DNA Replication:- E. coli Has at Least Five DNA Polymerases
More than 90% of the DNA polymerase activity observed in E. coli extracts can be accounted for by DNA polymerase I. Soon after the isolation of this enzyme in 1955, however, evidence began to accumulate that it is not suited for replication of the large E. coli chromosome. First, the rate at which it adds nucleotides (600 nucleotides/min) is too slow (by a factor of 100 or more) to account for the rates at which the replication fork moves in the bacterial cell. Second, DNA polymerase I has a relatively low processivity. Third, genetic studies have demonstrated that many genes, and therefore many proteins, are involved in replication: DNA polymerase I clearly does not act alone. Fourth, and most important, in 1969 John Cairns isolated a bacterial strain with an altered gene for DNA polymerase I that produced an inactive enzyme. Although this strain was ab normally sensitive to agents that damaged DNA, it was nevertheless viable! A search for other DNA polymerases led to the discovery of E. coli DNA polymerase II and DNA polymerase III in the early 1970s. DNA polymerase II is an enzyme involved in one type of DNA repair (Section 25.3). DNA polymerase III is the principal replication enzyme in E. coli. The properties of these three DNA polymerases are compared in Table 25–1. DNA polymerases IV and V, identified in 1999, are involved in an unusual form of DNA repair (Section 25.2). DNA polymerase I, then, is not the primary enzyme of replication; instead it performs a host of clean-up functions during replication, recombination, and repair. The polymerase’s special functions are enhanced by its 5→3 exonuclease activity. This activity, distinct from the 3→5 proofreading exonuclease , is located in a structural domain that can be separated from the enzyme by mild protease treatment. When the 5→3 exonuclease domain is removed, the remaining fragment (Mr68,000), the large fragmentor Klenow fragment (Fig. 1), retains the polymerization and proofreading activities. The 5→3 exonuclease activity of intact DNA polymerase I can replace a segment of DNA (or RNA) paired to the template strand, in a process known as nick translation (Fig.2). Most other DNA polymerases lack a 5→3 exonuclease activity.
DNA polymerase III is much more complex than DNA polymerase I, having ten types of subunits (Table 25–2). Its polymerization and proofreading activities re side in its and (epsilon) subunits, respectively. The Ө subunit associates with and to form a core polymerase, which can polymerize DNA but with limited processivity. Two core polymerases can be linked by
another set of subunits, a clamp-loading complex, or complex, consisting of five subunits of four different types, ℸ2ϒδδ . The core polymerases are linked through the (tau) subunits. Two additional subunits, (chi) and (psi), are bound to the clamp-loading complex. The entire assembly of 13 protein subunits (nine different types) is called DNA polymerase III . DNA polymerase III* can polymerize DNA, but with a much lower processivity than one would expect for the organized replication of an entire chromosome. The necessary increase in processivity is provided by the addition of the subunits β, four of which complete the DNA polymerase III holoenzyme. The β subunits associate in pairs to form donut-shaped structures that encircle the DNA and act like clamps . Each dimer as sociates with a core subassembly of polymerase III* (one dimeric clamp per core subassembly) and slides along the DNA as replication proceeds. The sliding clamp prevents the dissociation of DNA polymerase III from DNA, dramatically increasing processivity—to greater than 500,000 (Table 25–1).
FIGURE 1 Large (Klenow) fragment of DNA polymerase I. This polymerase is widely distributed in bacteria. The Klenow fragment, produced by proteolytic treatment of the polymerase, retains the poly merization and proofreading activities of the enzyme. The Klenow fragment shown here is from the thermophilic bacterium Bacillus stearothermophilus (PDB ID 3BDP). The active site for addition of nucleotides is deep in the crevice at the far end of the bound DNA. The dark blue strand is the template.
FIGURE 2 Nick translation. In this process, an RNA or DNA strand paired to a DNA template is simultaneously degraded by the 5n3 exonuclease activity of DNA polymerase I and replaced by the polymerase activity of the same enzyme. These activities have a role in both DNA repair and the removal of RNA primers during replication (both described later). The strand of nucleic acid to be removed (ei ther DNA or RNA) is shown in green, the replacement strand in red. DNA synthesis begins at a nick (a broken phosphodiester bond, leaving a free 3 hydroxyl and a free 5 phosphate). Polymerase I extends the nontemplate DNA strand and moves the nick along the DNA—a process called nick translation. A nick remains where DNA polymerase I dissociates, and is later sealed by another enzyme.
