Human Biochemistry, Volume 2 - Murray R. 1993

Structure, Function, and Replication of Information Macromolecules
DNA Organization and Replication
Histones and Nucleosomes

The term "Histones" refers to a group of closely related basic Proteins. H1 histones are the most loosely bound to Chromatin and are readily extracted in a salt solution, a Treatment that renders chromatin soluble. Isolated nucleosome cores consist of four classes of histones: H2A, H2B, H3, and H4. The Structure of the moderately Lysine-rich histones H2A and H2B is notably conservative, while the structure of H3 and H4 (which are rich in Arginine) is even more conserved. The high evolutionary conservation of histone structure indicates that the Functions of these proteins are identical across all eukaryotes. The C-terminal region of their molecules has a conventional Amino Acid Composition, whereas the N-terminal third consists predominantly of basic Amino Acids. The four groups of histones listed above undergo five types of covalent modification: Acetylation, methylation, phosphorylation, ADP-ribosylation, and covalent attachment (in H2A only) to ubiquitin (a nuclear protein). These modifications likely influence chromatin Structure and function, although this question requires further study.

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Fig. 38.1. Electron micrograph of nucleosomes connected by a DNA strand; the white bar corresponds to 2.5 µm. (Reproduced with permission, from P. Oudet, M. Gross-Bellard, P. Chambon: Electron microscopic and biochemical evidence that Chromatin Structure is a repeating unit Cell 1975, 4: 281.)

Histones isolated from chromatin interact with one another. Histones H3 and H4 aggregate to form tetramers consisting of two molecules of each type (H3₂-H4₂). Histones H2A and H2B form either dimers (H2A-H2B) or oligomeric complexes [H2A-H2B]_n. The H3₂-H4₂ tetramer does not interact with the H2A-H2B dimer or oligomer. H1 histones do not associate with other histones in solution.

Interestingly, a mixture of H3₂-H4₂ and H2A-H2B with purified double-stranded DNA yields an X-Ray Diffraction pattern characteristic of freshly isolated chromatin. Cytology/cytology/93.html">ELECTRON MICROGRAPHS OF such preparations reveal newly formed nucleosomes. Moreover, the in vitro formation of nucleosomes from DNA and histones H2A, H2B, H3, and H4 is independent of the Organism or cell type from which the mixture's components were isolated. Neither H1 histones nor nonhistone proteins are required to form the nucleosome core.

Within nucleosomes, DNA is supercoiled around The surface of the disc-shaped histone octamer into a left-handed helix. The octamer comprises a central H3₂-H4₂ tetramer and two H2A-H2B dimers (Fig. 38.2). The histone core of the nucleosome interacts with the inner surface of the superhelix without protruding beyond its boundaries.

The H3₂-H4₂ tetramer is capable of conferring a nucleosome-like structure on DNA and thus plays a central role in its assembly. Two additional H2A-H2B dimers stabilize the primary particle and firmly bind the two DNA half-turns that were previously weakly associated with the H3₂-H4₂ tetramer. Consequently, 1.75 superturns of DNA wrap around the histone octamer to form the nucleosome core (or minimal nucleosome), which "masks" 146 Base Pairs of DNA (Fig. 38.2). Along the helical path around the octamer, the DNA contacts histones in the following order:

H2A-H2B-H4-H3-H3-H4-H2B-H2A.

Fig. 38.2. Model of the nucleosome structure (left) and the nucleosome core (right), showing DNA wrapped around a protein cylinder containing two molecules each of histones H2A, H2B, H3, and H4. Histone H1 (shaded area) extends the region of masked DNA sequences. (Reproduced, with permission, from Laskey R. A. and Earnshaw W.C.: Nucleosome assembly. Nature 1980, 286: 763.)

Histone H1 binds to the nucleosome core at the DNA entry and exit sites, "clamping" 2 turns—i.e., 166 base pairs of the DNA superhelix—thus forming the mature nucleosome.

Nucleoplasmin, an anionic nuclear protein, is believed to participate in nucleosome assembly. Since histones are strong cations, they can nonspecifically bind to negatively charged DNA through salt bridges. Clearly, such nonspecific interactions could hinder nucleosome formation and chromatin function. Nucleoplasmin is an anionic pentameric protein that does not bind to DNA or chromatin, but it can reversibly associate with the histone octamer, preventing histones from nonspecifically "sticking" to negatively charged structures like DNA. Nucleoplasmin apparently establishes a specific ionic environment within The Nucleus that facilitates histone-DNA interactions and nucleosome assembly. Once assembly is complete, nucleoplasmin dissociates from the histone complex. Nucleoplasmin exhibits selectivity for specific regions of DNA. The Molecular Basis of this non-random distribution, termed phasing, remains unknown; it may be related to the relative physical plasticity of certain nucleotide sequences capable of supercoiling.

The packing of nucleosomes within the nucleus apparently depends on the interaction of H1 histones with the stretches of double-stranded DNA connecting the nucleosomes. The topology of this interaction, which leads to The formation of internucleosomal spacer regions, is not yet fully understood.

In addition to nucleosomes, Electron Microscopy of chromatin has revealed two higher-order structures: 10 nm fibrils and 25–30 nm fibers. The disc-shaped nucleosomes (see above) have a diameter of 10 nm and a height of 5 nm. The 10 nm fibrils apparently consist of a series of nucleosomes touching edge-to-edge, with their flat surfaces oriented along the fibril axis (Fig. 38.3). These fibrils likely coil into a helix containing 6–7 nucleosomes per turn, resulting in the formation of a 30 nm chromatin fiber (Fig. 38.4). The turns of this "superhelix" must be relatively flat, with the flat surfaces of nucleosomes in adjacent turns oriented parallel to one another. H1 histones most likely stabilize the fiber structure, but their exact arrangement, as well as the length of the DNA spacer regions, remains undetermined. Nucleosomes likely form several other compact superstructures as well. To produce a mitotic chromosome of normal size, the 30 nm fiber must undergo additional compaction, reducing its resulting length by another 100-fold (see below).

In interphase Chromosomes, chromatin fibers are organized into domains or loops consisting of 30,000 to 100,000 base pairs, anchored to an intranuclear supporting matrix. The distribution of genomic regions within the domain structure of chromatin is presumably non-random. It can be hypothesized that each loop-forming chromatin domain contains both coding and non-coding regions of genes corresponding to a specific genetic function.

Fig. 38.3. Structure of a 10 nm chromatin fibril composed of disc-like nucleosomes. THE POSITION OF histone H1 is not shown.

Fig. 38.4. Structure of a 30 nm chromatin fiber composed of supercoiled 10 nm fibrils. The fiber axis is perpendicular to the page.

Active Chromatin

As a rule, every cell of a multicellular organism contains the same Genetic information in the form of identical DNA sequences. Consequently, differences between cell types in a given organism must be explained by the differential expression of this shared genetic information. Chromatin containing active genes (transcriptionally active chromatin) differs in several features from inactive chromatin. The nucleosomal structure of active chromatin is modified or, in particularly active regions, entirely absent. DNA in active chromatin contains long stretches (approximately 100,000 base pairs) sensitive to Nucleases (such as DNase I). Sensitivity to DNase I indicates transcriptional competence and in some cases correlates with the absence of 5-methyldeoxycytidine in the corresponding region of DNA.

Within large domains of active chromatin, short regions (100–300 NUCLEOTIDES) have been discovered that exhibit an even higher—by an order of magnitude—sensitivity to DNase I. These so-called hypersensitive sites apparently arise from conformational changes that create exceptionally favorable conditions for nuclease action on DNA. Such sites are typically located immediately upstream of active genes and may be caused by enhancer elements that stimulate METABOLISM/31.html">Transcription (see chapters 39 and 41). There is reason to believe that in many cases, a Gene's transcriptional activity is associated with the presence of a DNase-hypersensitive site immediately adjacent to THE START OF the gene. These sites likely ensure that the coding strand is accessible to proteins involved in transcription.

Electron microscopy of the interphase nucleus reveals that transcriptionally inactive chromatin (heterochromatin) is densely packed and therefore stains intensely. Regions of transcriptionally active chromatin (euchromatin) stain much more weakly. Overall, during the mammalian Cell Cycle (see below), euchromatin replicates earlier than heterochromatin.

There are two types of heterochromatin: constitutive heterochromatin and facultative heterochromatin. Constitutive heterochromatin is permanently condensed and therefore inactive. It is found in regions close to the centromeres and telomeres of chromosomes. Facultative heterochromatin is condensed at certain times and decondensed at others; it is actively transcribed and thus resembles euchromatin. Of the two X chromosomes in female mammals, one is virtually entirely transcriptionally inactive—i.e., it displays The properties of heterochromatin. However, during gametogenesis and early Embryogenesis, this heterochromatic X chromosome becomes transcriptionally active and consequently exhibits the properties of facultative heterochromatin.

Certain insect Cells, such as Chironomus, contain giant chromosomes formed As a result of the failure of daughter chromatids to separate after undergoing ~10 rounds of Replication. DNA copies lying side-by-side in precise alignment with their localized genes form a chromosome with a distinctly banded pattern of condensed and less dense chromatin.

Transcriptionally active regions of such polytene chromosomes are distinguished by particularly pronounced decondensation—they form so-called "puffs" which, as established, harbor transcription system Enzymes and serve as sites of RNA Synthesis (Fig. 38.5).

Chromosomes

In metaphase, mammalian chromosomes exhibit second-order twofold Symmetry and consist of identical sister chromatids joined at the centromere, the position of which is characteristic of each individual chromosome (Fig. 38.6). Each sister chromatid contains a single double-stranded DNA molecule. During interphase, the packaging of the DNA molecule is less dense than in metaphase. Metaphase chromosomes are transcriptionally inactive.

The human haploid genome consists of 3.5∙10⁹ base pairs and approximately 1.7∙10⁷ nucleosomes. Consequently, each of the 23 chromatids of the human haploid genome contains an average of 1.5∙10⁸ nucleotides within a single double-stranded DNA molecule. Thus, during the formation of a condensed metaphase chromosome, the linear dimension of each DNA molecule must be reduced 8,000-fold! In metaphase chromosomes, chromatin fibers (25–30 nm in length) also fold into a series of loop-like domains, the proximal regions of which are anchored to an intranuclear protein (non-histone) scaffold. The packing ratios characterizing each ordered Introduction/20.html">DNA Structure are presented in Table 38.1.

Fig. 38.5. Correlation between RNA polymerase II activity and RNA synthesis. Upon heat Shock (39° C, 30 min), a series of genes are activated in Chironomus tentans larvae. A. Distribution of RNA polymerase B (type II) along the length of the fourth chromosome from salivary gland cells. The enzyme was detected by indirect immunofluorescence using Antibodies against the polymerase. 5C and BR3 are specific segments of chromosome IV. Arrows indicate puffs. B. Autoradiograph of chromosome IV incubated with H³-uridine for RNA labeling. The distribution of immunofluorescence signals and autoradiographic grains along the chromosome coincides. (Reproduced, with permission, from Sass H. RNA polymerase B in polytene chromosomes. Cell 1982: 28: 274. Copyright 1982 by the Massachusetts Institute of Technology.)

The packaging of Nucleoproteins into chromatids occurs in a non-random manner, as evidenced by the characteristic banding pattern on chromosomes stained with quinacrine mustard or Giemsa (Fig. 38.7).

Table 38.1. Packing ratios for various types of superstructured DNA

The distribution of stained bands in chromosomes is highly reproducible across preparations from different individuals of the same species, yet varies markedly among chromosomes of different, even closely related, species. Consequently, the packaging of nucleoproteins into chromosomes in higher eukaryotes must depend in a specific manner on the species-specific Structural Features of the DNA molecules themselves.