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In other words, part of the new strand must already be in place; the polymerase can only add nucleotides to a preexisting strand. This has proven to be the case for all DNA polymerases,Chapter 24 DNA Metabolismand this discovery provided an interesting wrinkle in the DNA replication story. No DNA-synthesizing enzyme can initiate synthesis of anew DNA strand. As we will see later in this chapter, enzymes thatsynthesize RNA do have the capability of initiating synthesis, and as aconsequence, primers are often oligonucleotides of RNA.After a nucleotide is added to a growing DNA strand, the DNApolymerase must either dissociate or move along the template and addanother nucleotide. Dissociation and reassociation of the polymerasecan limit the overall reaction rate, thus the rate generally increases ifa polymerase adds additional nucleotides without dissociating fromthe template.
The number of nucleotides added, on average, before apolymerase dissociates is defined as its processivity. DNA polymerases vary greatly in processivity, with some adding just a few nucleotides and others adding many thousands before dissociation occurs.Polymerization Is a Thermodynamically Favorable ReactionThroughout this book we have emphasized the importance of noncovalent as well as covalent interactions in biochemical processes.
A discussion of the energetics of the polymerization reaction can be deceptive ifonly the covalent bonds are considered. The rearrangement of covalentbonds is straightforward: one phosphoric anhydride bond (in thedNTP) is hydrolyzed and one phosphodiester bond (in the DNA) isformed. This results in a slightly positive (unfavorable) change in standard free energy (AG°' ~ 2 kJ/mol) for the overall reaction shown inEquation 24-1. Hydrolysis of the pyrophosphate to two molecules ofinorganic phosphate by the pyrophosphatases present in all cells yieldsa AG°' of - 3 0 kJ/mol, and by coupling these two reactions the cell canprovide a strong thermodynamic pull in the direction of polymerization, with a net AG°' of - 2 8 kJ/mol.
This is important to the cell, but inthis case it is not the whole story. If this calculation were complete,polymerases would tend to catalyze DNA degradation in the absence ofpyrophosphate hydrolysis. Purified DNA polymerases, however, carryout polymerization very efficiently in vitro in the absence of pyrophosphatases. The explanation of this seeming paradox now is clear: noncovalent interactions not considered in the calculation above make animportant thermodynamic contribution to the polymerization reaction.Every new nucleotide added to the growing chain is held there not justby the new phosphodiester bond but also by hydrogen bonds to itspartner in the template and base-stacking interactions with the adjacent nucleotide in the same chain (p.
330). The additional energy released by these multiple weak interactions helps drive the reaction inthe direction of polymerization.DNA Polymerases Are Very AccurateReplication must proceed with a very high degree of fidelity. In E. coli,a mistake is made only once for every 109 to 1010 nucleotides added. Forthe E. coli chromosome of about 4.7 x 106 base pairs, this means thatan error will be made only once per 1,000 to 10,000 replications. Duringpolymerization, discrimination between correct and incorrect nucleotides relies upon the hydrogen bonds that specify the correct pairingbetween complementary bases.
Incorrect bases will not form the correct hydrogen bonds and can be rejected before the phosphodiesterbond is formed. The accuracy of the polymerization reaction itself,821Part IV Information Pathways822DNA polymerase I- DNA polymeraseactive site- 3'—>5' (proofreading)exonucleaseactive siteOHQf 0 01I§ is a rare tautomericform of cytosine (C*)that pairs with A andis incorporated intothe growing strand.Before the polymerasemoves on, the cytosineundergoes a tautomericshift from C* to C. Thenew nucleotide is nowmispaired.The mispaired 3'-OHend of the growingstrand blocks furtherelongation.
DNApolymerase slides backto position themispaired base in the3'—>5' exonucleaseactive site.The mispairednucleotide is removed.DNA polymerase slidesforward and resumes itspolymerization activity.Figure 24—6 An example of error correction by the3'-»5' exonuclease activity of DNA polymerase I.Structural analysis has located the exonuclease activity ahead of the polymerization activity as theenzyme is oriented in its movement along the DNA.A mismatched base (here, a C-A mismatch) impedes translocation of the enzyme to the next site.Sliding backward, the enzyme corrects the mistakewith its S'—>5' exonuclease activity, then resumesits polymerase activity in the 5'^>3' direction.however, is insufficient to account for the high degree of fidelity inreplication.
Careful measurements in vitro have shown that DNA polymerases insert one incorrect nucleotide for every 104 to 105 correctones. These mistakes sometimes occur because a base is briefly in anunusual tautomeric form (see Fig. 12-9), allowing it to hydrogen-bondwith an incorrect partner. The error rate is reduced further in vivo byadditional enzymatic mechanisms.One mechanism intrinsic to virtually all DNA polymerases is aseparate 3'-»5' exonuclease activity that serves to double-check eachnucleotide after it is added. This nuclease activity permits the enzymeto remove a nucleotide just added and is highly specific for mismatchedbase pairs (Fig. 24-6).
If the wrong nucleotide has been added, translocation of the polymerase to the position where the next nucleotide is tobe added is inhibited. The 3'-»5' exonuclease activity removes the mispaired nucleotide, and the polymerase begins again. This activity,called proofreading, is not simply the reverse of the polymerizationreaction, because pyrophosphate is not involved. The polymerizing andproofreading activities of a DNA polymerase can be measured separately.
Such measurements have shown that proofreading improvesthe inherent accuracy of the polymerization reaction by 102- to 103-fold.The discrimination between correct and incorrect bases duringproofreading depends on the same base-pairing interactions that areused during polymerization. This strategy of enhancing fidelity byusing complementary noncovalent interactions for discriminationtwice in successive steps is common in the synthesis of informationcontaining molecules. A similar strategy is used to ensure the fidelityof protein synthesis (Chapter 26).Overall, a DNA polymerase makes about one error for every 106 to810 bases added.
The measured accuracy of replication in E. coli cells,however, is still higher. The remaining degree of accuracy is accountedfor by a separate enzyme system that repairs mismatched base pairsremaining after replication. This process, called mismatch repair, isdescribed with other DNA repair processes later in this chapter.E. coli Has at Least Three DNA PolymerasesMore than 90% of the DNA polymerase activity in E.
coli extracts canbe accounted for by DNA polymerase I. Nevertheless, almost immediately after the isolation of this enzyme in 1955, evidence began to accumulate that it is not suited for replication of the large E. coli chromosome. First, the rate at which nucleotides are added by this enzyme(600 nucleotides/min) is too slow, by a factor of 20 or more, to accountfor observed rates of fork movement in the bacterial cell. Second, DNApolymerase I has a relatively low processivity; only about 50 nucleotides are added before the enzyme dissociates. Third, genetic studieshave shown that many genes, and therefore many proteins, are involved in replication: DNA polymerase I clearly does not act alone.Finally, and most important, in 1969 John Cairns isolated a bacterialstrain in which the gene for DNA polymerase I was altered, inactivating the enzyme. This strain was nevertheless viable!A search for other DNA polymerases led to the discovery of E.
coliDNA polymerase II and DNA polymerase III in the early 1970s.DNA polymerase II appears to have a highly specialized DNA repairfunction (described later in this chapter). DNA polymerase III is theChapter 24 DNA Metabolism823primary replication enzyme in E.
coli. Properties of the three DNApolymerases are compared in Table 24-1. DNA polymerase III is amuch more complex enzyme than polymerase I. It is a multimeric enzyme with at least ten different subunits (Table 24-2). Notably, thepolymerization and proofreading activities of DNA polymerase III arelocated in separate subunits. The /3 subunit of this complex enzyme hasbeen crystallized. Its structure is depicted in Fig.
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