B. Alberts, A. Johnson, J. Lewis и др. - Molecular Biology of The Cell (6th edition) (1120996), страница 93
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Because both daughterDNA strands are polymerized in the 5ʹ-to-3ʹ direction, the DNA synthesized on the lagging strandmust be made initially as a series of short DNA molecules, called Okazaki fragments, named afterthe scientist who discovered them. Right, the same fork a short time later. On the lagging strand,the Okazaki fragments are synthesized sequentially, with those nearest the fork being the mostrecently made.MBoC6 m5.07/5.07DNA polymerase performsthe first proofreading step just before a new nucleotide is covalently added to the growing chain. Our knowledge of this mechanismcomes from studies of several different DNA polymerases, including one produced by a bacterial virus, T7, that replicates inside E. coli. The correct nucleotidehas a higher affinity for the moving polymerase than does the incorrect nucleotide, because the correct pairing is more energetically favorable.
Moreover, afternucleotide binding, but before the nucleotide is covalently added to the growing chain, the enzyme must undergo a conformational change in which its “grip”tightens around the active site (see Figure 5–4). Because this change occurs morereadily with correct than incorrect base-pairing, it allows the polymerase to “double-check” the exact base-pair geometry before it catalyzes the addition of thenucleotide. Incorrectly paired nucleotides are harder to add and therefore morelikely to diffuse away before the polymerase can mistakenly add them.The next error-correcting reaction, known as exonucleolytic proofreading,takes place immediately after those rare instances in which an incorrect nucleotide is covalently added to the growing chain.
DNA polymerase enzymes arehighly discriminating in the types of DNA chains they will elongate: they requirea previously formed, base-paired 3ʹ-OH end of a primer strand (see Figure 5–4).Those DNA molecules with a mismatched (improperly base-paired) nucleotideat the 3ʹ-OH end of the primer strand are not effective as templates because thepolymerase has difficulty extending such a strand. DNA polymerase moleculescorrect such a mismatched primer strand by means of a separate catalytic site(either in a separate subunit or in a separate domain of the polymerase molecule,depending on the polymerase). This 3ʹ-to-5ʹ proofreading exonuclease clips off anyunpaired or mispaired residues at the primer terminus, continuing until enoughnucleotides have been removed to regenerate a correctly base-paired 3ʹ-OH terminus that can prime DNA synthesis.
In this way, DNA polymerase functions as a“self-correcting” enzyme that removes its own polymerization errors as it movesalong the DNA (Figure 5–8 and Figure 5–9).The self-correcting properties of the DNA polymerase depend on its requirement for a perfectly base-paired primer terminus, and it is apparently not possible for such an enzyme to start synthesis de novo, without an existing primer.By contrast, the RNA polymerase enzymes involved in gene transcription do notneed such an efficient exonucleolytic proofreading mechanism: errors in makingRNA are not passed on to the next generation, and the occasional defective RNAmolecule that is produced has no long-term significance.
RNA polymerases arethus able to start new polynucleotide chains without a primer.Figure 5–8 Exonucleolytic proofreading by DNA polymerase during DNAreplication. In this example, a C is accidentally incorporated at the growing3ʹ-OH end of a DNA chain. The part of DNA polymerase that removes themisincorporated nucleotide is a specialized member of a large class ofenzymes, known as exonucleases, that cleave nucleotides one at a time fromthe ends of polynucleotides.OHprimerstrand5′3′TTTTTAAAAtemplatestrandOHAOHCAAAAC transiently base-pairs with Aand is incorporated by DNApolymerase into the primerstrandOHTTTTCAAAAAAAAAunpaired 3′-OH end of primerblocks further elongation ofprimer strand by DNApolymeraseOHCTTTTCAAAAAAATOHAA3′-to-5′ exonuclease activityattached to DNA polymerasechews back to create a basepaired 3′-OH end on the primerstrandOHTTTTAAAAOHAAAAADNA polymerase resumesthe process of addingnucleotides to the base-paired3′-OH end of the primer strandOHTTTTTTAAAAAOHAAAA244Chapter 5: DNA Replication, Repair, and Recombination5′templatestrand3′P5′PEEnewlysynthesizedDNAPOLYMERIZINGEDITINGFigure 5–9 Editing by DNA polymerase.
DNA polymerase complexed with the DNA template inthe polymerizing mode (left) and the editing mode (right). The catalytic sites for the exonucleolytic (E)and the polymerization (P) reactions are indicated. In the editing mode, the newly synthesized DNAtransiently unpairs from the template and enters the editing site where the most recently addednucleotide is catalytically removed.MBoC6 m5.09/5.09There is an error frequency of about one mistake for every 104 polymerizationevents both in RNA synthesis and in the separate process of translating mRNAsequences into protein sequences.
This error rate is over 100,000 times greaterthan that in DNA replication, where, as we have seen, a series of proofreadingprocesses makes the process unusually accurate (Table 5–1).Only DNA Replication in the 5ʹ-to-3ʹ Direction Allows Efficient ErrorCorrectionThe need for accuracy probably explains why DNA replication occurs only in the5ʹ-to-3ʹ direction.
If there were a DNA polymerase that added deoxyribonucleoside triphosphates in the 3ʹ-to-5ʹ direction, the growing 5ʹ end of the chain, ratherthan the incoming mononucleotide, would have to provide the activating triphosphate needed for the covalent linkage. In this case, the mistakes in polymerization could not be simply hydrolyzed away, because the bare 5ʹ end of the chainthus created would immediately terminate DNA synthesis (see Figure 5–3). It istherefore possible to correct a mismatched base only if it has been added to the3ʹ end of a DNA chain.
Although the backstitching mechanism for DNA replication seems complex, it preserves the 5ʹ-to-3ʹ direction of polymerization that isrequired for exonucleolytic proofreading.Despite these safeguards against DNA replication errors, DNA polymerasesoccasionally make mistakes. However, as we shall see later, cells have yet anotherTABLE 5–1 The Three Steps That Give Rise to High-Fidelity DNA SynthesisReplication stepErrors per nucleotide added5ʹ → 3ʹ polymerization1 in 1053ʹ → 5ʹ exonucleolytic proofreading1 in 102Strand-directed mismatch repair1 in 103Combined1 in 1010The third step, strand-directed mismatch repair, is described later in this chapter.
For thepolymerization step, “errors per nucleotide added” describes the probability that an incorrectnucleotide will be added to the growing chain. For the other two steps, “errors per nucleotideadded” describes the probability that an error will not be corrected. Each step therefore reducesthe chance of a final error by the factor shown.DNA REPLICATION MECHANISMSFigure 5–10 RNA primer synthesis. A schematic view of the reactioncatalyzed by DNA primase, the enzyme that synthesizes the short RNAprimers made on the lagging strand using DNA as a template. Unlike DNApolymerase, this enzyme can start a new polynucleotide chain by joiningtwo nucleoside triphosphates together.
The primase synthesizes a shortpolynucleotide in the 5ʹ-to-3ʹ direction and then stops, making the3ʹ end of this primer available for the DNA polymerase.chance to correct these errors by a process called strand-directed mismatch repair.Before discussing this mechanism, however, we describe the other types of proteins that function at the replication fork.A Special Nucleotide-Polymerizing Enzyme Synthesizes ShortRNA Primer Molecules on the Lagging StrandFor the leading strand, a primer is needed only at the start of replication: oncea replication fork is established, the DNA polymerase is continuously presentedwith a base-paired chain end on which to add new nucleotides. On the laggingside of the fork, however, each time the DNA polymerase completes a short DNAOkazaki fragment (which takes a few seconds), it must start synthesizing a completely new fragment at a site further along the template strand (see Figure 5–7).A special mechanism produces the base-paired primer strand required by theDNA polymerase molecules.
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