B. Alberts, A. Johnson, J. Lewis и др. - Molecular Biology of The Cell (6th edition) (1120996), страница 92
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Themutation rate, approximately one nucleotide change per 1010 nucleotides each timethe DNA is replicated, is roughly the same for organisms as different as bacteria andhumans. Because of this remarkable accuracy, the sequence of the human genome(approximately 3.2 × 109 nucleotide pairs) is unchanged or changed by only a fewnucleotides each time a typical human cell divides. This allows most humans topass accurate genetic instructions from one generation to the next, and also to avoidthe changes in somatic cells that lead to cancer.DNA REPLICATION MECHANISMSAll organisms duplicate their DNA with extraordinary accuracy before each celldivision.
In this section, we explore how an elaborate “replication machine”achieves this accuracy, while duplicating DNA at rates as high as 1000 nucleotidesper second.Base-Pairing Underlies DNA Replication and DNA RepairAs introduced in Chapter 1, DNA templating is the mechanism the cell uses to copythe nucleotide sequence of one DNA strand into a complementary DNA sequence(Figure 5–2).
This process requires the separation of the DNA helix into two template strands, and entails the recognition of each nucleotide in the DNA templatestrands by a free (unpolymerized) complementary nucleotide. The separation oftemplate S strand5′S strand5′3′3′S′ strand5′3′3′new S′ strand5′new S strand5′3′parent DNA double helix3′template S′ strand5′Figure 5–2 The DNA double helix actsas a template for its own duplication.Because the nucleotide A will pairsuccessfully only with T, and G only withC, each strand of DNA can serve asa template to specify the sequence ofnucleotides in its complementary strand byDNA base-pairing. In this way, a doublehelical DNA molecule can be copiedprecisely.Chapter 5: DNA Replication, Repair, and Recombination240the DNA helix exposes the hydrogen-bond donor and acceptor groups on eachDNA base for base-pairing with the appropriate incoming free nucleotide, aligning it for its enzyme-catalyzed polymerization into a new DNA chain.The first nucleotide-polymerizing enzyme, DNA polymerase, was discoveredin 1957.
The free nucleotides that serve as substrates for this enzyme were foundto be deoxyribonucleoside triphosphates, and their polymerization into DNArequired a single-strand DNA template. Figure 5–3 and Figure 5–4 illustrate thestepwise mechanism of this reaction.The DNA Replication Fork Is AsymmetricalDuring DNA replication inside a cell, each of the two original DNA strands servesas a template for the formation of an entire new strand.
Because each of the twodaughters of a dividing cell inherits a new DNA double helix containing one original and one new strand (Figure 5–5), the DNA double helix is said to be replicated“semiconservatively.” How is this feat accomplished?3′ end of strandO5′ end of strandOO P O_OOH2CCH2OGOCO_P OOPRIMERSTRANDTEMPLATESTRANDOATOOH2CCH2OO P OO_O_P OOOHOGOOCH2OO_CO3′ end of strandO P O P O P O CH2 O___OOO_P OOpyrophosphateOHCH2OAOOincoming deoxyribonucleoside triphosphate_P OOCH2OOT_O P OO5′ end of strandFigure 5–3 The chemistry of DNA synthesis. The addition of a deoxyribonucleotide to the3ʹ end of a polynucleotide chain (the primer strand) is the fundamental reaction by which DNA issynthesized. As shown, base-pairing between an incoming deoxyribonucleoside triphosphate andan existing strand of DNA (the template strand) guides the formation of the new strand of DNAand causes it to have a complementary nucleotide sequence.
The way in which complementarynucleotides base-pair is shown in Figure 4–4.241DNA REPLICATION MECHANISMS5′ triphosphateprimerstrand5′OH+pyrophosphate3′HO3′templatestrand5′OH5′3′3′5′-to-3′direction ofchain growthOH5′(A)(B)templatestrand5′3′5′primerstrandDNApolymeraseCORRECTPOSITIONINGOF INCOMINGDEOXYRIBONUCLEOSIDETRIPHOSPHATENUCLEOTIDEINCORPORATIONFOLLOWED BY DNATRANSLOCATIONP(C)PFigure 5–4 DNA synthesis catalyzed by DNA polymerase. (A) DNA polymerase catalyzes the stepwise addition of adeoxyribonucleotide to the 3ʹ-OH end of a polynucleotide chain, the growing primer strand that is paired to an existing templatestrand.
The newly synthesized DNA strand therefore polymerizes in the 5ʹ-to-3ʹ direction as shown also in the previous figure.Because each incoming deoxyribonucleoside triphosphate must pair with the template strand to be recognized by the DNApolymerase, this strand determines which of the four possible deoxyribonucleotides (A, C, G, or T) will be added. The reactionis driven by a large, favorable free-energy change, caused by the release of pyrophosphate and its subsequent hydrolysisto two molecules of inorganic phosphate. (B) Structure of DNA polymerase complexed wth DNA (orange), as determinedby x-ray crystallography (Movie 5.1). The template DNA strand is the longer strand and the newly synthesized DNA is theshorter. (C) Schematic diagram of DNA polymerase, based on the structure in (B).
The proper base-pair geometry of a correctincoming deoxyribonucleoside triphosphate causes the polymerase to tighten around the base pair, thereby initiating theMBoC6 m5.04/5.04nucleotide addition reaction (middle diagram (C)). Dissociation of pyrophosphate relaxes the polymerase, allowing translocationof the DNA by one nucleotide so the active site of the polymerase is ready to receive the next deoxyribonucleoside triphosphate.Analyses carried out in the early 1960s on whole replicating chromosomesrevealed a localized region of replication that moves progressively along theparental DNA double helix.
Because of its Y-shaped structure, this active regionis called a replication fork (Figure 5–6). At the replication fork, a multienzymecomplex that contains the DNA polymerase synthesizes the DNA of both newdaughter strands.Initially, the simplest mechanism of DNA replication seemed to be the continuous growth of both new strands, nucleotide by nucleotide, at the replicationfork as it moves from one end of a DNA molecule to the other. But because ofthe antiparallel orientation of the two DNA strands in the DNA double helix (seeFigure 5–2), this mechanism would require one daughter strand to polymerizein the 5ʹ-to-3ʹ direction and the other in the 3ʹ-to-5ʹ direction.
Such a replicationfork would require two distinct types of DNA polymerase enzymes. However, asattractive as this model might be, the DNA polymerases at replication forks cansynthesize only in the 5ʹ-to-3ʹ direction.How, then, can a DNA strand grow in the 3ʹ-to-5ʹ direction? The answerwas first suggested by the results of an experiment performed in the late 1960s.Researchers added highly radioactive 3H-thymidine to dividing bacteria for afew seconds, so that only the most recently replicated DNA—that just behind thereplication fork—became radiolabeled.
This experiment revealed the transientexistence of pieces of DNA that were 1000–2000 nucleotides long, now commonlyknown as Okazaki fragments, at the growing replication fork. (Similar replication242Chapter 5: DNA Replication, Repair, and RecombinationFigure 5–5 The semiconservative nature of DNA replication. In a roundof replication, each of the two strands of DNA is used as a template for theformation of a complementary DNA strand. The original strands thereforeremain intact through many cell generations.intermediates were later found in eukaryotes, where they are only 100–200 nucleotides long.) The Okazaki fragments were shown to be polymerized only in the5ʹ-to-3ʹ chain direction and to be joined together after their synthesis to createlong DNA chains.A replication fork therefore has an asymmetric structure (Figure 5–7).The DNA daughter strand that is synthesized continuously is known as theleading strand.
Its synthesis slightly precedes the synthesis of the daughter strandthat is synthesized discontinuously, known as the lagging strand. For the laggingstrand, the direction of nucleotide polymerization is opposite to the overall direction of DNA chain growth. The synthesis of this strand by a discontinuous “backstitching” mechanism means that DNA replication requires only the 5ʹ-to-3ʹ typeof DNA polymerase.REPLICATIONREPLICATIONREPLICATIONThe High Fidelity of DNA Replication Requires SeveralProofreading MechanismsAs discussed above, the fidelity of copying DNA during replication is such that onlyabout one mistake occurs for every 1010 nucleotides copied. This fidelity is muchhigher than one would expect from the accuracy of complementary base-pairing.The standard complementary base pairs (see Figure 4–4) are not the only onespossible.
For example, with small changes in helix geometry, two hydrogen bondscan form between G and T in DNA. In addition, rare tautomeric forms of the fourDNA bases occur transiently in ratios of 1 part to 104 or 105. These forms mispairwithout a change in helix geometry: the rare tautomeric form of C pairs with Ainstead of G, for example.If the DNA polymerase did nothing special when a mispairing occurredbetween an incoming deoxyribonucleoside triphosphate and the DNA template,the wrong nucleotide would often be incorporated into the new DNA chain,producing frequent mutations. The high fidelity of DNA replication, however,depends not only on the initial base-pairing but also on several “proofreading”mechanisms that act sequentially to correct any initial mispairings that mighthave occurred.replicationforks1 µmMBoC6 m5.05/5.05Figure 5–6 Two replication forks movingin opposite directions on a circularchromosome.
An active zone of DNAreplication moves progressively alonga replicating DNA molecule, creating aY-shaped DNA structure known as areplication fork: the two arms of each Yare the two daughter DNA molecules,and the stem of the Y is the parental DNAhelix. In this diagram, parental strands areorange; newly synthesized strands are red.(Micrograph courtesy of Jerome Vinograd.)DNA REPLICATION MECHANISMS3′5′3′5′5′3′5′leading strand3′5′5′3′3′lagging strand withOkazaki fragments243most recentlysynthesizedDNA5′3′5′3′5′3′3′5′Figure 5–7 The structure of a DNA replication fork. Left, replication fork with newly synthesizedDNA in red and arrows indicating the 5ʹ-to-3ʹ direction of DNA synthesis.
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