Hartl, Jones - Genetics. Principlers and analysis - 1998 (522927), страница 62
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How they work is suggested in Figure 5.13, which shows thestructure of part of the topoisomerase I enzyme from E. coli when it is in contact with duplex DNA. Note that themolecule is wrapped completely around the DNA molecule, which passes through the enzyme like a train througha tunnel.The position along a molecule at which DNA replication begins is called a replication origin, and the region inwhich parental strands are separating and new strands are being synthesized is called a replication fork. Theprocess of generating a new replication fork is initiation.
In some organisms, replication is initiated at manypositions; in others, the origin of replication may be a unique site. In most bacteria, bacteriophage, and viruses,DNA replication is initiated at a unique origin of replication. Furthermore, with only a few exceptions, tworeplication forks move in opposite directions from the origin (FigureFigure 5.10Autoradiogram of the intact replicating chromosome of an E.
coli cell thathas grown in a medium containing 3H-thymine for slightly lessthan two generations. The continuous lines of dark grains were produced byelectrons emitted by decaying 3H atoms in the DNA molecule.The pattern is seen by light microscopy. [From J. Cairns, Cold Spring Harbor Symp. Quant. Biol. 1963. 28:44].Figure 5.11Electron micrograph of a small circular DNA molecule replicating by the θ mode.The parental and daughter segments are shown in the drawing.[Electron micrograph courtesy of Donald Helinski.]Page 188Figure 5.12Replication of a circular DNA molecule. (A) The unwinding motion of the branches of a replicating circle, without positionsat which free rotation can occur, causes overwinding of the unreplicated part.
(B) Mechanism by which a single-strand break (a nick)ahead of a replication fork allows rotation.Figure 5.13Proposed structure of an association between duplex DNA and topoisomerase I in which theDNA passes through a hole formed in the enzyme. The two views are perpendicular to eachother. The structure of the enzyme includes a pair of jaw-like projections that can open andclose. The intermediate shown here is created when the enzyme closes its jaws around a DNAmolecule, completely enclosing it.
What happens then is that one DNA strand is cleaved,swiveled, and reconnected, whereupon the jaws open and the DNA molecule is released.[Courtesy of C. D. Lima, J. C. Wang, and A. Móndragon. From C. D. Lima, J. C. Wang, and A.Móndragon, Nature 1994. 367:138.]Page 189Figure 5.14The distinction between unidirectional and bidirectional DNA replication. In unidirectional replication, there is onlyone replication fork; bidirectional replication requires two replication forks. The curved arrows indicatethe direction of movement of the forks. Most DNA replicates bidirectionally.5.14); that is, DNA almost always replicates bidirectionally in both prokaryotes and eukaryotes.Multiple Origins of Replication in Eukaryotes The DNA duplex in a eukaryotic chromosome is linear andreplicates bidirectionally.
Furthermore, replication is initiated at many sites in the DNA. The structures that resultfrom the numerous origins are seen in electron micrographs as multiple loops along a DNA molecule (Figure 5.15).Multiple initiation is a means of reducing the total replication time of a large molecule. In eukaryotic cells,movement of each replication fork proceeds at a rate of approximately 10 to 100 nucleotide pairs per second. Forexample, in D. melanogaster, the rate of replication is about 50 nucleotide pairs per second at 25°C.
Because theDNA molecule in the largest chromosome in Drosophila contains about 7 × 107 nucleotide pairs, replication from asingle bidirectional origin of replication would take about 8 days. Developing Drosophila embryos actually useabout 8500 replication origins per chromosome, which reduces the replication time to a few minutes.
In a typicaleukaryotic cell, origins are spaced about 40,000 nucleotide pairs apart, which allows each chromosome to replicatein 15 to 30 minutes. Because chromosomes do not all replicate simultaneously, complete replication of allchromosomes in eukaryotes usually takes from 5 to 10 hours.DNA replication is much faster in prokaryotes than in eukaryotes. In E. coli, for example, replication of a DNAmolecule takes place at a rate of approximately 1500 nucleotide pairs per second; similar rates are found in thereplication of all phage DNA molecules studied. Complete replication of the E. coli DNA molecule from its singlebidirectional origin of replication requires approximately 30 minutes because the molecule contains about 4.7 × 106nucleotide pairs. Yet under optimal culture conditions, an E.
coli cell can divide every 20 minutes. How is thispossible? It is possible because a new round of replication is started at the origin every 20 minutes, even though theprevious round of replication has not yet been completed. In a rapidly dividing culture of E. coli cells, the DNAmolecule passed to each daughter cell in division is already in the process of replicating in preparation for the nextdivision.Rolling-Circle Replication Some circular DNA molecules, including those of a number of bacterial andeukaryotic viruses, replicate by a process that does not include a θ-shaped intermediate.
This replication mode iscalled rolling-circle replication.Page 190Figure 5.15Replicating DNA of Drosophila melanogaster. (A) An electron micrograph of a 30,000-nucleotide-pair segmentshowing seven replication loops. (B) An interpretive drawing showing how loops merge. Two replication originsare shown in the drawing.
The small arrows indicate the direction of movement of the replication forks.[Electron micrograph courtesy of David Hogness.]In this process, replication starts with a cut at a specific sugar-phosphate bond in a double-stranded circle (Figure5.16). This cut produces two chemically distinct ends: a 3' end (at which the nucleotide has a free 3'-OH group) anda 5' end (at which the nucleotide has a free 5'-P group). The DNA is synthesized by the addition ofdeoxynucleotides to the 3' end with simultaneous displacement of the 5' end from the circle. As replicationproceeds around the circle, the 5' end rolls out as a tail of increasing length.In most cases, as the tail is extended, a complementary chain is synthesized, which results in a double-strandedDNAPage 191Figure 5.16Rolling-circle replication.
Newly synthesized DNA is in red. The displaced strand is replicated in short fragments, asexplained in Section 5.6.tail. Because the displaced strand is chemically linked to the newly synthesized DNA in the circle, replication doesnot terminate, and extension proceeds without interruption, forming a tail that may be many times longer than thecircumference of the circle.
Rolling-circle replication is a common feature in late stages of replication of doublestranded DNA phages that have circular intermediates. An important example of rolling-circle replication will alsobe seen in Chapter 8, where matings between donor and recipient E. coli cells are described.So far, we have considered only certain geometrical features of DNA replication. In the next section, the enzymesand protein factors used in DNA replication ar described.5.5—DNA SynthesisNucleic acids are synthesized in chemical reactions controlled by enzymes, as is the case with most metabolicreactions in living cells.
An enzyme that forms the sugar-phosphate bond (the phosphodiester bond) betweenadjacent nucleotides in a nucleic acid chain is called a DNA polymerase. A variety of DNA polymerases havebeen purified, and DNA synthesis has been carried out in vitro in a cell-free system prepared by disrupting cellsand combining purified components in a test tube under precisely defined conditions.Three principal requirements must be met for DNA polymerases to catalyze synthesis of DNA.• The 5'-triphosphates of the four deoxynucleosides must be present. These are the compounds denoted in Table 5.1as dATP, dGTP, dTTP, and dCTP, which contain the bases adenine, guanine, thymine, and cytosine, respectively.Details of the structures of dCTP and dGTP are shown in Figure 5.17, in which the phosphate groups cleaved offduring DNA synthesis are indicated. DNA synthesis requires all four nucleoside 5'-triphosphates and does not takeplace if any of them is omitted.• A preexisting single strand of DNA to be replicated must be present.
Such a strand is called a template strand.• A nucleic acid segment, which may be very short, must be present and must be hydrogen-bonded to the templatestrand. This segment is called a primer. No known DNA polymerase is able to initiate chains, so the presence of aprimer chain with a free 3'-OH group is absolutely essential for the initiation of replication. In living cells, theprimer isPage 192Figure 5.17Two deoxynucleoside triphosphates used in DNA synthesis. The outertwo phosphate groups are removed during synthesis.a short segment of RNA; in cell-free replication in vitro, the primer may be either RNA or DNA.The reaction catalyzed by the DNA polymerases is the formation of a phosphodiester bond between the free 3'-OHgroup of the chain being extended and the innermost phosphorus atom of the nucleoside triphosphate beingincorporated at the 3' end (Figure 5.18).
The result is as follows:DNA synthesis proceeds by the elongation of primer chains, always in the 5' to 3' direction.Recognition of the appropriate incoming nucleoside triphosphate in replication depends on base pairing with theopposite nucleotide in the template chain. DNA polymerase will usually catalyze the polymerization reaction thatincorporates the new nucleotide at the primer terminus only when the correct base pair is present. They same DNApolymerase is used to add each of the four deoxynucleoside phosphates to the 3'-OH terminus of the growingstrand.Two DNA polymerases are needed for DNA replication in E. coli: DNA polymerase I, which is often written PolI, and DNA polymerase III (Pol III). Polymerase III is the major replication enzyme.
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