CHAPTER 24 GENES AND CHROMOSOMES tertiary structure. Chromosomes in all cells are maintained in a state of torsional stress. DNA is underwound relative to the stable B- form structure, facilitating both the packaging of DNA and access to the genetic information contained within it. Specialized proteins and RNAs maintain chromosome structure. Topoisomerases control DNA underwinding. Histones, condensins, cohesins, and other DNA-binding proteins provide scaffolds to organize chromosome structure. Certain long, noncoding RNAs also play important roles in chromosome structure and function. FIGURE 24-1 Bacteriophage T2 protein coat surrounded by its single, linear molecule of DNA. The DNA was released by lysing the bacteriophage particle in distilled water and allowing the DNA to spread on the water surface. An undamaged T2 bacteriophage particle consists of a head structure that tapers to a tail by which the bacteriophage attaches itself to the outer surface of a bacterial cell. All the DNA shown in this electron micrograph is normally packaged inside the phage head. [Republished with permission of Elsevier, from A. K. Kleinschmidt et al. (1962), “Preparation and length measurements of the total deoxyribonucleic acid content of T2 bacteriophages,” Biochim. Biophys. Acta 61:857–864; permission conveyed through Copyright Clearance Center, Inc.] The chapter begins with an examination of the elements that make up viral and cellular chromosomes, and then considers chromosomal size and organization. We then discuss DNA topology, describing the coiling and supercoiling of DNA molecules. Finally, we consider the protein-DNA interactions that organize chromosomes into compact structures. 24.1 Chromosomal Elements Cellular DNA contains genes and intergenic regions, both of which may serve functions vital to the cell. The more complex genomes, such as those of eukaryotic cells, demand increased levels of chromosomal organization, and this is reflected in the structural features of the chromosomes. We begin by considering the different types of DNA sequences and structural elements within chromosomes. Genes Are Segments of DNA That Code for Polypeptide Chains and RNAs Our understanding of genes has evolved tremendously over the past century. Classically, a gene was defined as a portion of a chromosome that determines or affects a single character or phenotype (visible property), such as eye color. George Beadle and Edward Tatum proposed a molecular definition of a gene in 1940. A er exposing spores of the fungus Neurospora crassa to x- rays and other agents now known to damage DNA and cause alterations in DNA sequence (mutations), they detected mutant fungal strains that lacked one or another specific enzyme, sometimes resulting in the failure of an entire metabolic pathway. Beadle and Tatum concluded that a gene is a segment of genetic material that determines, or codes for, one enzyme: the one gene–one enzyme hypothesis. Later this concept was broadened to one gene–one polypeptide, because many genes code for a protein that is not an enzyme or for one polypeptide of a multisubunit protein. The modern biochemical definition of a gene is even more precise. A gene is all the DNA that encodes the primary sequence of some final gene product, which can be either a polypeptide or an RNA with a structural or catalytic function. DNA also contains other segments or sequences that have a purely regulatory function. Regulatory sequences provide signals that may denote the beginning or the end of genes, or influence the transcription of genes, or function as initiation points for replication or recombination (Chapter 28). Some genes can be expressed in different ways to generate multiple gene products from a single segment of DNA; the special transcriptional and translational mechanisms that allow this are described in Chapters 26 through 28. We can estimate directly the minimum overall size of genes that encode proteins. As described in detail in Chapter 27, each amino acid of a polypeptide chain is coded for by a sequence of three consecutive nucleotides in a single strand of DNA (Fig. 24-2), with these “codons” arranged in a sequence that corresponds to the sequence of amino acids in the polypeptide that the gene encodes. A polypeptide chain of 350 amino acid residues (an average-size chain) corresponds to 1,050 base pairs (bp) of coding DNA. Many genes in eukaryotes and a few in bacteria and archaea are interrupted by noncoding DNA segments and are therefore considerably longer than this simple calculation would suggest.
FIGURE 24-2 Colinearity of the coding nucleotide sequences of DNA and mRNA and the amino acid sequence of a polypeptide chain. The triplets of nucleotide units in DNA determine the amino acids in a protein through the intermediary mRNA. One of the DNA strands serves as a template for synthesis of mRNA, which has nucleotide triplets (codons) complementary to those of the DNA. In some bacterial and many eukaryotic genes, coding sequences are interrupted at intervals by regions of noncoding sequences (called introns). How many genes are in a single chromosome? The Escherichia coli chromosome is a circular DNA molecule (in the sense of an endless loop rather than a perfect circle) with 4,641,652 bp. These base pairs encode about 4,300 genes for proteins and more than 200 genes for structural or catalytic RNA molecules. Among eukaryotes, the approximately 3.1 billion base pairs of the human genome include approximately 20,000 genes on the 24 different chromosomes. DNA Molecules Are Much Longer than the Cellular or Viral Packages That Contain Them Chromosomal DNAs are o en many orders of magnitude longer than the cells or viruses in which they are located (Fig. 24-1; Table 24-1). This is true of every class of organism or viral parasite. TABLE 24-1 The Sizes of DNA and Viral Particles for Some Bacterial Viruses (Bacteriophages) Virus Size of viral DNA (bp) Length of viral DNA (nm) Long dimension of viral particles (nm) ϕX174 5,386 1,939 25 T7 39,936 14,377 78 λ (lambda) 48,502 17,460 190 T4 168,889 60,800 210 Note: Data on size of DNA are for the replicative form (double-stranded). The contour length is calculated assuming that each base pair occupies a length of 3.4 Å (see Fig. 8-13). Viruses Viruses are not free-living organisms; rather, they are infectious parasites that use the resources of a host cell to carry out many of the processes they require to propagate. Many viral particles consist of no more than a genome (usually a single RNA or DNA molecule) surrounded by a protein coat. Almost all plant viruses and some bacterial and animal viruses have RNA genomes. These genomes tend to be particularly small. For example, the genomes of mammalian retroviruses such as HIV consist of 9,000 nucleotides of single-stranded RNA. The genomes of DNA viruses vary greatly in size. Many viral DNAs are circular for at least part of their life cycle. During viral replication within a host cell, specific types of viral DNA called replicative forms may appear; for example, many linear DNAs become circular and all single-stranded DNAs become double- stranded. A typical medium-size DNA virus is bacteriophage λ (lambda), which infects E. coli. In its replicative form inside cells, λ DNA is a circular double helix. This double-stranded DNA contains 48,502 bp and has a contour length of 17.5 μ m. Bacteriophage ϕX174 is much smaller; the DNA in the viral particle is a single-stranded circle, and the double-stranded replicative form contains 5,386 bp. Although viral genomes are small, the contour lengths of their DNAs are typically hundreds of times longer than the long dimensions of the viral particles that contain them (Table 24-1). Bacteria A single E. coli cell contains almost 100 times as much DNA as a bacteriophage λ particle. The chromosome of an E. coli cell is a single, double-stranded circular DNA molecule. Its 4,641,652 bp have a contour length of about 1.7 mm, some 850 times the length of the E. coli cell (Fig. 24-3). In addition to the very large, circular DNA chromosome in their nucleoid, many bacteria contain one or more small circular DNA molecules that are free in the cytosol. These extrachromosomal elements are called plasmids (Fig. 24-4; see also p. 305). Most plasmids are only a few thousand base pairs long, but some contain up to 400,000 bp. They carry genetic information and undergo replication to yield daughter plasmids, which pass into the daughter cells at cell division. Plasmids have been found in yeast and other fungi as well as in bacteria. FIGURE 24-3 A bacterial cell and its DNA. The length of the E. coli chromosome (1.7 mm), depicted in linear form, relative to the length of a typical E. coli cell (2 μ m). FIGURE 24-4 DNA from a lysed E. coli cell. In this electron micrograph, several small, circular plasmid DNAs are indicated by white arrows. The black spots and white specks are artifacts of the preparation. In many cases plasmids confer no obvious advantage on their host, and their sole function seems to be self-propagation. However, some plasmids carry genes that are useful to the host bacterium. For example, some plasmid genes make a host bacterium resistant to antibacterial agents. Plasmids carrying the gene for the enzyme β -lactamase confer resistance to β -lactam antibiotics such as penicillin, ampicillin, and amoxicillin (see Fig. 6-34). These and similar plasmids may pass from an antibiotic- resistant cell to an antibiotic-sensitive cell of the same or another bacterial species, making the recipient cell antibiotic-resistant. The extensive use of antibiotics in some human populations and in animal feeds has served as a strong selective force, encouraging the spread of antibiotic resistance–coding plasmids (as well as transposable elements, described below, that harbor similar genes) in disease-causing bacteria. Eukaryotes A yeast cell, one of the simplest eukaryotes, has 2.6 times more DNA in its genome than an E. coli cell (Table 24-2). Cells of Drosophila, the fruit fly used in classical genetic studies, contain more than 35 times as much DNA as E. coli cells, and human cells have almost 700 times as much. The cells of many plants and amphibians contain even more. The genetic material of eukaryotic cells is apportioned into chromosomes, the diploid (2n) number depending on the species. A human somatic cell, for example, has 46 chromosomes (Fig. 24-5). Each chromosome of a eukaryotic cell, such as that shown in Figure 24-5a, contains a single, very large, duplex DNA molecule. The DNA molecules in the 24 different types of human chromosomes (22 matching pairs of autosomes plus the X and Y sex chromosomes) vary in length over a 25-fold range. Each type of chromosome in eukaryotes carries a characteristic set of genes. TABLE 24-2 DNA, Gene, and Chromosome Content in Some Genomes Total DNA (bp) Number of chromosomes Approximate number of protein- coding genes Escherichia coli K12 4,641,652 1 4,494 a b (bacterium) Saccharomyces cerevisiae (yeast) 12,157,105 16 6,600 Caenorhabditis elegans (nematode) 100,286,401 12 20,191 Arabidopsis thaliana (plant) 119,667,750 10 27,655 Drosophila melanogaster (fruit fly) 143,726,002 18 13,931 Oryza sativa (rice) 375,049,285 24 37,849 Mus musculus (mouse) 2,730,871,774 40 22,480 Homo sapiens (human) 3,096,649,726 46 20,454 Note: This information is constantly being refined. For the most current information, consult the websites for the individual genome projects. [Data from ensembl.org. Accessed April 21, 2020.] The diploid chromosome number is given for all eukaryotes except yeast. Includes known RNA-coding genes. Haploid chromosomes number. Wild yeast strains generally have eight (octoploid) or more sets of these chromosomes. Number for females, with two X chromosomes. Males have an X but no Y, thus 11 chromosomes in all. When known genes encoding functional RNAs are included, this number rises to more than 43,000. c d e a b c d e FIGURE 24-5 Eukaryotic chromosomes. (a) A pair of linked and condensed sister chromatids from a Chinese hamster ovary cell. Eukaryotic chromosomes are in this state a er replication at metaphase during mitosis. (b) A complete set of chromosomes from a leukocyte from one of the authors. There are 46 chromosomes in every normal human somatic cell. DNA molecules of one human genome (22 chromosomes plus X and Y), placed end to end, would extend for about a meter. Most human cells are diploid, so each cell contains a total of 2 m of DNA. An adult human body contains approximately 1014 cells and thus a total DNA length of 2× 1011 km. Compare this with the circumference of the earth (4× 104 km) or the distance between the earth and the sun (1.5× 108 km) — a dramatic illustration of the extraordinary degree of DNA compaction in our cells. Eukaryotic cells also have organelles, mitochondria and chloroplasts, that contain DNA. Mitochondrial DNA (mtDNA) molecules are much smaller than the nuclear chromosomes. In animal cells, mtDNA contains fewer than 20,000 bp (16,569 bp in human mtDNA) and is a circular duplex. Each mitochondrion typically has 2 to 10 copies of this mtDNA molecule, and the number can rise to hundreds in certain cells of an embryo that is undergoing cell differentiation. Plant cell mtDNA ranges in size from 200,000 to 2,500,000 bp. Chloroplast DNA (cpDNA) also exists as circular duplexes and ranges in size from 120,000 to 160,000 bp. Mitochondrial and chloroplast DNAs have an evolutionary origin in the chromosomes of ancient bacteria that gained access to the cytoplasm of host cells and became the precursors of these organelles (see Fig. 1-37). Mitochondrial DNA codes for the mitochondrial tRNAs and rRNAs and for a few mitochondrial proteins. More than 95% of mitochondrial proteins are encoded by nuclear DNA. Mitochondria and chloroplasts divide when the cell divides. Their DNA is replicated before and during division, and the daughter DNA molecules pass into the daughter organelles. Eukaryotic Genes and Chromosomes Are Very Complex Many bacterial species have only one chromosome per cell and, in nearly all cases, each chromosome contains only one copy of each gene. A very few genes, such as those for rRNAs, are repeated several times. Genes and regulatory sequences account for almost all the DNA in bacteria. Moreover, almost every gene is precisely colinear with the amino acid sequence (or RNA sequence) it encodes throughout its entire length (Fig. 24-2). The organization of genes in eukaryotic DNA is structurally and functionally much more complex. Studies of eukaryotic chromosome structure and the sequencing of entire eukaryotic genomes have yielded many surprises. Many, if not most, eukaryotic genes have a distinctive structural feature: the colinearity of the DNA and amino acid sequence is periodically broken by intervening segments of DNA that do not code for the amino acid sequence of the polypeptide product. Such nontranslated DNA segments in genes are called introns, and the coding segments are called exons. Few bacterial genes contain introns. In higher eukaryotes, the typical gene has much more intron sequence than sequences devoted to exons. For example, in the gene coding for the single polypeptide chain of ovalbumin, an avian egg protein (Fig. 24-6), the introns are much longer than the exons; altogether, the seven introns make up 85% of the gene’s DNA. The gene encoding the hemoglobin β subunit has only two introns, but again they are larger than the exons. Genes for histones seem to have no introns. In many cases the function of introns is not clear. In total, only about 1.5% of human DNA is “coding” or exon DNA, carrying sequence information for protein products. However, when the much larger introns are included as functional elements in the gene and their length is included, as much as 30% of the human genome consists of protein-coding genes. FIGURE 24-6 Introns in two eukaryotic genes. The gene for ovalbumin has seven introns (A to G), splitting the coding sequences into eight exons (L, and 1 to 7). The gene for the β subunit of hemoglobin has two introns and three exons, including one intron that alone contains more than half the base pairs of the gene. A great deal of work remains to be done to understand the genomic sequences that do not correspond to protein-encoding genes. Much of the DNA that is not within genes is made up of repeated sequences of several kinds. These include transposable elements (transposons), molecular parasites that account for nearly half of the DNA in the human genome (see Fig. 9-25a and Chapters 25 and 26), and genes encoding functional RNA molecules of many types. Approximately 3% of the human genome consists of highly repetitive sequences, also referred to as simple-sequence DNA or simple sequence repeats (SSR). These short sequences, generally less than 10 bp long, are sometimes repeated millions of times per cell. The simple-sequence DNA is also called satellite DNA, so named because its unusual base composition o en causes it to migrate as “satellite” bands (separated from the rest of the DNA) when fragmented cellular DNA samples are centrifuged in a cesium chloride density gradient. Studies suggest that simple- sequence DNA does not encode proteins or RNAs. The functional importance of the highly repetitive DNA has been defined in at least some cases. Much of it is associated with two crucial features of eukaryotic chromosomes: centromeres and telomeres. The centromere (Fig. 24-7) is a sequence of DNA that functions during cell division as an attachment point for proteins that link the chromosome to the mitotic spindle. This attachment is essential for the equal and orderly segregation of chromosome sets to daughter cells. The centromeres of Saccharomyces cerevisiae have been isolated and studied. The sequences essential to centromere function are about 130 bp long and are very rich in A═T pairs. The centromeric sequences of higher eukaryotes are much longer and, unlike those of yeast, generally consist of thousands of tandem copies of one or several sequences of 5 to 10 bp, in the same orientation. FIGURE 24-7 Important structural elements of a yeast chromosome. Telomeres (Greek telos, “end”) are sequences at the ends of eukaryotic chromosomes that help stabilize the chromosome. Telomeres end with multiple repeated sequences of the form (5′)(TxGy)n (3′)(AxCy)n where x and y are generally between 1 and 4 (Table 24-3). The number of telomere repeats, n, is in the range of 20 to 100 for most single-celled eukaryotes and is generally more than 1,500 in mammals. The ends of a linear DNA molecule cannot be routinely replicated by the cellular replication machinery (which may be one reason why bacterial DNA molecules are circular). Repeated telomeric sequences are added to eukaryotic chromosome ends primarily by the enzyme telomerase (see Fig. 26-35). TABLE 24-3 Telomere Sequences Organism Telomere repeat sequence Homo sapiens (human) (TTAGGG)n Tetrahymena thermophila (ciliated protozoan) (TTGGGG )n Saccharomyces cerevisiae (yeast) (T(G)1–3(TG)2–3)n Arabidopsis thaliana (plant) (TTTAGGG)n Artificial chromosomes (Chapter 9) have been constructed as a means of better understanding the functional significance of many structural features of eukaryotic chromosomes. A reasonably stable artificial linear chromosome requires only three components: a centromere, a telomere at each end, and sequences that allow the initiation of DNA replication. Yeast artificial chromosomes (YACs; see Fig. 9-6) have been developed as a research tool in biotechnology. Similarly, human artificial chromosomes (HACs) are being developed for the treatment of genetic diseases. These may eventually provide a new path to the intracellular replacement of missing or defective gene products, or somatic gene therapy. SUMMARY 24.1 Chromosomal Elements Genes are segments of a chromosome that contain the information for a functional polypeptide or RNA molecule. In addition to genes, chromosomes contain a variety of regulatory sequences involved in replication, transcription, and other processes. Genomic DNA and RNA molecules are generally orders of magnitude longer than the viral particles or cells that contain them. Many genes in eukaryotic cells (but few in bacteria and archaea) are interrupted by noncoding sequences, or introns. The coding segments separated by introns are called exons. Only about 1.5% of human genomic DNA encodes proteins; even when introns are included, less than one-third of human genomic DNA consists of genes. Much of the remainder consists of repeated sequences of various types. Nucleic acid parasites known as transposons account for about half of the human genome. Eukaryotic chromosomes have two important special-function repetitive DNA sequences: centromeres, which are attachment points for the mitotic spindle, and telomeres, located at the ends of chromosomes. 24.2 DNA Supercoiling How is cellular or viral DNA compacted into the cells or viral coats that contain it in a way that still permits access to the information in the DNA? The extreme compaction implies a high degree of structural organization. First, the many negative charges of the phosphoryl groups in the DNA backbone must be neutralized. Cations, particularly M g2+ ions, and a class of molecules called polyamines provide multiple positive counterions to permit DNA compaction. Polyamines are derived from the amino acid ornithine (see Box 6-1). The second key to DNA compaction is a DNA structural alteration known as supercoiling. All cells maintain their DNA in a state that is underwound — having fewer right-handed helical turns per given length of DNA — than B-form DNA. The underwinding places structural strain on the DNA, causing it to twist upon itself. Supercoiling affects and is affected by processes such as replication and transcription. We introduce it here as a prelude to a broader discussion of DNA metabolism. “Supercoiling” means the coiling of a coil. An old- fashioned telephone cord, for example, was typically a coiled wire. The path taken by the wire between the base of the phone and the receiver o en included one or more supercoils (Fig. 24- 8). DNA is coiled in the form of a double helix, with both strands of the DNA coiling around an axis. The further coiling of that axis upon itself (Fig. 24-9) produces DNA supercoiling. As detailed below, DNA supercoiling is generally a manifestation of structural strain. When there is no net bending of the DNA axis upon itself, the DNA is said to be in a relaxed state. As we shall see, the supercoiling that occurs in cells reflects underwinding of the DNA, facilitating the separation of strands required for the processes of replication and transcription (Fig. 24-10). FIGURE 24-8 Supercoils. An old-fashioned phone cord is coiled like a DNA helix, and the coiled cord can itself coil in a supercoil. Examining phone cords helped lead Jerome Vinograd and his colleagues to the insight that many properties of small circular DNAs can be explained by supercoiling. They first detected DNA supercoiling — in small circular viral DNAs — in 1965. FIGURE 24-9 Supercoiling of DNA. When the axis of the DNA double helix is coiled on itself, it forms a new helix (superhelix). The DNA superhelix is usually called a supercoil.
FIGURE 24-10 The effects of replication and transcription on DNA supercoiling. Because DNA is a double-helical structure, strand separation leads to added stress and supercoiling if the DNA is constrained (not free to rotate) ahead of the strand separation. (a) The general effect can be illustrated by twisting two strands of a rubber band about each other to form a double helix. If one end is constrained, separating the two strands at the other end will lead to twisting. (b) In a DNA molecule, the progress of a DNA polymerase or RNA polymerase (as shown here) along the DNA involves separation of the strands. As a result, the DNA becomes overwound ahead of the enzyme (upstream) and underwound behind it (downstream). Red arrows indicate the direction of winding. That a DNA molecule would bend on itself and become supercoiled in tightly packaged cellular DNA would seem logical, and perhaps even trivial, were it not for one additional fact: many circular DNA molecules remain highly supercoiled even a er they are extracted and purified, 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. Several measurable properties of supercoiling have been established, and the study of supercoiling has provided many insights into DNA structure and function. 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 due to interaction with proteins or other molecules; discontinuous deformations involve DNA strand breakage. For circular DNA molecules, a topological property is one that is unaffected by deformations 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. In aqueous solutions, DNA is most stable — that is, it is in its lowest free-energy form — in the B-form structure. If the DNA of a closed-circular molecule conforms closely to the B-form structure (Watson-Crick structure; see Fig. 8-13), with one turn of the double helix per 10.5 bp, the DNA is relaxed rather than supercoiled (Fig. 24-11). Supercoiling results when DNA is subject to some form of structural strain such that it is overwound or underwound. 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 supercoiling. DNA structure is therefore strained in a manner that is regulated by the cell to induce the supercoiling. FIGURE 24-11 Relaxed and supercoiled plasmid DNAs. The molecule in the le most electron micrograph is relaxed; the degree of supercoiling increases from le to right. In almost every instance, the strain is a result of underwinding of the DNA double helix in the closed circle. 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 24-12. An 84 bp segment of a circular DNA in the relaxed state would contain eight double-helical turns, one for every 10.5 bp. If one of these turns were removed, there would be (84 bp)/7= 12.0 bp per turn, rather than the 10.5 found in B-DNA (Fig. 24-12b). This is a deviation from the most stable DNA form, and the molecule is thermodynamically strained as a result. Generally, much of this strain would be accommodated by coiling the axis of the DNA on itself to form a supercoil (Fig. 24-12c; some of the strain in this 84 bp segment would simply become dispersed in the untwisted structure 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. 24-12d). In isolated closed-circular DNA, strain introduced by underwinding 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 and disrupting the base stacking that stabilizes paired bases. Note, however, that the underwinding of DNA in vivo makes separation of the DNA strands easier, facilitating access to the information they contain. FIGURE 24-12 Effects of DNA underwinding. (a) A segment of DNA in 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 somewhat easier. In principle, each turn of underwinding should facilitate strand separation over about 10 bp, as shown here. 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. Every cell actively underwinds its DNA with the aid of enzymatic processes (described below), and the resulting strained state represents a form of stored energy. Cells maintain DNA in an underwound state to facilitate 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 isolated, protein-free circular DNA, free rotation at that point will cause the underwound DNA to revert spontaneously 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 some ideas that are useful to the discussion of DNA supercoiling, particularly the concept of linking number. Linking number is a topological property 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 24-13. As we shall see, all cells have enzymes called topoisomerases that catalyze changes in the linking number. Because of this critical role, topoisomerases are the targets of many antibiotics and cancer chemotherapy agents.
FIGURE 24-13 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. 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 24-13a, 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 the soap film framed by the loop of a bubble wand before you blow a bubble). The linking number can be defined as the number of times the second strand pierces this surface. For the molecule in Figure 24-13a, Lk= 1; for that in Figure 24-13b, 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 le -handed helix, the linking number is negative (−). Negative linking numbers are, for all practical purposes, not encountered in DNA. We can now extend these ideas to a closed-circular DNA with 2,100 bp (Fig. 24-14a). 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. The linking number in relaxed DNA is designated Lk0. For a circular DNA molecule to have a topological property such as linking number, both strands must be intact, without a break. If there is a break in either strand, the two strands can, in principle, be unraveled and separated completely. In this case, no topological bond exists and Lk is undefined (Fig. 24-14b). FIGURE 24-14 Linking number applied to closed-circular DNA molecules. 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 molecule generally exists as a supercoiled molecule, but underwinding also facilitates the separation of DNA strands. We can now describe DNA underwinding in terms of changes in the linking number. For the molecule shown in Figure 24-14a, Lk0 = 200; if two turns are removed from this molecule, Lk= 198. The change can be described by the equation Δ Lk= Lk− Lk0 = 198− 200=−2 (24-1) It is o en 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 (σ ), is a measure of the number of turns removed relative to the number present in relaxed DNA: σ= (24-2) In the example in Figure 24-14c, σ= 0.01, which means that 1% (2 of 200) of the helical turns present in the DNA (in its B form) have been removed. The degree of underwinding in cellular DNAs generally falls in the range of 5% to 7%; that is, σ=−0.05 to −0.07. The negative sign indicates that the change in linking number is Δ Lk Lk0 due to underwinding of the DNA. The supercoiling induced by underwinding is therefore defined as negative supercoiling. Conversely, under some conditions DNA can be overwound, resulting in positive supercoiling. Note that the twisting path taken by the axis of the DNA helix when the DNA is underwound (negative supercoiling) is the mirror image of that taken when the DNA is overwound (positive supercoiling) (Fig. 24-15). 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. An increase in superhelical density brings about an increase in DNA compaction. FIGURE 24-15 Negative and positive supercoils. For the relaxed DNA molecule of Figure 24-14a, underwinding or overwinding by two helical turns (Lk= 198 or 202) will produce negative or positive supercoiling, respectively. Notice that the DNA axis twists in opposite directions in the two cases. 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. WORKED EXAMPLE 24-1 Calculation of Superhelical Density What is the superhelical density (σ ) of a closed-circular DNA with a length of 4,200 bp and a linking number (Lk) of 374? What is the superhelical density of the same DNA when Lk= 412? Are these molecules negatively or positively supercoiled? SOLUTION: First, calculate Lk0 by dividing the length of the closed-circular DNA (in bp) by 10.5 bp/turn: (4,200 bp)/(10.5 bp/turn)= 400. We can now calculate Δ Lk from Equation 24-1: Δ Lk= Lk− Lk0 = 374 − 400=−26. Substituting the values for Δ Lk and Lk0 into Equation 24-2: σ=Δ Lk/Lk0 =−26/400=−0.065. Because the superhelical density is negative, this DNA molecule is negatively supercoiled. When the same DNA molecule has an Lk of 412, Δ Lk= 412− 400= 12, and σ= 12/400= 0.03. The superhelical density is positive, and the molecule is positively supercoiled. In addition to causing supercoiling and making strand separation somewhat easier, the underwinding of DNA facilitates structural changes in the molecule. These are of less physiological importance but they help illustrate the effects of underwinding. Recall that a cruciform (see Fig. 8-19) generally contains a few unpaired bases; DNA underwinding helps to maintain the required strand separation (Fig. 24-16). Underwinding of a right- handed DNA helix also enables the formation of short stretches of le -handed Z-DNA in regions where the base sequence is consistent with the Z form (see Chapter 8). FIGURE 24-16 Promotion of cruciform structures by DNA underwinding. In principle, cruciforms can form at palindromic sequences (see Fig. 8-19), but they seldom occur in relaxed DNA because the linear DNA accommodates more paired bases than does the cruciform structure. Underwinding of the DNA facilitates the partial strand separation needed to promote cruciform formation at appropriate sequences. 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 decrease the extent of DNA underwinding are topoisomerases; the property of DNA that they change is the linking number. These enzymes play an especially important 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. When a circular DNA is supercoiled, it is twisted upon itself and therefore is more compact than when it is relaxed. The supercoiled molecule will thus migrate faster in a gel matrix (Fig. 24-17). A population of identical plasmid DNAs with the same linking number migrates as a discrete band during agarose gel electrophoresis. 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 readily detected. FIGURE 24-17 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 molecules are more compact than relaxed molecules, they migrate more rapidly during gel electrophoresis. The gels shown here separate topoisomers (moving from top to bottom) over a limited range of superhelical 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; Lk changes by 1 from one band to the next. E. coli has at least four individual topoisomerases (I through IV). Those of type I (topoisomerases 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 24-18. A bacterial type II enzyme, called either topoisomerase II or DNA gyrase, can introduce 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 overall degree of supercoiling of bacterial DNA is maintained by regulation of the net activity of topoisomerases I and II. Topoisomerases III and IV have more-specialized roles in DNA metabolism. MECHANISM FIGURE 24-18 The type I topoisomerase reaction. Bacterial topoisomerase I increases Lk by breaking one DNA strand, passing the unbroken strand through the break, then resealing the break. Nucleophilic attack by the active-site Tyr residue breaks one DNA strand. The ends are ligated by a second nucleophilic attack. At each step, one high-energy bond replaces another. [Information from J. J. Champoux, Annu. Rev. Biochem. 70:369, 2001, Fig. 3.] Eukaryotic cells also have type I and type II topoisomerases. The type I enzymes are topoisomerases I and III. The single type II enzyme has two isoforms in vertebrates, called IIα and IIβ . Most type II enzymes, including a DNA gyrase in archaea, are related and define a family called type IIA. The eukaryotic type II topoisomerases cannot underwind DNA (introduce negative supercoils), but they can relax both positive and negative supercoils (Fig. 24-19a). The capacity of type II topoisomerases to pass one duplex DNA segment through a double-strand break in another duplex allows these enzymes to untangle catenanes, DNA circles that are topologically linked (Fig. 24-19b). Some topoisomerases are specialized for decatenation functions. For example, the bacterial type II enzyme called topoisomerase IV is involved in chromosome untangling during cell division (Chapter 25). FIGURE 24-19 Alteration of the linking number by a eukaryotic type IIα topoisomerase. (a) The general mechanism features passage of one intact duplex DNA segment through a transient double-strand break in another segment. The DNA segment enters and leaves the topoisomerase through gated cavities, called the N gate and the C gate, above and below the bound DNA. Two ATPs are bound and hydrolyzed during this cycle. The enzyme structure and use of ATP are specific to this reaction. (b) When topologically linked as shown, two DNA circles are referred to as a catenane. By cleaving both strands of one circle and passing a segment of the second circle through the break, a type II topoisomerase can decatenate the circles. [(a) Information from J. J. Champoux, Annu. Rev. Biochem. 70:369, 2001, Fig. 11.] Archaea have an unusual enzyme, topoisomerase VI, which alone defines the type IIB family. The full diversity of DNA topoisomerases is illustrated in Table 24-4. As we shall show in the next few chapters, topoisomerases play a critical role in every aspect of DNA metabolism, making them important drug targets for the treatment of bacterial infections and cancer. TABLE 24-4 Diversity in DNA Topoisomerases Type Mechanism Family (defined by Domain(s) Notes structural class) IA Strand passage Topoisomerase I Bacteria, Eukarya Relaxes (−) Topoisomerase III Bacteria, Eukarya Relaxes (−) Reverse gyrase Archaea, Bacteria Uses ATP to introduce positive supercoils; thermophilic bacteria and archaea only IB Swivelase Topoisomerase IB Bacteria, Eukarya A few bacteria; all eukaryotes IC Swivelase Topoisomerase V Archaea Methanopyrus only IIA Strand passage Topoisomerase II (DNA gyrase) Archaea, Bacteria Introduces negative supercoils (ATPase) Topoisomerase IIα Eukarya Relaxes (+ or −) Topoisomerase IIβ Eukarya Relaxes (+ or −) Topoisomerase IV Bacteria Decatenase IIB Strand passage Topoisomerase VI Archaea, Bacteria, Eukarya Among eukaryotes, plants, algae, and protists only See Figure 24-18. A nick is made in one strand, and the other strand is allowed to rotate to relieve topological strain. See Figure 24-19a. a b c d a b c See Figure 24-19b. DNA Compaction Requires a Special Form of Supercoiling Supercoiled DNA molecules are uniform in several respects. The supercoils are right-handed in a negatively supercoiled DNA molecule (Fig. 24-15), and they tend to be extended and narrow rather than compacted, o en with multiple branches (Fig. 24-20). At the superhelical 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”). The term can be applied 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. FIGURE 24-20 Plectonemic supercoiling. (a) Electron micrograph of plectonemically supercoiled plasmid DNA and (b) an interpretation of the d observed structure. The dotted lines show the axis of the supercoil; notice the branching of the supercoil. (c) An idealized representation of this structure. [(a, b) Republished with permission of Elsevier, from T. C. Boles et al. (1990), “Structure of plectonemically supercoiled DNA,” J. Mol. Biol. 213:931–951, Fig. 2; permission conveyed through Copyright Clearance Center, Inc.] Plectonemic supercoiling, the form observed in isolated DNAs in the laboratory, does not produce sufficient compaction to package DNA in the cell. A second form of supercoiling, solenoidal (Fig. 24-21), can be adopted by an underwound DNA. Instead of the extended right-handed supercoils characteristic of the plectonemic form, solenoidal supercoiling involves tight le - 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 supercoiling that can be taken up by the same segment of underwound DNA. The two forms are readily interconvertible. Although the plectonemic form is more stable in solution, the solenoidal form can be stabilized by protein binding, as it is in eukaryotic chromosomes; it provides a much greater degree of compaction. Solenoidal supercoiling is a primary mechanism by which underwinding contributes to DNA compaction in cells. FIGURE 24-21 Plectonemic and solenoidal supercoiling of the same DNA molecule, drawn to scale. Plectonemic supercoiling takes the form of extended right-handed coils. Solenoidal negative supercoiling takes the form of tight le -handed turns about an imaginary tubelike structure. The two forms are readily interconverted, although the solenoidal form is generally not observed unless certain proteins are bound to the DNA. Solenoidal supercoiling provides a much greater degree of compaction. SUMMARY 24.2 DNA Supercoiling Most cellular DNAs are supercoiled. Underwinding decreases the total number of helical turns in 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, or superhelical density, σ , which is (Lk− Lk0)/Lk0. For cellular DNAs, σ 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. Topoisomerases, enzymes that underwind and/or relax DNA, 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. Supercoiled DNA in solution, unconstrained by proteins, takes on a plectonemic supercoiling structure. When supercoiled DNA is wrapped around specialized DNA-binding proteins, it forms solenoidal supercoils. 24.3 The Structure of Chromosomes The term “chromosome” is used to refer to a nucleic acid molecule that is the repository of genetic information in a virus, a bacterium, an archaeon, a eukaryotic cell, or an organelle. It also refers to the densely colored bodies seen in the nuclei of dye- stained eukaryotic cells undergoing mitosis, as visualized using a light microscope. Chromatin Consists of DNA, Proteins, and RNA The eukaryotic cell cycle produces remarkable changes in the structure of chromosomes (Fig. 24-22). In nondividing eukaryotic cells (in the G0 phase) and those in interphase (G1, S, and G2), the chromosomal material, chromatin, is amorphous. In the S phase of interphase, the DNA in this amorphous state replicates, each chromosome producing two sister chromosomes (called sister chromatids) that remain associated with each other a er replication is complete. The chromosomes become much more condensed during prophase of mitosis, taking the form of a species-specific number of well-defined pairs of sister chromatids (Fig. 24-5). FIGURE 24-22 Changes in chromosome structure during the eukaryotic cell cycle. The relative lengths of the phases shown here are arbitrary. The duration of each phase varies with cell type and with growth conditions (for single-celled organisms) or metabolic state (for multicellular organisms); mitosis is typically the shortest phase. Cellular DNA is uncondensed throughout interphase, as shown in the cartoons of the nucleus. The interphase period can be divided into the G1 (gap) phase; the S (synthesis) phase, when the DNA is replicated; and the G2 phase, throughout which the replicated chromosomes (chromatids) cohere to one another. Mitosis can be divided into four stages. The DNA undergoes condensation in prophase. During metaphase, the condensed chromosomes line up in pairs along the plane halfway between the spindle poles. The two chromosomes of each pair are linked to different spindle poles 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. The process is completed in telophase. A er cell division, the chromosomes decondense and the cycle begins anew. Chromatin consists of fibers containing protein and DNA in approximately equal proportions (by mass), along with a significant amount of associated 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. 24-23). Also found in chromatin are many nonhistone proteins, some of which help maintain chromosome structure and others that regulate the expression of specific genes (Chapter 28). Beginning with nucleosomes, eukaryotic chromosomal DNA is packaged into a succession of higher-order structures that ultimately 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 packaging of DNA in bacterial cells. FIGURE 24-23 Nucleosomes. (a) Regularly spaced nucleosomes consist of core histone proteins bound to DNA. (b) In this electron micrograph, the DNA-wrapped histone octamer structures are clearly visible. [(b) J. Bednar et al., Nucleosomes, linker DNA, and linker histone form a unique structural motif that directs the higher-order folding and compaction of chromatin, Proc. Natl. Acad. Sci. USA vol. 95 no. 24: 14173–14178, November 1998 Cell Biology Fig. 1. © 1998 National Academy of Sciences, U.S.A.] 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 lysine (together these make up about one-fourth of the amino acid residues). All eukaryotic cells have five major classes of histones, differing in molecular weight and amino acid composition (Table 24-5). 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 across eukaryotic species. TABLE 24-5 Types and Properties of the Common Histones Histones Molecular weight Number of amino acid residues Content of basic amino acids (% of total) Lys Arg H1 21,130 223 29.5 11.3 H2A 13,960 129 10.9 19.3 H2B 13,774 125 16.0 16.4 H3 15,273 135 19.6 13.3 H4 11,236 102 10.8 13.7 The sizes of these histones vary somewhat from species to species. The numbers given here are for bovine histones. Each type of histone is subject to enzymatic modification by methylation, acetylation, ADP-ribosylation, phosphorylation, glycosylation, SUMOylation, or ubiquitination (p. 216 and Fig. 6- 38). Such modifications affect the net electric charge, shape, and other properties of histones, as well as the structural and a a a a functional properties of the chromatin. The modifications play a role in the regulation of transcription and in chromatin structure at different stages of the cell cycle. In addition, eukaryotes generally have several variant forms of certain histones, most notably histones H2A and H3, described in more detail below. The variant forms, along with their modifications, have specialized roles in DNA metabolism. 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 μ m long into a cell nucleus that is typically 5 to 10 μ m in diameter. This 10,000-fold compaction is achieved by means of several levels of highly organized folding. Subjection of chromosomes to treatments that partially unfold them reveals a structure in which the DNA is bound tightly to beads of protein, o en regularly spaced. The beads in this “beads-on-a-string” arrangement are complexes of histones and DNA. The bead plus the connecting DNA that leads to the next bead form the nucleosome, the fundamental unit of organization on which the higher-order packing of chromatin is built (Fig. 24-24). 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 repeating 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 treatment of chromatin with enzymes that digest DNA causes the linker DNA to degrade preferentially, releasing histone particles containing 146 bp of bound DNA that is protected from digestion. FIGURE 24-24 DNA wrapped around a histone core. (a) The simplified structure of a nucleosome octamer (le ), with DNA wrapped around the histone core (right). (b) A ribbon representation of the nucleosome from the African frog Xenopus laevis. Different colors represent the different histones, matching the colors in (a). (c) Surface representation of the nucleosome. The view in (c) is rotated relative to the view in (b) to match the orientation shown in (a). A 146 bp segment of DNA in the form of a le -handed solenoidal supercoil wraps around the histone complex 1.67 times. (d) Two views of histone amino-terminal tails protruding from between the two DNA duplexes that supercoil around the nucleosome core. Some tails pass between the supercoils, through holes formed by alignment of the minor grooves of adjacent helices. The H3 and H2B tails emerge between the two coils of DNA wrapped around the histone; the H4 and H2A tails emerge between adjacent histone subunits. (e) The amino-terminal tails of one nucleosome protrude from the particle and interact with adjacent nucleosomes, helping to define higher-order DNA packaging. [(b–d) Data from PDB ID 1AOI, K. Luger et al., Nature 389:251, 1997.] Researchers have crystallized 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 the core in the form of a le -handed solenoidal supercoil (Fig. 24-24; see also Fig. 24-21). Extending out from the nucleosome core are the amino-terminal tails of the histones, which are intrinsically disordered (Fig. 24-24d). Most of the histone modifications occur in these tails. The tails play a key role in forming contacts between nucleosomes in the chromatin (Fig. 24-24e). As the nucleosomes are approximately 10 to 11 nm in diameter, this simple beads-on-a-string structure is sometimes called the 10 nm fiber. A close inspection of the nucleosome structure reveals why eukaryotic 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 underwound (negatively supercoiled) DNA. The tight wrapping of DNA around the histone core requires the removal 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 region of the DNA (Fig. 24-25). As mentioned earlier, eukaryotic topoisomerases can relax positive supercoils. Relaxing the unbound positive supercoil leaves the negative supercoil fixed (through its binding to the nucleosome’s histone core) and results in an overall decrease in linking number. Indeed, topoisomerases have proved necessary for assembling chromatin from purified histones and closed-circular DNA in vitro.
FIGURE 24-25 Chromatin assembly. (a) Relaxed closed-circular DNA. (b) Binding of a histone core to form a nucleosome induces one negative supercoil. In the absence of any strand breaks, a positive supercoil must form elsewhere in the DNA (ΔLk= 0). (c) Relaxation of this positive supercoil by a topoisomerase leaves one net negative supercoil (ΔLk=−1). Another factor that affects the binding of DNA to histones in nucleosome cores is the sequence of the bound DNA. Histone cores do not bind at random positions on the DNA; rather, some locations are more likely to be bound than others. This positioning is not fully understood, but in some cases it seems to depend on a local abundance of A═T base pairs in the DNA helix where it is in contact with the histones (Fig. 24-26). A cluster of two or three A═T base pairs facilitates the compression of the minor groove that is needed for the DNA to wrap tightly around the nucleosome’s histone core. Nucleosomes bind particularly well to sequences where AA or AT or TT dinucleotides are staggered at 10 bp intervals, an arrangement that can account for up to 50% of the positions of bound histones in vivo. FIGURE 24-26 The effect of DNA sequence on nucleosome binding. Runs of two or more A═T base pairs facilitate the bending of DNA, whereas runs of two or more G≡C base pairs have the opposite effect. When spaced at about 10 bp intervals, consecutive A═T base pairs help bend DNA into a circle. When consecutive G≡C base pairs are spaced 10 bp apart, offset by 5 bp from runs of A═T base pairs, DNA binding to the nucleosome core is facilitated. [Data from PDB ID 1AOI, K. Luger et al., Nature 389:251, 1997.] Nucleosome cores are deposited on DNA during replication or following other processes that require a transient displacement of nucleosomes. Other, nonhistone proteins are required for the positioning of some nucleosome cores. In several organisms, certain proteins bind to a specific DNA sequence and facilitate the formation of a nucleosome core nearby. Nucleosome cores seem to be deposited stepwise. A tetramer of two H3 and two H4 histones binds first, followed by two H2A–H2B dimers. The incorporation of nucleosomes into chromosomes a er chromosomal replication is mediated by a complex of histone chaperones conserved in all eukaryotes and described here for the yeast system. These include the proteins CAF1 (chromatin assembly factor 1), RTT106 and RTT109 (regulation of Ty1 transposition), and ASF1 (anti-silencing factor 1). ASF1 binds to newly synthesized H3–H4 dimers and facilitates RTT109-mediated acetylation at Lys56 (K56) of histone H3 (H3K56). The H3K56 modification increases the affinity of H3 for CAF1 and RTT, which in turn promote the deposition of H3-containing histone complexes on the DNA a er replication. CAF1 binds directly to a key component of the replication complex called PCNA (see Chapter 25), so that nucleosome deposition is closely coordinated with replication. Some of the same histone chaperones, or different ones, may help assemble nucleosomes a er DNA repair, transcription, or other processes. Histone exchange factors permit the substitution of histone variants for core histones in contexts other than postreplication. Proper placement of these variant histones is important. Studies show that mice lacking one of the variant histones die as early embryos (Box 24-1). Precise positioning of nucleosome cores also plays a role in the expression of some eukaryotic genes (Chapter 28). BOX 24-1 METHODS Epigenetics, Nucleosome Structure, and Histone Variants Information that is passed from one generation to the next — to daughter cells at cell division or from parent to offspring — but is not encoded in DNA sequences is referred to as epigenetic information. Much of it is in the form of covalent modification of histones and/or the placement of histone variants in chromosomes. Understanding that placement in the context of a chromosome encompassing millions of base pairs is the focus of some powerful technologies. Chromatin regions where active gene expression (transcription) is occurring tend to be partially decondensed and are called euchromatin. In these regions, histones H3 and H2A are o en replaced by the histone variants H3.3 and H2AZ, respectively (Fig. 1). The complexes that deposit nucleosome cores containing histone variants on the DNA are similar to those that deposit nucleosome cores with the more common histones. Nucleosome cores containing histone H3.3 are deposited by a complex in which CAF1 (chromatin assembly factor 1) is replaced by the protein HIRA (a name derived from a class of proteins called HIR, for histone repressor). Both CAF1 and HIRA can be considered histone chaperones, helping to ensure the proper assembly and placement of nucleosomes. Histone H3.3 differs in sequence from H3 by only four amino acid residues, but these residues all play key roles in histone deposition. FIGURE 1 Shown here are the standard histones H3, H2A, and H2B and a few of the known variants. Sites of Lys/Arg residue methylation and Ser phosphorylation are indicated. HFD denotes the histone-fold domain, a structural domain common to all standard histones. Regions denoted in other colors define sequence and structural homologies. [Information from K. Sarma and D. Reinberg, Nat. Rev. Mol. Cell Biol. 6:139, 2005.] Like histone H3.3, H2AZ is associated with a distinct nucleosome deposition complex, and it is generally associated with chromatin regions involved in active transcription. Incorporation of H2AZ stabilizes the nucleosome octamer, but it impedes some cooperative interactions between nucleosomes that are needed to compact the chromosome. This leads to a more open chromosome structure that enables the expression of genes in the region where H2AZ is located. The gene encoding H2AZ is essential in mammals. In fruit flies, loss of H2AZ prevents development beyond the larval stage. Another H2A variant is H2AX, which is associated with DNA repair and genetic recombination. In mice, the absence of H2AX results in genome instability and male infertility. Modest amounts of H2AX seem to be scattered throughout the genome. When a double-strand break occurs, nearby molecules of H2AX become phosphorylated at Ser139 in the carboxyl-terminal region. If this phosphorylation is blocked experimentally, formation of the protein complexes necessary for DNA repair is inhibited. The H3 histone variant known as CENPA (centromere protein A) is associated with the repeated DNA sequences in centromeres. The chromatin in the centromere region contains the histone chaperones CAF1 and HIRA, and both proteins could be involved in the deposition of nucleosome cores containing CENPA. Elimination of the gene for CENPA is lethal in mice. The function and positioning of the histone variants can be studied by an application of technologies used in genomics. One useful technology is chromatin immunoprecipitation, or chromatin IP (ChIP). Nucleosomes containing a particular histone variant are precipitated by an antibody that binds specifically to this variant. These nucleosome cores can be studied in isolation from their DNA, but more commonly the DNA associated with them is included in the study to determine where the nucleosome cores of interest bind. The DNA can be sequenced, yielding a map of genomic sequences to which those particular nucleosome cores bind. This technique is called a ChIP- Seq experiment (Fig. 2). FIGURE 2 A ChIP-Seq experiment is designed to reveal the genomic DNA sequences to which a particular histone variant binds. (a) A histone variant with an epitope tag (a protein or chemical structure recognized by an antibody; see Chapters 5 and 9) is introduced into a particular cell type, where it is incorporated into nucleosomes. (In some cases, an epitope tag is unnecessary because antibodies are available that bind directly to the histone modification of interest.) Chromatin is isolated from the cells and digested briefly with micrococcal nuclease (MNase). The DNA bound in nucleosomes is protected from digestion, but the linker DNA is cleaved, releasing segments of DNA bound to one or two nucleosomes. An antibody that binds to the epitope tag is added, and the nucleosomes containing the epitope-tagged histone variant are selectively precipitated. The DNA in these nucleosomes is extracted from the precipitate and sequenced in depth to reveal the locations where the nucleosomes are bound. (b) In this example, the binding of different modified versions of histone H3 is characterized along the gene YLR249W from the yeast S. cerevisiae. Numbers along the bottom correspond to numbered nucleotide positions in this chromosomal segment. The top and bottom panels show the distributions of H3K4me3 (H3 with Lys4 methylated three times) and H3K4me1, respectively. SPMR is sequence tags per million reads. [Information from L. M. Soares et al., Mol. Cell 68:773, 2017, Fig. 1D.] The histone variants, along with the many covalent modifications that histones undergo, help define and isolate the functions of chromatin. They mark the chromatin, facilitating or suppressing specific functions such as chromosome segregation, transcription, and DNA repair. The histone modifications do not disappear at cell division or during meiosis, and thus they become part of the information transmitted from one generation to the next in all eukaryotic organisms. Nucleosomes Are Packed into Highly Condensed Chromosome Structures Wrapping of DNA around a nucleosome core compacts the DNA length about sevenfold. The overall compaction in a chromosome, however, is greater than 10,000-fold — ample evidence for even higher orders of structural organization. The condensation does not follow a rigid organization, but it is also not random and occurs so as to avoid the formation of knots. The higher levels of folding are not yet fully understood, but certain regions of DNA seem to associate with a chromosomal scaffold (Fig. 24-27) that contains many proteins. Given the need to fold and compact the chromosome without creating knots, topoisomerase II is one of the most abundant proteins in the chromosome, further emphasizing the relationship between DNA underwinding and chromatin structure. Topoisomerase II is so important to the maintenance of chromatin structure that inhibitors of this enzyme can kill rapidly dividing cells. Several drugs used in cancer chemotherapy are topoisomerase II inhibitors that allow the enzyme to promote strand breakage but not the resealing of the breaks (Box 24-2, p. 906). FIGURE 24-27 Loops of DNA attached to a chromosomal scaffold. (a) A swollen mitotic chromosome, produced in a buffer of low ionic strength, as seen in the electron microscope. Notice the appearance of chromatin loops at the margins. (b) Extraction of the histones leaves a proteinaceous chromosomal scaffold surrounded by naked DNA. (c) The DNA appears to be organized in loops attached at their base to the scaffold in the upper le corner; scale bar= 1 μm. The three images are at different magnifications. BOX 24-2 MEDICINE Curing Disease by Inhibiting Topoisomerases The topological state of cellular DNA is intimately connected with its function. Without topoisomerases, cells cannot replicate or package their DNA, or express their genes — and they die. Inhibitors of topoisomerases have therefore become important pharmaceutical agents, targeted at infectious agents and malignant cells. Two classes of bacterial topoisomerase inhibitors have been developed as antibiotics. The coumarins, including novobiocin and coumermycin A1, are natural products derived from Streptomyces species. They inhibit the ATP binding of the bacterial type II topoisomerases, DNA gyrase and topoisomerase IV. These antibiotics are not o en used to treat infections in humans, but research continues to identify clinically effective variants. The quinolone antibiotics, also inhibitors of bacterial DNA gyrase and topoisomerase IV, first appeared in 1962 with the introduction of nalidixic acid. This compound had limited effectiveness and is no longer used clinically in the United States, but the continued development of this class of drugs eventually led to the introduction of the fluoroquinolones, exemplified by ciprofloxacin (Cipro). The quinolones act by blocking the last step of the topoisomerase reaction, the resealing of the DNA strand breaks. Ciprofloxacin is a broad- spectrum antibiotic. It is one of the few antibiotics reliably effective in treating anthrax infections and is considered a valuable agent in protection against possible bioterrorism. Quinolones are selective for the bacterial topoisomerases, inhibiting the eukaryotic enzymes only at concentrations several orders of magnitude greater than the therapeutic doses. Some of the most important chemotherapeutic agents used in cancer treatment are inhibitors of human topoisomerases. Topoisomerases are generally present at elevated levels in tumor cells, and agents targeted to these enzymes are much more toxic to the tumors than to most other tissue types. Inhibitors of both type I and type II topoisomerases have been developed as anticancer drugs. Camptothecin, isolated from a Chinese ornamental tree and first tested clinically in the 1970s, is an inhibitor of eukaryotic type I topoisomerases. Clinical trials indicated limited effectiveness, despite its early promise in preclinical work on mice. However, two effective derivatives, irinotecan (Campto) and topotecan (Hycamtin) — used to treat colorectal cancer and ovarian cancer, respectively — were developed in the 1990s. All of these drugs act by trapping the topoisomerase-DNA complex in which the DNA is cleaved, inhibiting religation. The human type II topoisomerases are targeted by a variety of antitumor drugs, including doxorubicin (Adriamycin), etoposide (Etopophos), and ellipticine. Doxorubicin, effective against several kinds of human tumors, is an anthracycline in clinical use. Most of these drugs stabilize the covalent topoisomerase-DNA (cleaved) complex. All of these anticancer agents generally increase the levels of DNA damage in the targeted, rapidly growing tumor cells. However, noncancerous tissues can also be affected, leading to a more general toxicity and unpleasant side effects that must be managed during therapy. As cancer therapies become more effective and survival statistics for cancer patients improve, the independent appearance of new tumors is becoming a greater problem. In the continuing search for new cancer therapies, topoisomerases are likely to remain prominent targets for research. There are additional layers of organization in the eukaryotic nucleus. Just before cell division during mitosis, chromosomes can be seen as highly condensed and organized structures (Figure 24-28a). During interphase, chromosomes appear dispersed (Fig. 24-28b, top), but they do not meander randomly in nuclear space (Fig. 24-28b, bottom). Each chromosome appears to be organized with two sets of compartments, one set that is transcriptionally active and the other that is transcriptionally inactive. The level of chromatin condensation is reduced in the transcriptionally active compartments. The highly condensed DNA in transcriptionally inactive regions or in regions lacking genes is also called heterochromatin. Within each compartment, large segments of DNA are organized in loops called topologically associating domains, or TADs. The TADs, which typically average about 800,000 bp, are o en bordered by DNA sites recognized by the CCCTC-binding factor (CTCF). The binding by CTCF brings together sites that are otherwise quite distant in the linear DNA sequence, tethering the base of the loop (Fig. 24-28c).
FIGURE 24-28 Chromosomal organization in the eukaryotic nucleus. (a) Condensed chromosomes at the mitotic anaphase in cells of the bluebell (Endymion sp.). (b) Interphase nuclei of human breast epithelial cells. The nucleus on the bottom has been treated so that its two copies of chromosome 11 fluoresce green. (c) Chromosomes are organized into active compartments, in which actively transcribed genes are clustered, and inactive compartments, made of heterochromatin within which genes are silenced. In both cases, the compartments feature large DNA loops called topologically associating domains (TADs), many constrained at their bases by DNA-binding proteins such as CTCF. Certain lncRNAs (Fig. 24-29) also play a role in defining loops within chromatin. Constraining a loop at its base not only provides a boundary for the loop, but also allows supercoiling within the loop to be controlled. Another important component defining the structure of chromosomes is RNA, particularly a class of RNAs called long noncoding RNAs (lncRNAs). RNA has the potential to take up a variety of structures (see Chapter 8), and can interact with DNA, proteins, or other RNA molecules. The lncRNAs, as the name implies, are functional RNAs, generally over 200 nucleotides long, that do not necessarily encode proteins. Many lncRNAs are now known and more are being discovered rapidly. Many of them provide a scaffold for proteins that both bind to the RNA and affect chromosome structure and function. Some proteins that bind to lncRNAs also have binding sites on DNA, and the RNAs provide a link that tethers distant parts of the chromosome together (Fig. 24-29). Other proteins that bind to lncRNAs help to position nucleosomes, modify histones, methylate DNA at various locations to alter gene transcription, and generally affect chromosome structure in many different ways. Some well-studied examples include particular lncRNAs that play a major role in X chromosome inactivation in mammals (Box 24-3). FIGURE 24-29 Effects of lncRNAs on chromosome architecture and gene expression. (a) Various lncRNAs can interact with DNA-binding proteins and in some cases with DNA to tether otherwise distant segments of DNA. (b) A transcribed lncRNA can interact with multiple proteins that have gene regulatory roles, suppressing or activating transcription of nearby genes. [Information from J. M. Engreitz et al., Nat. Rev. Mol. Cell Biol. 17:756, 2016, Fig. 5.] BOX 24-3 X Chromosome Inactivation by an lncRNA: Preventing Too Much of a Good (or Bad) Thing In higher eukaryotes, the Y chromosome contains only a few genes. The X chromosome, in contrast, o en contains more than a thousand genes, many essential for cell function and organismal development. Males have just one X chromosome, whereas females have two. Females thus could end up with a double dose of gene products, leading to a variety of potentially toxic outcomes. Mechanisms of gene dosage compensation vary among different groups of eukaryotes. In most mammals, dosage compensation is accomplished by X chromosome inactivation, randomly affecting just one of the two X chromosomes in a given cell. The effects of this can be seen in calico cats, all of which are female. Fur pigmentation genes are found on the X chromosome. When a cat inherits chromosomes with different pigmentation alleles on its two X chromosomes, its coloration patterns reflect the random inactivation of one or the other X chromosome in different sets of cells. A calico cat. An lncRNA called Xist (X-inactive specific transcript) is uniquely expressed from its gene on the inactive X chromosome. Xist is approximately 17,000 nucleotides long, encodes no proteins, and is expressed only in cells with two X chromosomes (not in males). Xist is an essential component of the X inactivation process. This lncRNA interacts with many proteins and transcription factors (Fig. 1). As it migrates in the region immediately proximal to the gene from which it is transcribed, Xist gradually encompasses more and more DNA as that X chromosome undergoes a major condensation, giving rise to the inactive form called a Barr body.
FIGURE 1 (a) As Xist RNA is transcribed, it migrates to nearby regions within an X chromosome, (b) binding to proteins including SAFA (scaffold attachment factor A). Xist binding spreads through the chromosome, leading to condensation and other architectural changes to form a Barr body. Other than the Xist gene itself, gene expression is suppressed throughout the chromosome. (c) The effects are quite local; Xist does not spread within the nucleus beyond the chromosome territory occupied by the inactivated X chromosome. [Information from J. M. Engreitz et al., Nat. Rev. Mol. Cell Biol. 17:756, 2016, Fig. 5.] The active X chromosome also transcribes the gene that produces Xist, but it does so in the opposite direction to produce a longer lncRNA called Tsix (Xist spelled backward). Tsix is synthesized using a different RNA polymerase binding site (promoter; Chapter 26) and is 40,000 nucleotides long. A large part of Tsix is perfectly complementary to Xist, and it antagonizes the function of any Xist that may appear near the active X chromosome. Random X inactivation occurs in the cells of a female embryo long before birth. The processes that initiate the inactivation of one, but not both, of the X chromosomes in a particular cell are still being elucidated. The entire structure of each chromosome is constrained within a subnuclear domain called a chromosome territory (Fig. 24-30). There is little or no intermingling of chromosomal DNA in different territories. The exact location of chromosome territories varies from cell to cell in an organism, but some spatial patterns are evident. Some chromosomes have a higher density of genes than others (for example, human chromosomes 1, 16, 17, 19, and 22), and these tend to have territories in the center of the nucleus. Chromosomes with more heterochromatin tend to be located on the nuclear periphery. Spaces between chromosomes are o en sites where transcriptional machinery and transcriptionally active genes on adjacent chromosomes are concentrated. FIGURE 24-30 Chromosome territories. Cartoon showing chromosome territories in a eukaryotic nucleus. The interchromatin compartments are enriched in transcriptional machinery and have abundant actively transcribed genes. The nucleolus is a suborganelle within the nucleus where ribosomes are synthesized and assembled (Chapter 27). Condensed Chromosome Structures Are Maintained by SMC Proteins SMC proteins (structural maintenance of chromosomes), the third major class of chromatin protein in addition to the histones and topoisomerases, are responsible for maintaining the structure and integrity of chromosomes following replication. The primary structure of SMC proteins consists of five distinct domains (Fig. 24-31a). The amino- and carboxyl-terminal globular domains, N and C, each of which contains part of an ATP-hydrolytic site, are connected by two regions of α -helical coiled-coil motifs (see Fig. 4-10) joined by a hinge domain. The proteins are generally dimeric, forming a V-shaped complex that is thought to be tied together through the protein’s hinge domains. One N domain and one C domain come together like tweezers to form a complete ATP-hydrolytic site at each free end of the V (Fig. 24-31b). FIGURE 24-31 Structure of SMC proteins. (a) SMC proteins have five domains. (b) Each SMC polypeptide is folded so that the two coiled-coil domains wrap around each other and the N and C domains come together to form a complete ATP-binding site. Two polypeptides are linked at the hinge region to form the dimeric V-shaped SMC molecule. (c) Bacterial SMC proteins form a homodimer. The six different eukaryotic SMC proteins form heterodimers. Cohesins are made up of SMC1–SMC3 pairs, and condensins consist of SMC2–SMC4 pairs. The SMC5–SMC6 pair is involved in DNA repair. (d) Electron micrographs of SMC dimers from the bacterium Bacillus subtilis. [(a–c) Information from T. Hirano, Nat. Rev. Mol. Cell Biol. 7:311, 2006, Fig. 1. (d) Harold P. Erickson, Duke University Medical Center, Department of Cell Biology.] Proteins in the SMC family are found in all types of organisms, from bacteria to humans. Eukaryotes have two major types, cohesins and condensins, both of which are bound by regulatory and accessory proteins (Fig. 24-31c, d). Cohesins play a substantial role in linking together sister chromatids immediately a er replication and keeping them together as the chromosomes condense to metaphase. This linkage is essential if chromosomes are to segregate properly at cell division. Cohesins, along with a third protein, kleisin, are thought to form a ring around the replicated chromosomes that ties them together until separation is required. The ring may expand and contract in response to ATP hydrolysis. Condensins are essential to the condensation of chromosomes as cells enter mitosis. In the laboratory, condensins bind to DNA in a manner that creates positive supercoils; that is, condensin binding causes the DNA to become overwound, in contrast to the underwinding induced by the binding of nucleosomes. A model for the role of condensins in chromatin compaction is presented in Figure 24-32. In brief, as DNA is compacted to form tighter and tighter loops, the condensins stabilize the loops by binding at the base of each one. Cohesins and condensins are essential in orchestrating the many changes in chromosome structure during the eukaryotic cell cycle (Fig. 24-33). FIGURE 24-32 Two current models of the possible role of condensins in chromatin condensation. Initially, the DNA is bound at the hinge region of the SMC protein, in the interior of what can become an intramolecular SMC ring. ATP binding leads to head-to-head association, forming supercoiled loops in the bound DNA. Subsequent rearrangement of the head-to-head interactions to form rosettes condenses the DNA. Condensins may organize the looping of the chromosome segments in several ways. [Information from T. Hirano, Nat. Rev. Mol. Cell Biol. 7:311, 2006, Fig. 6.] FIGURE 24-33 The roles of cohesins and condensins in the eukaryotic cell cycle. Cohesins are loaded onto the chromosomes during G1 (see Fig. 24-22), tying the sister chromatids together during replication. At the onset of mitosis, condensins bind and maintain the chromatids in a condensed state. During anaphase, the enzyme separase removes the cohesin links. Once the chromatids separate, condensins begin to unload and the daughter chromosomes return to the uncondensed state. [Information from D. P. Bazett-Jones et al., Mol. Cell 9:1183, 2002, Fig. 5.] Bacterial DNA Is Also Highly Organized We now turn briefly to the structure of bacterial chromosomes. Bacterial DNA is compacted in a structure called the nucleoid, which can occupy a significant fraction of the cell volume (Fig. 24-34). The DNA seems to be 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 of eukaryotic chromatin, but a complex organization is slowly being revealed. In E. coli, a scaffoldlike structure seems to organize the circular chromosome into a series of about 500 looped domains, each encompassing, on average, 10,000 bp (Fig. 24-35), as described above for chromatin. The domains are topologically constrained; for example, if the DNA is cleaved in one domain, only the DNA within that domain will be relaxed. The domains do not have fixed end points. Instead, the boundaries are most likely in constant motion along the DNA, coordinated with DNA replication. FIGURE 24-34 E. coli nucleoids. The DNA of these cells is stained with a dye that fluoresces blue when exposed to UV light. The blue areas define the nucleoids. Notice that some cells have replicated their DNA but have not yet undergone cell division and hence have multiple nucleoids. FIGURE 24-35 Looped domains of the E. coli chromosome. Each domain is about 10,000 bp long. The domains are not static but move along the DNA as replication proceeds. Barriers at the boundaries of the domains, of unknown composition, prevent the relaxation of DNA beyond the boundaries of the domain where a strand break occurs. The putative boundary complexes are shown as gray-shaded ovoids. The arrows denote movement of DNA through the boundary complexes. Bacterial DNA does not seem to have any structure comparable to the local organization provided by nucleosomes in eukaryotes. Histonelike proteins are abundant in E. coli — the best- characterized example is a two-subunit protein called HU (Mr 19,000) — but these proteins bind and dissociate within minutes, and no regular, stable DNA-histone structure has been found. The dynamic structural changes in the bacterial chromosome may reflect a requirement for more ready access to its genetic information. The bacterial cell division cycle can be as short as 15 min, whereas a typical eukaryotic cell may not divide for hours or even months. In addition, a much greater fraction of bacterial DNA is used to encode RNA and/or protein products. Higher rates of cellular metabolism in bacteria mean that a much higher proportion of the DNA is being transcribed or replicated at a given time than in most eukaryotic cells. SUMMARY 24.3 The Structure of Chromosomes A eukaryotic chromosome is made of DNA, protein, and RNA, forming a structure called chromatin. Histones are small, basic DNA-binding proteins. Complexes of histones form nucleosomes, the fundamental structural unit of chromatin. The nucleosome consists of histones and a 200 bp segment of DNA. A core protein particle containing eight histone molecules (two copies each of histones H2A, H2B, H3, and H4) is encircled by a segment of DNA (about 146 bp) in the form of a le -handed solenoidal supercoil. Higher-order folding of chromosomes involves attachment to a chromosomal scaffold. Transcriptionally active and inactive regions of chromosomes are separated into compartments, each featuring large loops of DNA, each loop constrained at its base by proteins and lncRNAs. Individual chromosomes are constrained within nuclear subdomains called territories. Histone H1, topoisomerase II, and SMC proteins play organizational roles in chromosomes. The SMC proteins, principally cohesins and condensins, have important roles in keeping the chromosomes organized during each stage of the cell cycle. The bacterial chromosome is extensively compacted into the nucleoid, but the chromosome seems to be much more dynamic and irregular in structure than eukaryotic chromatin, reflecting the shorter cell cycle and very active metabolism of a bacterial cell. Chapter Review KEY TERMS Terms in bold are defined in the glossary. chromosome phenotype mutation gene regulatory sequence plasmid intron exon simple-sequence DNA satellite DNA centromere telomere supercoil relaxed DNA topology linking number specific linking difference superhelical density (σ ) topoisomers topoisomerases catenane plectonemic solenoidal chromatin histones nucleosome histone exchange factors heterochromatin topologically associating domains (TADs) long noncoding RNA (lncRNA) epigenetic euchromatin chromosome territory Barr body SMC proteins cohesins condensins nucleoid PROBLEMS 1. Packaging of DNA in a Virus Bacteriophage T2 has a DNA of molecular weight 120× 106 contained in a head about 210 nm long. Calculate the length of the DNA and compare it with the length of the T2 head. Assume the molecular weight of a nucleotide base pair is 650 and that the DNA is in the B form and relaxed. 2. The DNA of Phage M13 The base composition of the DNA within a bacteriophage M13 viral particle is A, 23%; T, 36%; G, 21%; C, 20%. What does this tell you about this DNA molecule? 3. The Mycoplasma Genome The complete genome of the simplest bacterium known, Mycoplasma genitalium, is a circular DNA molecule with 580,070 bp. Calculate the molecular weight and contour length (when relaxed) of this molecule. What is Lk0 for the Mycoplasma chromosome? If σ=−0.06, what is Lk? 4. Size of Eukaryotic Genes An enzyme isolated from rat liver has 192 amino acid residues and is encoded by a gene with 1,440 bp. Explain the relationship between the number of amino acid residues in the enzyme and the number of nucleotide pairs in its gene. 5. Linking Number A closed-circular DNA molecule in its relaxed form has an Lk of 500. Approximately how many base pairs are in this DNA? How is Lk altered (increases, decreases, doesn’t change, becomes undefined) when a. a protein complex binds to form a nucleosome, b. one DNA strand is broken, c. DNA gyrase and ATP are added to the DNA solution, or d. the double helix is denatured by heat? 6. DNA Topology In the presence of a eukaryotic condensin and a bacterial type II topoisomerase, the Lk of a relaxed closed-circular DNA molecule does not change. However, the DNA becomes highly knotted. Formation of the knots requires breakage of the DNA, passage of a segment of DNA through the break, and religation by the topoisomerase. Given that every reaction of the topoisomerase would be expected to result in a change in linking number, how can Lk remain the same? 7. Superhelical Density Bacteriophage λ infects E. coli by integrating its DNA into the bacterial chromosome. The success of this recombination depends on the topology of the E. coli DNA. When the superhelical density (σ ) of the E. coli DNA is greater than −0.045, the probability of integration is less than 20%; when σ is less than −0.06, the probability is >70%. Plasmid DNA isolated from an E. coli culture is found to have a length of 13,800 bp and an Lk of 1,222. Calculate σ for this DNA and predict the likelihood that bacteriophage λ will be able to infect this culture. 8. Altering Linking Number a. What is the Lk of a 5,000 bp circular duplex DNA molecule with a nick in one strand? b. What is the Lk of the molecule in (a) when the nick is sealed (relaxed)? c. How would the Lk of the molecule in (b) be affected by the action of a single molecule of E. coli topoisomerase I? d. What is the Lk of the molecule in (b) a er eight enzymatic turnovers by a single molecule of DNA gyrase in the presence of ATP? e. What is the Lk of the molecule in (d) a er four enzymatic turnovers by a single molecule of bacterial type I topoisomerase? f. What is the Lk of the molecule in (d) a er binding of one nucleosome core? 9. Chromatin The agarose gel shown, in which the thick bands represent DNA, helped researchers define nucleosome structure. They generated this result by briefly treating chromatin with an enzyme that degrades DNA, then removing all protein and subjecting the purified DNA to electrophoresis. Numbers at the side of the gel denote the position to which a linear DNA of the indicated size would migrate. What does this gel demonstrate about chromatin structure? Why are the DNA bands thick and spread out rather than sharply defined?
10. DNA Structure Explain how the underwinding of a B-DNA helix might facilitate or stabilize the formation of Z-DNA (see Fig. 8-17). 11. Maintaining DNA Structure a. Describe two structural features required for a DNA molecule to maintain a negatively supercoiled state. b. List three structural changes that become more favorable when a DNA molecule is negatively supercoiled. c. What E. coli enzyme, with the aid of ATP, can generate negative superhelicity in DNA? d. Describe the physical mechanism by which this enzyme acts. 12. Yeast Artificial Chromosomes (YACs) Researchers use YACs to clone large pieces of DNA in yeast cells. What three types of DNA sequence do researchers require to ensure proper replication and propagation of a YAC in a yeast cell? 13. Nucleoid Structure in Bacteria In bacteria, DNA topology affects the transcription of a subset of genes, with expression increasing or (more o en) decreasing when the DNA is relaxed. Following cleavage of a bacterial chromosome at a specific site by a restriction enzyme (one that cuts at a long, and thus rare, sequence), only nearby genes (within 10,000 bp) exhibit either an increase or a decrease in expression. The transcription of genes elsewhere in the chromosome is unaffected. Explain. (Hint: See Fig. 24-35.) 14. DNA Topology When DNA is subjected to electrophoresis in an agarose gel, shorter molecules migrate faster than longer ones. Closed-circular DNAs of the same size but with different linking numbers also can be separated on an agarose gel: topoisomers that are more supercoiled, and thus more condensed, migrate faster through the gel. In the gel shown, purified plasmid DNA has migrated from top to bottom. There are two bands, with the faster band much more prominent.
a. What are the DNA species in the two bands? b. If topoisomerase I were added to a solution of this DNA, what would happen to the upper and lower bands a er electrophoresis? c. If DNA ligase were added to the DNA, would the appearance of the bands change? Explain your answer. d. If DNA gyrase plus ATP were added to the DNA a er the addition of DNA ligase, how would the band pattern change? 15. DNA Topoisomers When DNA is subjected to electrophoresis in an agarose gel, shorter molecules migrate faster than longer ones. Closed-circular DNAs of the same size but different linking number also can be separated on an agarose gel: topoisomers that are more supercoiled, and thus more condensed, migrate faster through the gel — from top to bottom in the two gels shown. An investigator added a dye, chloroquine, to these gels. Chloroquine intercalates between base pairs and stabilizes a more underwound DNA structure. When the dye binds to a relaxed closed-circular DNA, the DNA is underwound where the dye binds, and unbound regions take on positive supercoils to compensate. In the experiment shown here, an investigator used topoisomerases to make preparations of the same DNA circle with different superhelical densities (σ ). Completely relaxed DNA migrated to the position labeled N (for nicked), and highly supercoiled DNA (above the limit where individual topoisomers can be distinguished) migrated to the position labeled X. a. In gel A, why does the σ= 0 lane (i.e., DNA prepared so that σ= 0, on average) have multiple bands? b. In gel B, is the DNA from the σ= 0 preparation positively or negatively supercoiled in the presence of the intercalating dye? c. In both gels, the σ=−0.115 lane has two bands, one a highly supercoiled DNA and one relaxed. Propose a reason for the presence of relaxed DNA in these lanes (and others). d. The native DNA (le most lane in each gel) is the same DNA circle isolated from bacterial cells and untreated. What is the approximate superhelical density of this native DNA? 16. Nucleosomes The human genome comprises just over 3.1 billion base pairs. Assuming it contains nucleosomes that are spaced as described in this chapter, how many molecules of histone H2A are present in one somatic human cell? (Ignore reductions in H2A due to its replacement in some regions by H2A variants.) How would the number change a er DNA replication but before cell division? 17. Bacterial DNA Topoisomerase IV The gene encoding topoisomerase IV in E. coli is essential, even though another type II topoisomerase (topoisomerase II or gyrase) is present. Suggest a reason for the requirement for topoisomerase IV. 18. Chromosome Topology Eukaryotic chromosomes are linear DNA molecules, yet the DNA of a chromosome retains a high level of underwinding (supercoiling) throughout its length. How does the organization of chromosomal DNA into loops called TADs contribute to the maintenance of supercoiling? DATA ANALYSIS PROBLEM 19. Defining the Functional Elements of Yeast Chromosomes Figure 24-7 shows the major structural elements of a chromosome of budding yeast (S. cerevisiae). Heiter, Mann, Snyder, and Davis (1985) determined the properties of some of these elements. They based their study on the finding that in yeast cells, plasmids (which have genes and an origin of replication) act differently from chromosomes (which have these elements plus centromeres and telomeres) during mitosis. The plasmids are not manipulated by the mitotic apparatus and segregate randomly between daughter cells. Without a selectable marker to force the host cells to retain them (see Fig. 9-4), these plasmids are rapidly lost. In contrast, chromosomes, even without a selectable marker, are manipulated by the mitotic apparatus and are lost at a very low frequency (about 10−5 per cell division). Heiter and colleagues set out to determine the important components of yeast chromosomes by constructing plasmids with various parts of chromosomes and observing whether these “synthetic chromosomes” segregated properly during mitosis. To measure the frequencies of different types of failed chromosome segregation, the researchers needed a rapid assay to determine the number of copies of synthetic chromosomes present in different cells. The assay took advantage of the fact that wild-type yeast colonies are white whereas certain adenine-requiring (ade–) mutants yield red colonies on nutrient media; ade2− cells lack functional AIR carboxylase (the enzyme of step in Fig. 22-35) and accumulate AIR (5-aminoimidazole ribonucleotide) in their cytoplasm, and the excess AIR is converted to a conspicuous red pigment. The other part of the assay involved the gene SUP11, which encodes an ochre suppressor (a type of nonsense suppressor in which a termination codon specifies an amino acid) that suppresses the phenotype of some ade2− mutants. Heiter and coworkers started with a diploid strain of yeast homozygous for ade2−; these cells are red. When the mutant cells contain one copy of SUP11, the metabolic defect is partly suppressed and the cells are pink. When the cells contain two or more copies of SUP11, the defect is completely suppressed and the cells are white. The researchers inserted one copy of SUP11 into synthetic chromosomes containing various elements thought to be important in chromosome function, and then observed how well these chromosomes were passed from one generation to the next. These pink cells were plated on nonselective media, and the behavior of the synthetic chromosomes was observed. Heiter and coworkers looked for colonies in which the synthetic chromosomes segregated improperly at the first division a er plating, giving rise to a colony that was half one genotype and half the other. Because yeast cells are nonmotile, this would be a sectored colony, with one half one color and the other half another color. a. One way for the mitotic process to fail is nondisjunction: the chromosome replicates but the sister chromatids fail to separate, so both copies of the chromosome end up in the same daughter cell. Explain how nondisjunction of the synthetic chromosome would give rise to a colony that is half red and half white. b. Another way for the mitotic process to fail is chromosome loss: the chromosome does not enter the daughter nucleus or is not replicated. Explain how loss of the synthetic chromosome would give rise to a colony that is half red and half pink. By counting the frequency of the different colony types, Heiter and colleagues could estimate the frequency of these aberrant mitotic events with different types of synthetic chromosome. First, they explored the requirement for centromeric sequences by constructing synthetic chromosomes with DNA fragments of different sizes containing a known centromere. Their results are shown here. Synthetic chromosome Size of centromere- containing fragment (kbp) Chromosome loss (%) Nondisjunction (%) 1 None — >50 2 0.63 1.6 1.1 3 1.6 1.9 0.4 4 3.0 1.7 0.35 5 6.0 1.6 0.35 c. Based on these data, what can you conclude about the size of the centromere required for normal mitotic segregation? Explain your reasoning. d. All the synthetic chromosomes created in these experiments were circular and lacked telomeres. Explain how they could be replicated more-or-less properly. Heiter and colleagues next constructed a series of linear synthetic chromosomes that included the functional centromeric sequence and telomeres, and they measured the total mitotic error frequency (% loss + % nondisjunction) as a function of size. Synthetic chromosome Size (kbp) Total error frequency (%) 6 15 11.0 7 55 1.5 8 95 0.44 9 137 0.14 e. Based on these data, what can you conclude about the chromosome size required for normal mitotic segregation? Explain your reasoning. f. Normal yeast chromosomes are linear, range from 250 kbp to 2,000 kbp in length, and, as noted above, have a mitotic error frequency of about 10−5 per cell division. Extrapolating the results from (e), do the centromeric and telomeric sequences used in these experiments explain the mitotic stability of normal yeast chromosomes, or must other elements be involved? Explain your reasoning. (Hint: A plot of log of error frequency vs. length will be helpful.) Reference Heiter, P., C. Mann, M. Snyder, and R.W. Davis. 1985. Mitotic stability of yeast chromosomes: A colony color assay that measures nondisjunction and chromosome loss. Cell 40:381– 392.
Stems are from the chapter Problems section; correct choices are drawn from Abbreviated Solutions to Problems (Appendix B) in the same edition.
1. Packaging of DNA in a Virus Bacteriophage T2 has a DNA of molecular weight 120× 106 contained in a head about 210 nm long. Calculate the length of the DNA and compare it with the length of the T2 head. Assume the molecular weight of a nucleotide base pair is 650 and that the DNA is in the B form and relaxed.
2. The DNA of Phage M13 The base composition of the DNA within a bacteriophage M13 viral particle is A, 23%; T, 36%; G, 21%; C, 20%. What does this tell you about this DNA molecule?
3. The Mycoplasma Genome The complete genome of the simplest bacterium known, Mycoplasma genitalium, is a circular DNA molecule with 580,070 bp. Calculate the molecular weight and contour length (when relaxed) of this molecule. What is Lk0 for the Mycoplasma chromosome? If σ=−0.06, what is Lk?
4. Size of Eukaryotic Genes An enzyme isolated from rat liver has 192 amino acid residues and is encoded by a gene with 1,440 bp. Explain the relationship between the number of amino acid residues in the enzyme and the number of nucleotide pairs in its gene.
5. Linking Number A closed-circular DNA molecule in its relaxed form has an Lk of 500. Approximately how many base pairs are in this DNA? How is Lk altered (increases, decreases, doesn’t change, becomes undefined) when a. a protein complex binds to form a nucleosome, b. one DNA strand is broken, c. DNA gyrase and ATP are added to the DNA solution, or d. the double helix is denatured by heat?
6. DNA Topology In the presence of a eukaryotic condensin and a bacterial type II topoisomerase, the Lk of a relaxed closed-circular DNA molecule does not change. However, the DNA becomes highly knotted. Formation of the knots requires breakage of the DNA, passage of a segment of DNA through the break, and religation by the topoisomerase. Given that every reaction of the topoisomerase would be expected to result in a change in linking number, how can Lk remain the same?
7. Superhelical Density Bacteriophage λ infects E. coli by integrating its DNA into the bacterial chromosome. The success of this recombination depends on the topology of the E. coli DNA. When the superhelical density (σ ) of the E. coli DNA is greater than −0.045, the probability of integration is less than 20%; when σ is less than −0.06, the probability is >70%. Plasmid DNA isolated from an E. coli culture is found to have a length of 13,800 bp and an Lk of 1,222. Calculate σ for this DNA and predict the likelihood that bacteriophage λ will be able to infect this culture.
8. Altering Linking Number a. What is the Lk of a 5,000 bp circular duplex DNA molecule with a nick in one strand? b. What is the Lk of the molecule in (a) when the nick is sealed (relaxed)? c. How would the Lk of the molecule in (b) be affected by the action of a single molecule of E. coli topoisomerase I? d. What is the Lk of the molecule in (b) a er eight enzymatic turnovers by a single molecule of DNA gyrase in the presence of ATP? e. What is the Lk of the molecule in (d) a er four enzymatic turnovers by a single molecule of bacterial type I topoisomerase? f. What is the Lk of the molecule in (d) a er binding of one nucleosome core?
9. Chromatin The agarose gel shown, in which the thick bands represent DNA, helped researchers define nucleosome structure. They generated this result by briefly treating chromatin with an enzyme that degrades DNA, then removing all protein and subjecting the purified DNA to electrophoresis. Numbers at the side of the gel denote the position to which a linear DNA of the indicated size would migrate. What does this gel demonstrate about chromatin structure? Why are the DNA bands thick and spread out rather than sharply defined?
10. DNA Structure Explain how the underwinding of a B-DNA helix might facilitate or stabilize the formation of Z-DNA (see Fig. 8-17).
11. Maintaining DNA Structure a. Describe two structural features required for a DNA molecule to maintain a negatively supercoiled state. b. List three structural changes that become more favorable when a DNA molecule is negatively supercoiled. c. What E. coli enzyme, with the aid of ATP, can generate negative superhelicity in DNA? d. Describe the physical mechanism by which this enzyme acts.
12. Yeast Artificial Chromosomes (YACs) Researchers use YACs to clone large pieces of DNA in yeast cells. What three types of DNA sequence do researchers require to ensure proper replication and propagation of a YAC in a yeast cell?
13. Nucleoid Structure in Bacteria In bacteria, DNA topology affects the transcription of a subset of genes, with expression increasing or (more o en) decreasing when the DNA is relaxed. Following cleavage of a bacterial chromosome at a specific site by a restriction enzyme (one that cuts at a long, and thus rare, sequence), only nearby genes (within 10,000 bp) exhibit either an increase or a decrease in expression. The transcription of genes elsewhere in the chromosome is unaffected. Explain. (Hint: See Fig. 24-35.)
14. DNA Topology When DNA is subjected to electrophoresis in an agarose gel, shorter molecules migrate faster than longer ones. Closed-circular DNAs of the same size but with different linking numbers also can be separated on an agarose gel: topoisomers that are more supercoiled, and thus more condensed, migrate faster through the gel. In the gel shown, purified plasmid DNA has migrated from top to bottom. There are two bands, with the faster band much more prominent. a. What are the DNA species in the two bands? b. If topoisomerase I were added to a solution of this DNA, what would happen to the upper and lower bands a er electrophoresis? c. If DNA ligase were added to the DNA, would the appearance of the bands change? Explain your answer. d. If DNA gyrase plus ATP were added to the DNA a er the addition of DNA ligase, how would the band pattern change?
15. DNA Topoisomers When DNA is subjected to electrophoresis in an agarose gel, shorter molecules migrate faster than longer ones. Closed-circular DNAs of the same size but different linking number also can be separated on an agarose gel: topoisomers that are more supercoiled, and thus more condensed, migrate faster through the gel — from top to bottom in the two gels shown. An investigator added a dye, chloroquine, to these gels. Chloroquine intercalates between base pairs and stabilizes a more underwound DNA structure. When the dye binds to a relaxed closed-circular DNA, the DNA is underwound where the dye binds, and unbound regions take on positive supercoils to compensate. In the experiment shown here, an investigator used topoisomerases to make preparations of the same DNA circle with different superhelical densities (σ ). Completely relaxed DNA migrated to the position labeled N (for nicked), and highly supercoiled DNA (above the limit where individual topoisomers can be distinguished) migrated to the position labeled X. a. In gel A, why does the σ= 0 lane (i.e., DNA prepared so that σ= 0, on average) have multiple bands? b. In gel B, is the DNA from the σ= 0 preparation positively or negatively supercoiled in the presence of the intercalating dye? c. In both gels, the σ=−0.115 lane has two bands, one a highly supercoiled DNA and one relaxed. Propose a reason for the presence of relaxed DNA in these lanes (and others). d. The native DNA (le most lane in each gel) is the same DNA circle isolated from bacterial cells and untreated. What is the approximate superhelical density of this native DNA?
16. Nucleosomes The human genome comprises just over 3.1 billion base pairs. Assuming it contains nucleosomes that are spaced as described in this chapter, how many molecules of histone H2A are present in one somatic human cell? (Ignore reductions in H2A due to its replacement in some regions by H2A variants.) How would the number change a er DNA replication but before cell division?
17. Bacterial DNA Topoisomerase IV The gene encoding topoisomerase IV in E. coli is essential, even though another type II topoisomerase (topoisomerase II or gyrase) is present. Suggest a reason for the requirement for topoisomerase IV.
18. Chromosome Topology Eukaryotic chromosomes are linear DNA molecules, yet the DNA of a chromosome retains a high level of underwinding (supercoiling) throughout its length. How does the organization of chromosomal DNA into loops called TADs contribute to the maintenance of supercoiling? DATA ANALYSIS PROBLEM
19. Defining the Functional Elements of Yeast Chromosomes Figure 24-7 shows the major structural elements of a chromosome of budding yeast (S. cerevisiae). Heiter, Mann, Snyder, and Davis (1985) determined the properties of some of these elements. They based their study on the finding that in yeast cells, plasmids (which have genes and an origin of replication) act differently from chromosomes (which have these elements plus centromeres and telomeres) during mitosis. The plasmids are not manipulated by the mitotic apparatus and segregate randomly between daughter cells. Without a selectable marker to force the host cells to retain them (see Fig. 9-4), these plasmids are rapidly lost. In contrast, chromosomes, even without a selectable marker, are manipulated by the mitotic apparatus and are lost at a very low frequency (about 10−5 per cell division). Heiter and colleagues set out to determine the important components of yeast chromosomes by constructing plasmids with various parts of chromosomes and observing whether these “synthetic chromosomes” segregated properly during mitosis. To measure the frequencies of different types of failed chromosome segregation, the researchers needed a rapid assay to determine the number of copies of synthetic chromosomes present in different cells. The assay took advantage of the fact that wild-type yeast colonies are white whereas certain adenine-requiring (ade–) mutants yield red colonies on nutrient media; ade2− cells lack functional AIR carboxylase (the enzyme of step in Fig. 22-35) and accumulate AIR (5-aminoimidazole ribonucleotide) in their cytoplasm, and the excess AIR is converted to a conspicuous red pigment. The other part of the assay involved the gene SUP11, which encodes an ochre suppressor (a type of nonsense suppressor in which a termination codon specifies an amino acid) that suppresses the phenotype of some ade2− mutants. Heiter and coworkers started with a diploid strain of yeast homozygous for ade2−; these cells are red. When the mutant cells contain one copy of SUP11, the metabolic defect is partly suppressed and the cells are pink. When the cells contain two or more copies of SUP11, the defect is completely suppressed and the cells are white. The researchers inserted one copy of SUP11 into synthetic chromosomes containing various elements thought to be important in chromosome function, and then observed how well these chromosomes were passed from one generation to the next. These pink cells were plated on nonselective media, and the behavior of the synthetic chromosomes was observed. Heiter and coworkers looked for colonies in which the synthetic chromosomes segregated improperly at the first division a er plating, giving rise to a colony that was half one genotype and half the other. Because yeast cells are nonmotile, this would be a sectored colony, with one half one color and the other half another color. a. One way for the mitotic process to fail is nondisjunction: the chromosome replicates but the sister chromatids fail to separate, so both copies of the chromosome end up in the same daughter cell. Explain how nondisjunction of the synthetic chromosome would give rise to a colony that is half red and half white. b. Another way for the mitotic process to fail is chromosome loss: the chromosome does not enter the daughter nucleus or is not replicated. Explain how loss of the synthetic chromosome would give rise to a colony that is half red and half pink. By counting the frequency of the different colony types, Heiter and colleagues could estimate the frequency of these aberrant mitotic events with different types of synthetic chromosome. First, they explored the requirement for centromeric sequences by constructing synthetic chromosomes with DNA fragments of different sizes containing a known centromere. Their results are shown here. Synthetic chromosome Size of centromere- containing fragment (kbp) Chromosome loss (%) Nondisjunction (%) 1 None — >50 2 0.63 1.6 1.1 3 1.6 1.9 0
20. Packaging of DNA in a Virus Bacteriophage T2 has a DNA of molecular weight 120× 106 contained in a head about 210 nm long. Calculate the length of the DNA and compare it with the length of the T2 head. Assume the molecular weight of a nucleotide base pair is 650 and that the DNA is in the B form and relaxed.
21. The DNA of Phage M13 The base composition of the DNA within a bacteriophage M13 viral particle is A, 23%; T, 36%; G, 21%; C, 20%. What does this tell you about this DNA molecule?
22. The Mycoplasma Genome The complete genome of the simplest bacterium known, Mycoplasma genitalium, is a circular DNA molecule with 580,070 bp. Calculate the molecular weight and contour length (when relaxed) of this molecule. What is Lk0 for the Mycoplasma chromosome? If σ=−0.06, what is Lk?
23. Size of Eukaryotic Genes An enzyme isolated from rat liver has 192 amino acid residues and is encoded by a gene with 1,440 bp. Explain the relationship between the number of amino acid residues in the enzyme and the number of nucleotide pairs in its gene.
24. Linking Number A closed-circular DNA molecule in its relaxed form has an Lk of 500. Approximately how many base pairs are in this DNA? How is Lk altered (increases, decreases, doesn’t change, becomes undefined) when a. a protein complex binds to form a nucleosome, b. one DNA strand is broken, c. DNA gyrase and ATP are added to the DNA solution, or d. the double helix is denatured by heat?
25. DNA Topology In the presence of a eukaryotic condensin and a bacterial type II topoisomerase, the Lk of a relaxed closed-circular DNA molecule does not change. However, the DNA becomes highly knotted. Formation of the knots requires breakage of the DNA, passage of a segment of DNA through the break, and religation by the topoisomerase. Given that every reaction of the topoisomerase would be expected to result in a change in linking number, how can Lk remain the same?