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The type 2 enzymes in eukaryotic cellscannot underwind DNA (introduce negative supercoils), although bothtypes can relax both positive and negative supercoils. We will considerone probable origin of negative supercoils in eukaryotic cells in ourdiscussion of chromatin.Relaxed DNA —ALk = - 2HighlysupercoiledDNAFigure 23-20 E. coli topoisomerase II (DNA gyrase) alters the linking number of circular DNAmolecules by an unusual mechanism. Two regionsof a DNA molecule are overlaid in a specific configuration in the bound complex (a positive ( + ) node).A compensating (—) node forms spontaneously elsewhere in the DNA molecule.
As shown, bothstrands of one DNA segment are broken, the othersegment is passed through the break, and thebreak is then resealed. The product now containstwo minus nodes, and a comparison with Fig.23-16 shows that the DNA now contains two negative supercoils. The change in structure reflects achange in Lk of - 2 .Figure 23-19 Circular DNA molecules that differin linking number can be separated by gel electrophoresis. All the DNA molecules shown here havethe same number of base pairs. Because supercoiled DNA molecules are more compact, they migrate more rapidly in a gel than the correspondingrelaxed molecules. Gels such as those shown hereseparate topoisomers only over a limited range ofsuperhelical density, so that highly supercoiledDNA migrates in a single band (lane 1) eventhough many different topoisomers may be present.Lanes 2 and 3 illustrate the effect of treating thesupercoiled DNA with a type I topoisomerase (theDNA in lane 3 was treated for a longer time thanthat in lane 2).DNA Compaction Requires a Special Form of SupercoilingSupercoiled DNA molecules are remarkably uniform in many respects;the characteristic form is illustrated in Figure 23-21.
Supercoils areright-handed in a negatively supercoiled DNA molecule (Fig. 23-16).Supercoiled DNA also tends to be extended and narrow rather thancompacted, and it often exhibits multiple branches. At superhelicaldensities normally encountered in cells, the length of the supercoilaxis, including branches, is about 40% the length of the DNA itself.This type of supercoiling is referred to as plectonemic (from the Greekplektos, "twisted," and nema, "thread") supercoiling.Figure 23-21 (a) An electron micrograph of plectonemically supercoiled plasmid DNA with (b) aninterpretation of the observed structure.
The bluelines define the axis of the supercoil. Note thebranching of this molecule, (c) An idealized representation of this structure.Supercoil axis805Part IV Information Pathways806(a)(b)Figure 23-22 (a) Plectonemic and solenoidalforms of supercoiling. Solenoidal negative supercoiling takes the form of tight left-handed turns aboutan imaginary tubelike structure. The two forms arereadily interconverted, although the solenoidal formis generally not observed unless certain proteinsare bound to the DNA. (b) Plectonemic and solenoidal supercoiling of the same DNA molecule, drawnto scale. Note that solenoidal supercoiling providesa much greater degree of compaction.Histone coreof nucleosomeLinker DNA(a)Although plectonemic coiling is the form observed in underwoundDNAs in solution, it does not give the compaction required to packageDNA in the cell. A second form of supercoiling, called solenoidal supercoiling (Fig. 23-22), can be adopted by an underwound DNA.
Instead of the extended right-handed supercoils characteristic of theplectonemic form, solenoidal supercoiling involves tighter, left-handedturns. The structure is similar to that taken up by a garden hose neatlywrapped on a reel. Although their structures are dramatically different, plectonemic and solenoidal supercoiling represent two forms ofnegative supercoiling that can be taken up by the same underwoundDNA. The two forms are readily interconvertible.
Although the plectonemic form is more stable in solution, the solenoidal form can bestabilized by protein binding and is the form found in chromatin. Itprovides a much greater degree of compaction (Fig. 23-22b). Solenoidal supercoiling explains how underwinding contributes to actual DNAcompaction.Chromatin and Nucleoid StructureThe term chromosome today refers to the nucleic acid molecule that isthe repository of the genetic information of a virus, a bacterium, aeukaryotic cell, or an organelle. But the word chromosome was originally used in another sense, to refer to the densely colored bodies ineukaryotic nuclei that can be visualized with the light microscope afterthe cells are stained with a dye.
Eukaryotic chromosomes, in the original sense of the word, appear as sharply defined bodies in the nucleusduring the period just before and during mitosis, the process of nucleardivision in somatic cells (see Fig. 2-14). In nondividing eukaryoticcells, the chromosomal material, called chromatin, is amorphous andappears to be randomly dispersed throughout the nucleus. But whenthe cells prepare to divide, the chromatin condenses and assemblesitself into a species-specific number of well-defined chromosomes (seeFig. 23-4).Chromatin has been isolated and analyzed.
It consists of fibersthat contain protein and DNA in approximately equal masses, plus asmall amount of RNA. The DNA in the chromatin is very tightly associated with proteins called histones, which package and order the DNAinto structural units called nucleosomes (Fig. 23-23). Also found inchromatin are many nonhistone proteins, some of which regulate theexpression of specific genes (Chapter 27). Beginning with nucleosomes,eukaryotic chromosomal DNA is packaged into a succession of higherorder structures that ultimately yield the compact chromosome seenwith the light microscope.
We now turn to a description of this structure in eukaryotes, and compare the DNA packaging in bacterial cells.Histones Are Small, Basic Proteins50 nmFigure 23-23 Regularly spaced nucleosomes,consisting of histone complexes bound to DNA.(a) Schematic illustration; (b) electron micrograph.Found in the chromatin of all eukaryotic cells, histones have molecularweights of between 11,000 and 21,000 and are very rich in the basicamino acids arginine and lysine (together these make up about onefourth of the amino acid residues). Five major classes of histones arefound in all eukaryotic cells, differing in molecular weight and aminoacid composition (Table 23-3).
The H3 histones are nearly identical inamino acid sequence in all eukaryotes, as are the H4 histones, suggesting strict conservation of their functions. Comparing the 102 aminoChapter 23 Genes and ChromosomesThe DNA molecules in chromosomes are the largest macromolecules in cells. Many smaller DNAsalso occur in cells, in the form of viral DNAs, plasmids, and (in eukaryotes) mitochondrial or chloroplast DNAs. Many DNAs, especially those in bacteria, mitochondria, and chloroplasts, are circular.Viral and chromosomal DNAs have one major feature in common: they are generally much longerthan the viral particles or cells in which they arepackaged. The total DNA content of a eukaryoticcell is much greater than that of a bacterial cell.Genes are segments of a chromosome that contain the information for a functional polypeptide orRNA molecule.
In addition to these structuralgenes, chromosomes contain a variety of regulatory sequences involved in replication, transcription, and other processes. In eukaryotic chromosomes, there are two important special-functionrepetitive DNA sequences: centromeres, which areattachment points for the mitotic spindle, and telomeres, which occur at the ends of the linear chromosomes. Many genes in eukaryotic cells, andoccasionally in bacteria, are interrupted by noncoding sequences called introns.
The coding segments separated by introns are called exons.Most cellular DNAs are supercoiled. Supercoiling is a manifestation of structural strain impartedby the underwinding of the DNA molecule. Underwinding is a decrease in the total number of helicalturns in the DNA relative to the relaxed or B form.To maintain an underwound state, DNA must be aclosed circle or be bound with protein.
Supercoilsresulting from underwinding are defined as negative supercoils. Underwinding is quantified by atopological parameter called linking number, Lk.The linking number of a relaxed, closed-circularDNA is used as a reference {Lk0) and is equal to thenumber of base pairs divided by 10.5. Underwinding is measured in terms of the specific linking difference or cr, which equals (Lk - Lko)/Lko. For cellular DNAs, a typically equals -0.05 to -0.07,which means that approximately 5 to 7% of thehelical turns in the DNA have been removed.
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