Hartl, Jones - Genetics. Principlers and analysis - 1998 (522927), страница 71
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If the DNA molecule in human chromosome 1 (thePage 224longest chromosome) were a cooked spaghetti noodle 1 mm in diameter, it would stretch for 25 miles; inchromosome condensation, this noodle is gathered together, coil upon coil, until at metaphase it is a canoe-sizedtangle of spaghetti 16 feet long and 2 feet wide. After cell division, the noodle is unwound again. Although thegenomes of prokaryotes and viruses are much smaller than those of eukaryotes, they also are very compact.
Forexample, an E. coli chromosome, which contains a DNA molecule about 1500 µm long, is contained in a cell about2 µm long and 1 µm in diameter.6.2—The Supercoiling of DNAThe DNA of prokaryotic and eukaryotic chromosomes is supercoiled, which means that segments of doublestranded DNA are twisted around one another, analogous to the manner in which a telephone cord can be twistedaround itself.
The geometry of supercoiling can be illustrated by a simple example. Consider first a linear duplexDNA molecule whose ends are joined in such a way that each strand forms a continuous circle. Such a DNAmolecule is called a covalent circle, and it is said to be relaxed if no twisting is present other than the helicaltwisting (Figure 6.1A). The individual polynucleotide strands of a relaxed circle form the usual right-handed(positive) helical structure with ten nucleotide pairs per turn of the helix. Suppose you were to cut one strand in arelaxed circle and unwind it one complete rotation of 360° so as to undo one complete turn of the double helix.When the ends were rejoined again, the result would be a circular helix that is "underwound." Because a DNAmolecule has a strong tendency to maintain its standard helical form with ten nucleotide pairs per turn, the circularmolecule would respond to the underwinding in one of two ways: (1) by forming regions with "bubbles" in whichthe bases are unpaired (Figure 6.1B) or (2) by twisting the circular molecule in the opposite sense from thedirection of under-Figure 6.1Different states of a covalent circle.
(A) A nonsupercoiled (relaxed) covalent circle with 36 helical turns.(B) An underwound covalent circle with only 32 helical turns.(C) The molecule in part B, but with four twists toeliminate the underwinding. (D) Electron micrograph showing nicked circular and supercoiled DNA of phagePM2. Note that no bases are unpaired in part C. In solution, parts B and C would be in equilibrium[Electron micrograph courtesy of K. G. Murti.]Page 225winding (Figure 6.1C). This twisting is called supercoiling, and a molecule with this sense of twisting isnegatively supercoiled.
Examples of supercoiled molecules are shown in Figure 6.1C and D. The two responses tounderwinding are not independent, and underwinding is usually accommodated by a combination of the twoprocesses: An underwound molecule contains some bubbles of unpaired bases and some supercoiling, with thesupercoiling predominating. Although supercoiling occasionally plays a role in the expression of some genes, theoverall biological function of supercoiling is unknown.Topoisomerase EnzymesThe negative supercoiling of natural DNA molecules is produced by DNA topoisomerase enzymes (Chapter 5).The class of enzymes denoted topoisomerase I cause a single-stranded nick by breaking a phosphodiester bond inthe backbone of one of the DNA strands; the nick produces a gap through which the intact DNA strand is passed,and then the nick is resealed. Depending on conditions, topoisomerase I enzymes can either increase or decreasethe amount of supercoiling.
As we saw in Section 5.4, topoisomerase I enzymes are essential to relieve the coilingproduced by separation of the DNA strands in DNA replication.A second class of topoisomerase enzymes is called topoisomerase II. These enzymes work by producing a doublestranded gap in one molecule through which another double-stranded molecule is passed. Therefore, topoisomeraseII enzymes are able to pass one DNA duplex entirely through another or to separate two circular DNA moleculesthat are interlocked.
The mechanism of action of topoisomerase II from the yeast Saccharomyces cerevisiae isillustrated in Figure 6.2. The molecular structure of the enzyme includes two sets of "jaws" set approximately atright angles (Figure 6.2A). The inner set clamps one of the duplexes, the outer set the other (Figure 6.2B and C).To allow the outer DNA molecule to pass completely through the inner one, the inner duplex is first cleaved, andthen the outer duplex is passed through the gap (Figure 6.2D and E).
After passage, the gap is repaired and bothmolecules are released (Figure 6.2F and G).In a supercoiled DNA molecule free of proteins that maintain the supercoiling, any nick eliminates all supercoilingbecause the strain of underwinding is relaxed by free rotation of the intact strand about the sugar-phosphate bondopposite the break. Therefore, any treatment that nicks DNA relaxes the supercoiling. Single-stranded nicks can beproduced by any of a variety of enzymes, such as deoxyribonuclease (DNase), that cleaves sugar-phosphatebonds.6.3—The Structure of the Bacterial ChromosomeThe chromosome of E.
coli is a condensed unit called a nucleoid or folded chromosome, which is composed of asingle circular DNA molecule and associated proteins. The term chromosome is a misnomer for this structure,because it is not a true "chromosome" in the sense of a eukaryotic chromosome. The most striking feature of thebacterial nucleoid is that the DNA is organized into a set of looped domains (Figure 6.3). As isolated from bacterialcells, the nucleoid contains, in addition to DNA, small amounts of several proteins, which are thought to beresponsible in some way for the multiply looped arrangement of the DNA. The degree of condensation of theisolated nucleoid (that is, its physical dimensions) is affected by a variety of factors, and some controversy existsabout the state of the nucleoid within the cell.Figure 6.3 also shows that loops of the DNA of the E.
coli chromosome are supercoiled. Note that some loops arenot supercoiled; this is a result of the action of DNases during isolation, and it indicates that the loops are in someway independent of one another. In the preceding section, we stated that supercoiling is generally eliminated in aDNA molecule by one single-strand break. However, such a break in the E. coli chromosome does notPage 226Figure 6.2Topoisomerase II untangles a pair of DNA molecules by cleaving one DNA duplex and passing the other duplex through thegap.
The enzyme illustraed here, from the yeast Saccharomyces cerevisiae, has two sets of "jaws." (A through C)The inner jaws (green) trap one duplex. The outer jaws (red) trap the other duplex. (D and E) The duplex in theinner jaws is cleaved, and the second, uncleaved duplex is passed through. (F) The uncleavedduplex is expelled through an opening that forms in the basal part of the enzyme. (G) Thenthe gap in the cleaved duplex is repaired,and it is released. [After J. M.
Berger, S. J. Gamblin, S. C. Harrisonand J. C. Wang. 1996. Nature 379:225.]Page 227Figure 6.3An electron micrograph of an E. coli chromosome showingthe multiple loops emerging from a central region.[Courtesy of Ruth Kavenoff. Copyright 1983 by Designergenes Posters Ltd.]eliminate all supercoiling. If nucleoids, all of whose loops are supercoiled, are treated with a DNase and examinedat various times after treatment, it is observed that supercoiling is relaxed in one loop at a time, not in all loops atonce (Figure 6.4).
The loops must be isolated from one another in such a way that rotation in one loop is nottransmitted to other loops. The independence is probably the result of proteins that bind to the DNA in a way thatprevents rotation of the helix.Figure 6.4A schematic drawing of the folded supercoiled E.
coli chromosome, showing 11 of the 40 to 50 loops attached toa protein core (blue shaded area) and the opening of loops by nicks.Page 2286.4—The Structure of Eukaryotic ChromosomesA eukaryotic chromosome contains a single DNA molecule of enormous length. For example, the largestchromosome in the D. melanogaster genome has a DNA content of about 65,000 kb (6.5 × 107 nucleotide pairs),which is equivalent to a continuous linear duplex about 22 mm long. These long molecules usually fracture duringisolation, but some fragments that are recovered are still very long.
Figure 6.5 is an autoradiograph of radioactivelylabeled Drosophila DNA more than 36,000 kb in length.In organisms such as baker's yeast, Saccharomyces cerevisiae, which have small genomes, the DNA moleculespresent in the chromosomes can be separated by special types of electrophoretic methods. In conventionalelectrophoresis, which we examined in Section 5.7, the electric field is maintained in a constant state, usually atconstant voltage. The fragments move inFigure 6.5Autoradiogram of a DNA molecule fromD. melanogaster.
The molecule is 12 mmlong (approximately 36,000 kb).[From R. Kavenoff, L. C. Klotz, and B. H. Zimm.1974. Cold Spring Harbor Symp. Quant.Biol., 38:4.]response to the field according to their size, smaller fragments moving faster. However, conventionalelectrophoresis can separate only molecules smaller than about 20 kb. All molecules larger than about 20 kb havethe same electrophoretic mobility under these conditions and so form a single band in the gel. Simple modificationsof the electrophoresis result in separation among much larger DNA fragments. The most common modificationsalter the geometry of the electric field at periodic intervals during the course of the electrophoretic separation.Some electrophoretic apparatuses alternate the electric field between two sets of electrodes oriented at right anglesor among three sets of electrodes forming a hexagon.
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