DNA Polymerase III: Running Rings around the Fork (1996)

Metabolic processes are often orchestrated by the coordinated action of multiple protein components. Because of the complexity of such enzymatic mechanisms, the participant proteins are aptly referred to as constituting enzymatic “machinery.” Deciphering the inner workings of the multiprotein machines that mediate processes, such as DNA replication and transcription, is a major goal of biology, but is a technically demanding task owing to the difficulty in reassembling functional complexes from purified components outside of the cell. Since its discovery nearly 25 years ago, the replicase of Escherichia coli, DNA polymerase III (pol III) holoenzyme, has been extensively studied as a model replication machine (7Kornberg A. Baker T. DNA Replication, Second Edition. W.H. Freeman and Company, New York1992Google Scholar, 5Kelman Z. O'Donnell M. Annu. Rev. Biochem. 1995; 64: 171-200Crossref PubMed Scopus (361) Google Scholar). The 10 protein subunits of pol III holoenzyme function in cooperation with other replication proteins to carry out the duplication of the entire 4.4 Mb E. coli chromosome in 30–40 min. Over the past decade, work in several laboratories resulted in the identification of the genes encoding all 10 subunits and the high level expression of the corresponding gene products. This accomplishment has made possible elegant biochemical studies that have brought understanding of the structure and function of the pol III holoenzyme to a level of detail unmatched by other protein machines. At the E. coli replication fork, the DNA duplex is progressively unwound by the action of a DNA helicase, and the exposed single strands serve as templates for the synthesis of short RNA primer by the primase and associated proteins. The role of pol III holoenzyme is to elongate newly synthesized primer to generate the two progeny strands. Because of the antiparallel nature of the DNA duplex, two different modes of priming are required. Polymerization of one progeny strand (the “leading” strand) occurs in the same direction as the replication fork moves. Thus, only a single priming event is required, after which the leading strand is elongated continuously by pol III holoenzyme. Leading strand synthesis is highly processive owing to the presence of a “sliding clamp” subunit that tethers the polymerase to the template. Polymerization of the second progeny strand (the “lagging” strand) occurs in the direction opposite to replication fork movement. Thus, elongation of the lagging strand is a discontinuous process involving the repeated synthesis of RNA primer that are then extended into short DNA chains (Okazaki fragments) by pol III holoenzyme. Completion of the lagging strand requires a repair system to remove the primer, fill in the resulting gaps, and join together the short nascent DNA strands. It is likely that the synthesis of both the leading and the lagging strands at a chromosomal replication fork is mediated by a single pol III holoenzyme molecule that contains two identical DNA polymerase subunits (4Johanson K.O. McHenry C.S. J. Biol. Chem. 1984; 259: 4589-4595Abstract Full Text PDF PubMed Google Scholar) (see below). The synthesis of the lagging strand by pol III holoenzyme is a complex process that entails a number of discrete steps that must occur in an orderly and efficient fashion. To complete the synthesis of the chromosome within 30–40 min, RNA primer are generated on the lagging strand template every 1–2 s at average intervals of 1–2 kb. The elongation of each primer by pol III holoenzyme takes place at a rate of about 1000 nucleotides per second and is highly processive owing to the presence of the sliding clamp subunit. The discontinuous mode of replication demands that pol III must cycle to the next RNA primer upon completion of each Okazaki fragment. This raises two potential difficulties. First, the cycling process must be very rapid, occupying only a fraction of the total time devoted to polymerization. Rapid cycling is essential to ensure that the synthesis of the lagging strand keeps pace with the synthesis of the leading strand. Second, the requirement for cycling of pol III would appear, at least at first sight, to be at odds with the highly processive character of the polymerization process. Recent experiments by O'Donnell and colleagues suggest that these problems are solved by a remarkable mechanism that involves the partial disassembly and reassembly of the holoenzyme structure during the synthesis of each Okazaki fragment (15Stukenberg P.T. Turner J. O'Donnell M. Cell. 1994; 78: 877-887Abstract Full Text PDF PubMed Scopus (147) Google Scholar, 11Naktinis V. Turner J. O'Donnell M. Cell. 1996; 84 (this issue)Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar [this issue of Cell). The mechanism is powered by ATP hydrolysis and is controlled by specific protein–protein and protein–DNA interactions. Pol III holoenzyme is composed of 10 unique subunits and harbors at least three essential enzymatic activities (Table 1). The enzyme contains four distinct functional components: the core polymerase (αεθ), which contains both DNA polymerase (α) and proofreading exonuclease (ε) activities; the sliding clamp (β dimer), which confers processivity by tethering the holoenzyme to the template DNA; the clamp loader or γ complex (γ2δ1δ′1χ1ψ1), which assembles β clamps onto the DNA in an ATP-dependent reaction; the linker protein (τ2), which binds two core polymerase molecules and one γ complex. The structure of a stable subassembly of pol III, known as pol III*, has been studied in detail by a variety of methods. Pol III* contains two core polymerases, one τ dimer and one γ complex (Figure 1). The enzyme exhibits greatly reduced processivity relative to the holoenzyme because it lacks the β subunit, which readily dissociates from the holoenzyme during purification. Addition of the β subunit to pol III* regenerates the holoenzyme and restores processivity. The pol III* complex can be reconstituted from individual subunits, and a general picture of its overall organization has been deduced from detailed analysis of subunit–subunit interactions (12Onrust R. Finkelstein J. Turner J. Naktinis V. O'Donnell M. J. Biol. Chem. 1995; 270: 13366-13377Crossref PubMed Scopus (113) Google Scholarreferences therein) (Figure 1). As mentioned above, it has been proposed that the dual core polymerases in pol III holoenzyme mediate the coordinated synthesis of the leading and lagging strands at the replication fork (4Johanson K.O. McHenry C.S. J. Biol. Chem. 1984; 259: 4589-4595Abstract Full Text PDF PubMed Google Scholar, 17Wu C.A. Zechner E.L. Hughes A.J. Franden M.A. McHenry C.S. Marians K.J. J. Biol. Chem. 1992; 267: 4064-4073Abstract Full Text PDF PubMed Google Scholar). The single γ complex in pol III holoenzyme presumably serves to load β clamps onto both strands. It is likely that the loading of a single β clamp is sufficient for processive leading strand synthesis. However, as described in greater detail below, the cycling of core polymerase during lagging strand synthesis necessitates the loading of a clamp for each Okazaki fragment. Other stable subassemblies of pol III holoenzyme have also been purified from E. coli. These include the pol III core, the γ complex, and pol III′ (pol III* without the γ complex). It is not known whether these subassemblies represent artifacts of purification or whether they have specific roles in replication apart from those played by the holoenzyme. It is also possible that they function in other intracellular processes such as repair or recombination.Table 1Subunit composition of DNA Polymerase III holoenzyme Open table in a new tab One of the major advances in understanding holoenzyme function came from studies of the β processivity factor. When pol III is associated with the β clamp, its processivity increases from about 10 nucleotides polymerized per binding event to over 50,000 nucleotides polymerized per binding event (9Mok M. Marians K.J. J. Biol. Chem. 1987; 262: 16644-16654Abstract Full Text PDF PubMed Google Scholar). The overall rate of polymerization increases from ∼20 to ∼750 nucleotides per second (5Kelman Z. O'Donnell M. Annu. Rev. Biochem. 1995; 64: 171-200Crossref PubMed Scopus (361) Google Scholar). Thus, the presence of the β clamp is absolutely essential for the efficient duplication of the large E. coli chromosome. In an elegant series of biochemical experiments, it was established that the β clamp is associated with the DNA via a unique topological linkage. When β dimers were assembled onto singly nicked circular DNA, the resulting DNA–protein complexes were observed to be extremely stable, dissociating with a half time of 72 min under physiological conditions (14Stukenberg P.T. Studwell-Vaughan P.S. O'Donnell M. J. Biol. Chem. 1991; 266: 11328-11334Abstract Full Text PDF PubMed Google Scholar, 19Yao N. Turner J. Kelman Z. Stukenberg P.T. Dean F. Shechter D. Pan Z.-Q. Hurwitz J. O'Donnell M. Genes to Cell, in press. 1996; Google Scholar). However, linearization of the DNA with a restriction enzyme resulted in the rapid dissociation of β from the DNA, suggesting that β dimers are capable of sliding freely on the DNA and can slip off the ends of linear molecules. This general picture gained support by the observation that the stability of β dimers on linear DNA could be increased by the presence of sequence-specific DNA-binding proteins that blocked the path to the DNA ends (14Stukenberg P.T. Studwell-Vaughan P.S. O'Donnell M. J. Biol. Chem. 1991; 266: 11328-11334Abstract Full Text PDF PubMed Google Scholar). These and other experiments led to the prediction that the β dimer encircles the DNA. Subsequent X-ray diffraction studies showed that the β dimer is indeed ring-shaped and possesses a central cavity large enough to accommodate a DNA duplex (6Kong X.-P. Onrust R. O'Donnell M. Kuriyan J. Cell. 1992; 69: 425-437Abstract Full Text PDF PubMed Scopus (634) Google Scholar). Based upon biochemical studies, the processivity factors of the bacteriophage T4 and eukaryotic replication machines (gp45 and proliferating cell nuclear antigen [PCNA], respectively) were also predicted to be ring-shaped structures capable of sliding along the DNA (1Alberts B.M. Phil. Trans. R. Soc. (Lond.) B. 1987; 317: 395-420Crossref PubMed Scopus (67) Google Scholar, 3Herendeen D.R. Kassavetis G.A. Geiduschek E.P. Science. 1992; 256: 1298-1303Crossref PubMed Scopus (108) Google Scholar, 16Tinker R.L. Kassavetis G.A. Geiduschek E.P. EMBO J. 1994; 13: 5330-5337Crossref PubMed Scopus (57) Google Scholar, 19Yao N. Turner J. Kelman Z. Stukenberg P.T. Dean F. Shechter D. Pan Z.-Q. Hurwitz J. O'Donnell M. Genes to Cell, in press. 1996; Google Scholar). In the case of PCNA, this prediction has recently been confirmed by X-ray crystallographic studies (8Krishna T.S.R. Kong X.-P. Gary S. Burgers P. Kuriyan J. Cell. 1994; 79: 1233-1244Abstract Full Text PDF PubMed Scopus (752) Google Scholar). Figure 2 outlines the current picture of the elemental steps involved in the synthesis of an Okazaki fragment by the pol III replication machine. The first step in the sequence is the loading of the β clamp at a primer terminus. The γ complex, which functions as the clamp loader, has been reconstituted from purified components, and some aspects of its mechanism of action are beginning to emerge. It has been established that the δ subunit of the clamp loader is responsible for binding the β dimer during the loading process (10Naktinis V. Onrust R. Fang L. O'Donnell M. J. Biol. Chem. 1995; 270: 13358-13365Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). Interestingly, the isolated δ subunit can bind β in the absence of ATP, while binding of the complete γ complex to β is almost completely ATP dependent. This has led to the hypothesis that the δ subunit is normally buried, but becomes exposed for interaction with β as a result of an ATP-induced conformational change in the γ complex. The predicted conformational change has been detected by analyzing changes in the sensitivity of the γ complex to proteases upon binding ATP. The clamp loader specifically recognizes primer termini and transfers the bound β dimer onto the DNA in a reaction that requires ATP hydrolysis. It has been demonstrated that the clamp loader is a DNA-dependent ATPase whose activity is maximal in the presence of both β and a primer terminus (5Kelman Z. O'Donnell M. Annu. Rev. Biochem. 1995; 64: 171-200Crossref PubMed Scopus (361) Google Scholar). One reasonable model is that hydrolysis of ATP induced by DNA binding causes the δ subunit to retract again, releasing the β dimer onto the DNA. ATP hydrolysis might also reduce the affinity of the clamp loader for the DNA facilitating its dissociation from the primer terminus. How the ring shaped β dimer is slipped onto the DNA is an interesting problem that has not yet been solved. One possibility (depicted in Figure 2) is that the binding of the β dimer to the clamp loader breaks one (or both) sets of contacts that hold the two β subunits together, thus opening the protein ring. In this scenario, the ring would close again when β is released from the loader at the primer terminus. Alternatively, transient opening of the ring may be coupled directly to the hydrolysis of ATP. Given the molecular tools now available, the answer to this interesting mechanistic puzzle may soon be forthcoming. The second step in Okazaki strand synthesis is the association of pol III core in the holoenzyme with the β clamp to form a processive polymerase. In the absence of DNA, the core polymerase appears to have a relatively low affinity for β dimers. However, once the clamp is placed at the primer terminus, the stability of the complex between core polymerase and the clamp is dramatically increased (11Naktinis V. Turner J. O'Donnell M. Cell. 1996; 84 (this issue)Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar references therein). The basis for this enhanced stability of the core–β complex at a primer terminus is not fully understood, but could be due to extra contacts between polymerase and DNA or to a change in the structure of the polymerase that augments the favorable contacts between polymerase and clamp. Whatever its physical basis, it is the stability of this complex that explains the ability of pol III to polymerize thousands of nucleotides without dissociating from the template. Although tightly bound to the β clamp during processive DNA synthesis, the core polymerase suddenly loses its affinity for the β clamp when it reaches the end of the template and encounters the 5′ end of the previously synthesized Okazaki fragment. The polymerase then dissociates from the DNA leaving the β clamp behind (15Stukenberg P.T. Turner J. O'Donnell M. Cell. 1994; 78: 877-887Abstract Full Text PDF PubMed Scopus (147) Google Scholar). This third step in the reaction sequence is clearly central to the process of Okazaki strand synthesis, since it allows the polymerase to cycle to the next primer. It is not yet clear what signal is recognized by the polymerase to cause it to switch out of the processive protein configuration upon completion of an Okazaki fragment. However, in vitro studies indicate that this property is intrinsic to the pol III holoenzyme and does not require any accessory factors (15Stukenberg P.T. Turner J. O'Donnell M. Cell. 1994; 78: 877-887Abstract Full Text PDF PubMed Scopus (147) Google Scholar). The T4 DNA polymerase holoenzyme is similarly programmed for rapid disassembly upon completion of Okazaki strand synthesis (2Hacker K.J. Alberts B.M. J. Biol. Chem. 1994; 269: 24221-24228Abstract Full Text PDF PubMed Google Scholar; reviewed by13Stillman B. Cell. 1994; 78: 725-728Abstract Full Text PDF PubMed Scopus (222) Google Scholar). Presumably, some structural feature at a nick (e.g., the abutting 5′ terminus) induces a structural transition in the polymerase that breaks the contacts with the sliding clamp and facilitates dissociation from the DNA. What happens to the β clamps that are left behind on the DNA when pol III dissociates from the competed nascent strand? Given that the number of β dimers per cell is an order of magnitude less than the number of Okazaki fragments produced during the replication of the E. coli genome (7Kornberg A. Baker T. DNA Replication, Second Edition. W.H. Freeman and Company, New York1992Google Scholar), there must be a mechanism to reuse such abandoned clamps. In vitro studies suggest that the γ complex itself is capable of catalyzing the efficient dissociation of β dimers from the DNA (i.e., the clamp loader is also a clamp unloader) (15Stukenberg P.T. Turner J. O'Donnell M. Cell. 1994; 78: 877-887Abstract Full Text PDF PubMed Scopus (147) Google Scholar, 11Naktinis V. Turner J. O'Donnell M. Cell. 1996; 84 (this issue)Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). Whether the γ complex functions as a clamp loader or unloader is probably modulated by the DNA structure to which it is bound. When the γ complex is bound tightly to a primer terminus, it adopts a conformation capable of efficiently coupling ATP hydrolysis to the transfer of β clamps onto the DNA. When bound to other DNA structures or free in solution, the γ complex may adopt a different conformation in which this coupling is lost. The latter conformation may still be capable of catalyzing the opening and closing of the protein ring, thus allowing the rapid equilibration of β clamps on and off the DNA. The foregoing discussion suggests a possible paradox. If the γ complex can function as a clamp unloader, what prevents it from removing β clamps associated with pol III core during the processive stage of DNA synthesis? If such unloading events were to occur at a significant frequency, the overall efficiency of lagging strand synthesis would be greatly reduced. Work by 11Naktinis V. Turner J. O'Donnell M. Cell. 1996; 84 (this issue)Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar suggests that E. coli has evolved an economical solution to this problem. Using both biochemical and genetic approaches, Naktinis demonstrated that both the pol III core and the γ complex bind to the same of the β ring via contacts the of each β As a of this of binding pol III core and the γ complex be bound to the β clamp at the same processive DNA synthesis, the stable association of β with pol III core prevents by the γ complex and thus prevents unloading of the clamp. It is only after completion of an Okazaki when the pol III core dissociates from that the γ complex can the clamp and mediate It is from the foregoing discussion that the of the pol III replication machine upon a number of specific protein–protein and protein–DNA interactions. These interactions ensure that the events for synthesis of each Okazaki fragment place in the order and are One for the of the machine is that all of the components are in close by protein–protein interactions. Thus, the lagging strand polymerase must from the termini of Okazaki the pol III holoenzyme is at the fork by the leading strand which to the DNA via a β clamp chromosomal DNA The physical of the polymerase to a newly synthesized RNA primer facilitates the cycling of the polymerase. the presence of a γ complex within the holoenzyme the rapid of β clamps on newly synthesized primer, as as rapid disassembly from DNA strands. Although it has not yet been possible to the time for the cycling of pol III core from strand to nascent primer, it has been from in vitro studies of transfer that the cycle time is less than s (15Stukenberg P.T. Turner J. O'Donnell M. Cell. 1994; 78: 877-887Abstract Full Text PDF PubMed Scopus (147) Google Scholar). Given that it requires 1–2 s to complete the synthesis of an Okazaki it is clear that the of the machine is not by the time for polymerase of what has been of the mechanism of pol III action may be to other and to processes to DNA T4 and E. coli polymerases for coordinated synthesis of leading and lagging strand DNA (1Alberts B.M. Phil. Trans. R. Soc. (Lond.) B. 1987; 317: 395-420Crossref PubMed Scopus (67) Google Scholar, K.O. McHenry C.S. J. Biol. Chem. 1984; 259: 4589-4595Abstract Full Text PDF PubMed Google Scholar). of the eukaryotic replication may also physical coupling of the leading and lagging strand As both the T4 and eukaryotic replication machines of ring-shaped sliding clamps (gp45 and in overall structure to the β and both have clamp that function the γ complex (1Alberts B.M. Phil. Trans. R. Soc. (Lond.) B. 1987; 317: 395-420Crossref PubMed Scopus (67) Google Scholar, A. Baker T. DNA Replication, Second Edition. W.H. Freeman and Company, New York1992Google Scholar, B. Cell. 1994; 78: 725-728Abstract Full Text PDF PubMed Scopus (222) Google Scholar). Interestingly, work in the past several years has in which components of the the sliding with proteins not involved in DNA synthesis. In the case of bacteriophage for it has been demonstrated that the sliding clamps abandoned during DNA replication can serve as proteins to the RNA polymerase responsible for the genes of the D.R. Kassavetis G.A. Geiduschek E.P. Science. 1992; 256: 1298-1303Crossref PubMed Scopus (108) Google Scholar). This mechanism explains the switch from to gene expression during T4 is coupled to the of DNA In it has been that a significant fraction of in the cell is in complexes with and the suggesting that the protein may a role in DNA replication to other processes in the cell cycle D. Cell. 1992; Full Text PDF PubMed Scopus Google Scholar). Thus, it appears that have for the complex that evolved to the It would not be other are in the of studies of pol III