stable DNA-histone complexes have been detected. Dynamic Changes in the bacterial chromosome structure may be related to The Need for more rapid

access to genetic information. The Cell Cycle of a bacterium can take as little as 15 min, whereas a typical eukaryotic cell does not divide for several hours or even months. Furthermore, a much larger fraction of prokaryotic DNA is involved in encoding RNA and/or proteins. The more intense metabolic rate of bacteria means that, over the same time interval, a significantly greater proportion of DNA is transcribed or replicated in bacterial cells than in most eukaryotic cells.

Having examined the complex structure of DNA, we are now ready to move on to the next chapter, which discusses DNA metabolism.

Fig. 24-36. The E. coli nucleoid. The DNA molecules in these cells have been treated with a fluorescent dye under UV irradiation. The glowing regions represent the nucleoids. Note that the DNA in some cells has replicated, but the cells have not yet divided and therefore contain multiple nucleoids.

Fig. 24-37. Loops in the E. coli chromosome. Each such domain is approximately 10,000 bp in length. The domains are not static; rather, they shift along the DNA sequence as replication proceeds. DNA relaxation outside the BOUNDARIES OF THE domain in which a strand break has occurred is prevented by boundary elements of unknown composition located at the domain ends. These boundary complexes are shown as grey ovales. The arrows indicate the movement of DNA through the boundary complexes.

Summary of Section 24.3 Chromosome Structure

■ The fundamental unit of chromatin organization in eukaryotic cells is the nucleosome, which consists of histones and a DNA segment about 200 bp in length. The protein core of the nucleosome, containing eight histones (two copies each of histones H2A, H2B, H3, and H4), is wrapped with a DNA segment (approximately 146 bp) in the form of a left-handed solenoidal supercoil.

■ Nucleosomes form 30-nm fibers that are packaged to achieve the 10,000-fold compaction of DNA required to fit a typical eukaryotic chromosome into the cell nucleus. Further Levels of organization rely on attachment to a chromosome scaffold consisting of histone H1, topoisomerase II, and SMC proteins. SMC proteins, primarily cohesins and condensins, play a vital role in maintaining chromosome structure at every stage of the cell cycle.

■ Bacterial chromosomes within the nucleoid are also compactly packaged, but the prokaryotic chromosome has a much more dynamic and irregular structure than eukaryotic chromatin, reflecting the short CELL CYCLE AND High Metabolic Rate of bacteria.