Читать книгу Replicating And Repairing The Genome: From Basic Mechanisms To Modern Genetic Technologies - Kenneth N Kreuzer - Страница 19

Many features of T7 DNA polymerase are conserved in other replicative DNA polymerases, making the enzyme a good model. The enzyme tightly grips the primer-template using all three subdomains (palm, finger, and thumb; Figure 2.1A). As introduced in Chapter 1, nearly all DNA polymerases require a primer for extension, and the single-stranded template is used to determine which of the four DNA nucleotides will be added to the end of the primer by base pairing (Figure 1.4). The active site of the enzyme, where the new nucleotide is added, is within the palm subdomain near the base of the finger subdomain (Figure 2.1A). The active site region is tightly organized to strongly favor the correct base pairing of the incoming nucleotide and disfavor the three possible mispairs, contributing to the high fidelity of the enzyme.

The structure and geometry of the active site of the polymerase are very adept at aligning the incoming nucleotide for proper base pairing and excluding the three possible incorrect nucleotides (which don’t fit the active site region very well). Detailed biochemical and structural studies have provided beautiful insights into the details of this base selection process, which is beyond the scope of this chapter. The fidelity of the polymerase step is very high but not perfect, and an incorrect base is inserted roughly once every 20,000 or so incorporation cycles. Incorrect base insertion is probably impossible to avoid. For example, tautomer forms of the DNA bases exist transiently at a very low level and these can pair with the “wrong” template base and thereby fit reasonably well within the polymerase active site. Without some additional mechanism to correct these misinsertions, mutation rates would be very high and complex cells may never have evolved.

The T7 DNA polymerase illustrates one of the major correction mechanisms/pathways that explain the extremely low mutation rate seen in genome replication (see Chapter 1). The exonuclease domain of the polymerase, introduced above, plays the major function of correcting most of the misinsertions from the polymerization reaction, increasing the fidelity by one to two orders of magnitude (Figure 2.2). This activity is therefore referred to as “proofreading exonuclease.” As the “exo” in the name implies, exonucleases attack DNA from one of the two ends (from outside). The proofreading exonuclease in T7 and other replicative polymerases is a 3′ to 5′ exonuclease, because it attacks DNA from the 3′ end, which of course is the end of the growing strand found at the polymerase active site.

Figure 2.2.Excision of misincorporated residue by DNA polymerase. Replicative DNA polymerases generally have a 3′ to 5′ exonuclease activity that removes the vast majority of misincorporated residues. In the case of T7 DNA polymerase, the exonuclease activity resides in the same protein chain as the DNA polymerase activity, but the two active sites are some distance from each other. Upon misincorporation, the growing 3′ end is displaced from the polymerase active site to the exonuclease active site, where the 3′ terminal residue is excised (as a monophosphate). Synthesis resumes when the corrected 3′ end returns to the polymerase active site.

Notice in the polymerase structure (Figure 2.1A) that the exonuclease active site is far removed from the polymerase active site, and indeed the DNA primer-template within the complex must dramatically rearrange in order that a 3′ end relocates to the exonuclease active site and gets clipped. How does the enzyme favor exonuclease action when an incorrect base is incorporated? Incorporation of an incorrect base will invariably cause mispairing and thereby destabilize the double helix in its vicinity. For example, the end of the primer would become destabilized and fray when the rare tautomeric form of the incorporated base described above reverts to its normal form. Two forces then favor removal of the incorrect base. The exonuclease active site prefers a single-stranded end rather than one in a duplex primer-template, and the polymerase active site is inhibited from adding an additional base by a mispaired 3′ terminus in the primer-template (Figure 2.2). In this way, a misincorporated base has a high likelihood of ending up in the exonuclease active site, and once the terminal mismatched base is removed, the (correctly paired) duplex primer-template reforms and is favored to migrate back to the polymerase active site.

An important property of DNA polymerases, called processivity, is the ability to extend a primer (add a nucleotide residue) repeatedly without dissociating from the primer-template. A processive polymerase extends repeatedly, while a distributive polymerase incorporates a small number of nucleotide residues before dissociating and switching to a different primer-template. Processivity increases the rate of DNA replication because it eliminates the time-consuming process of recruiting a new polymerase whenever the replicating polymerase dissociates. It turns out that the processivity of all replicative polymerases is modulated extensively in order to carry out an efficient and concerted replication reaction. The most obvious need for modulating processivity is to accommodate the very different length requirements for replication on the leading versus the lagging strand.

The T7 DNA polymerase devoid of thioredoxin has quite a low processivity, incorporating only about 10–15 nucleotides before dissociating. However, the addition of thioredoxin dramatically increases processivity to an average of about 800 nucleotides per binding event. Concomitant with the increased processivity, the structure of the region of polymerase that binds thioredoxin is reorganized, and the polymerase complex interacts more extensively with the primer-template. Thioredoxin itself is situated over the duplex portion of the primer-template in the structure of the complex (Figure 2.1A). One model for the increased processivity conferred by thioredoxin is that the thumb domain with thioredoxin folds down over the duplex portion of the primer-template after nucleotide addition, essentially encircling the duplex region transiently. This mechanism would be somewhat analogous to that used in cellular DNA replication, where specialized proteins called sliding clamps tether the DNA polymerase to its template (see Chapters 3 and 4).

Although the increase in processivity conferred by thioredoxin is impressive, it is not sufficient to account for the rapid copying of the 40,000-base pair viral genome. Indeed, the processivity of T7 DNA polymerase is further enhanced by the helicase/primase hexamer when the proteins are appropriately arranged for leading-strand synthesis (Figure 2.3, also see below). In this case, DNA polymerase extending the leading strand continues for approximately 5000 nucleotides, on average, before dissociating from the nascent 3′ end. Remarkably, however, this dissociation does not significantly impede replication. There is another DNA polymerase binding site on the helicase/primase,² and the dissociating polymerase can transiently bind to this site and then quickly return to the leading-strand 3′ end. In all, the helicase/primase increases DNA polymerase processivity to greater than 17,000 nucleotides, likely sufficient to copy the entire T7 genome.

Figure 2.3.T7 replication fork, the “traditional” view. The T7 replication fork is depicted with two DNA polymerase molecules (blue), the combined helicase/primase (orange) and the ssDNA-binding protein (green). The direction of helicase movement is indicated by the dotted arrow.