Function Of Dna Polymerase 3
Metabolic processes are often orchestrated past the coordinated activity of multiple protein components. Considering of the complexity of such enzymatic mechanisms, the participant proteins are aptly referred to every bit constituting enzymatic "mechanism." 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 3) holoenzyme, has been extensively studied as a model replication car (
,
). The 10 poly peptide subunits of political leader III holoenzyme part in cooperation with other replication proteins to acquit out the duplication of the unabridged 4.4 Mb Eastward. coli chromosome in 30–40 min. Over the by decade, work in several laboratories resulted in the identification of the genes encoding all ten subunits and the high level expression of the corresponding factor products. This accomplishment has made possible elegant biochemical studies that have brought understanding of the structure and role of the political leader 3 holoenzyme to a level of detail unmatched by other poly peptide machines.
At the East. coli replication fork, the Dna duplex is progressively unwound past the action of a Deoxyribonucleic acid helicase, and the exposed single strands serve as templates for the synthesis of brusk 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, ii dissimilar modes of priming are required. Polymerization of one progeny strand (the "leading" strand) occurs in the same direction every bit the replication fork moves. Thus, only a unmarried priming upshot is required, after which the leading strand is elongated continuously past pol III holoenzyme. Leading strand synthesis is highly processive attributable to the presence of a "sliding clamp" subunit that tethers the polymerase to the template. Polymerization of the 2d 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 and so extended into brusk Deoxyribonucleic acid chains (Okazaki fragments) by pol III holoenzyme. Completion of the lagging strand requires a repair system to remove the primer, fill up in the resulting gaps, and join together the short nascent DNA strands. It is probable that the synthesis of both the leading and the lagging strands at a chromosomal replication fork is mediated past a single pol Three holoenzyme molecule that contains two identical DNA polymerase subunits (
) (see beneath).
The synthesis of the lagging strand by politico Three holoenzyme is a circuitous process that entails a number of discrete steps that must occur in an orderly and efficient mode. To complete the synthesis of the chromosome within xxx–twoscore min, RNA primer are generated on the lagging strand template every 1–2 s at average intervals of 1–two kb. The elongation of each primer by politician Three holoenzyme takes place at a rate of about thousand nucleotides per 2nd and is highly processive owing to the presence of the sliding clench 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. Get-go, the cycling process must be very rapid, occupying just 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 offset sight, to be at odds with the highly processive character of the polymerization procedure. Recent experiments by O'Donnell and colleagues suggest that these problems are solved by a remarkable mechanism that involves the fractional disassembly and reassembly of the holoenzyme structure during the synthesis of each Okazaki fragment (
,
[this issue of Cell). The mechanism is powered by ATP hydrolysis and is controlled past specific poly peptide–protein and protein–DNA interactions.
Pol Three holoenzyme is composed of 10 unique subunits and harbors at least 3 essential enzymatic activities (Table ane). The enzyme contains four distinct functional components: the core polymerase (αεθ), which contains both DNA polymerase (α) and proofreading exonuclease (ε) activities; the sliding clench (β dimer), which confers processivity by tethering the holoenzyme to the template DNA; the clamp loader or γ complex (γ2δ1δ′oneχ1ψ1), which assembles β clamps onto the DNA in an ATP-dependent reaction; the linker protein (τ2), which binds 2 core polymerase molecules and one γ complex. The structure of a stable subassembly of pol III, known as pol 3*, has been studied in detail by a diverseness of methods. Politico Three* contains ii cadre polymerases, i τ dimer and one γ complex (Effigy 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 political leader III* regenerates the holoenzyme and restores processivity. The pol Iii* circuitous can be reconstituted from individual subunits, and a general flick of its overall organization has been deduced from detailed assay of subunit–subunit interactions (
references therein) (Figure one). Equally 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 (
,
). The unmarried γ circuitous in politician 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. Yet, every bit 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 politico III core, the γ complex, and pol Three′ (politician Iii* without the γ complex). It is not known whether these subassemblies correspond artifacts of purification or whether they accept specific roles in replication apart from those played by the holoenzyme. Information technology is also possible that they function in other intracellular processes such as repair or recombination.
1 of the major advances in understanding holoenzyme function came from studies of the β processivity factor. When pol Iii is associated with the β clench, its processivity increases from nearly 10 nucleotides polymerized per bounden consequence to over 50,000 nucleotides polymerized per binding event (
). The overall rate of polymerization increases from ∼20 to ∼750 nucleotides per second (
). Thus, the presence of the β clamp is absolutely essential for the efficient duplication of the large East. 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 Deoxyribonucleic acid, the resulting Deoxyribonucleic acid–protein complexes were observed to be extremely stable, dissociating with a one-half fourth dimension of 72 min nether physiological conditions (
,
nineteen
- Yao Due north.
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- Kelman Z.
- Stukenberg P.T.
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- Hurwitz J.
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). Withal, linearization of the Dna with a brake enzyme resulted in the rapid dissociation of β from the Deoxyribonucleic acid, suggesting that β dimers are capable of sliding freely on the Deoxyribonucleic acid and tin can sideslip off the ends of linear molecules. This general picture gained support past the observation that the stability of β dimers on linear DNA could be increased by the presence of sequence-specific Dna-bounden proteins that blocked the path to the DNA ends (
). 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 cardinal cavity big enough to accommodate a DNA duplex (
). 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 exist band-shaped structures capable of sliding along the Dna (
,
,
,
nineteen
- Yao N.
- Turner J.
- Kelman Z.
- Stukenberg P.T.
- Dean F.
- Shechter D.
- Pan Z.-Q.
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- Google Scholar
). In the example of PCNA, this prediction has recently been confirmed by X-ray crystallographic studies (
).
Figure two outlines the electric current moving-picture show of the elemental steps involved in the synthesis of an Okazaki fragment past the pol III replication machine. The first stride 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 activeness are first to sally. Information technology has been established that the δ subunit of the clench loader is responsible for bounden the β dimer during the loading procedure (
). Interestingly, the isolated δ subunit can bind β in the absenteeism of ATP, while binding of the complete γ complex to β is about completely ATP dependent. This has led to the hypothesis that the δ subunit is normally buried, but becomes exposed for interaction with β equally a result of an ATP-induced conformational change in the γ complex. The predicted conformational change has been detected past 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. Information technology has been demonstrated that the clamp loader is a DNA-dependent ATPase whose action is maximal in the presence of both β and a primer terminus (
). 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 analogousness of the clamp loader for the DNA facilitating its dissociation from the primer terminus.
Effigy 2 Synthesis of Okazaki Fragments by Pol 3 Holoenzyme during Lagging Strand Replication
Evidence full explanation
For simplicity, the diagram shows but pol Three core and the γ circuitous. Other components of the pol III holoenzyme, including the τ subunit and the second pol III cadre molecule that mediates leading strand synthesis, are omitted. The timing of ATP hydrolysis is speculative.
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How the ring shaped β dimer is slipped onto the DNA is an interesting problem that has not withal been solved. One possibility (depicted in Effigy 2) is that the binding of the β dimer to the clamp loader breaks one (or both) sets of contacts that concur the two β subunits together, thus opening the protein ring. In this scenario, the ring would close once again when β is released from the loader at the primer terminus. Alternatively, transient opening of the band may exist coupled directly to the hydrolysis of ATP. Given the molecular tools now available, the answer to this interesting mechanistic puzzle may presently exist forthcoming.
The second footstep in Okazaki strand synthesis is the association of pol Three core in the holoenzyme with the β clamp to course a processive polymerase. In the absence of Deoxyribonucleic acid, the core polymerase appears to have a relatively depression analogousness for β dimers. However, once the clench is placed at the primer terminus, the stability of the complex between core polymerase and the clamp is dramatically increased (
references therein). The basis for this enhanced stability of the cadre–β complex at a primer terminus is not fully understood, just could be due to extra contacts betwixt 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 Deoxyribonucleic acid synthesis, the core polymerase suddenly loses its affinity for the β clench when it reaches the terminate of the template and encounters the v′ end of the previously synthesized Okazaki fragment. The polymerase then dissociates from the Dna leaving the β clamp backside (
). This third footstep in the reaction sequence is clearly cardinal to the process of Okazaki strand synthesis, since it allows the polymerase to cycle to the side by side primer. Information technology is not even so clear what signal is recognized by the polymerase to crusade it to switch out of the processive protein configuration upon completion of an Okazaki fragment. However, in vitro studies indicate that this belongings is intrinsic to the pol III holoenzyme and does non require any accessory factors (
). The T4 Deoxyribonucleic acid polymerase holoenzyme is similarly programmed for rapid disassembly upon completion of Okazaki strand synthesis (
; reviewed by
). Presumably, some structural feature at a nick (east.chiliad., 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 Deoxyribonucleic acid 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 (
), there must be a machinery to reuse such abandoned clamps. In vitro studies propose that the γ complex itself is capable of catalyzing the efficient dissociation of β dimers from the Deoxyribonucleic acid (i.e., the clamp loader is also a clamp unloader) (
,
). Whether the γ complex functions every bit a clench loader or unloader is probably modulated past the DNA construction 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 Deoxyribonucleic acid structures or costless in solution, the γ complex may adopt a different conformation in which this coupling is lost. The latter conformation may nonetheless 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 part equally a clamp unloader, what prevents it from removing β clamps associated with politician III cadre 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 profoundly reduced. Piece of work by
suggests that Eastward. coli has evolved an economic solution to this problem. Using both biochemical and genetic approaches, Naktinis et al. demonstrated that both the politician Iii core and the γ complex bind to the same confront of the β ring via contacts nigh the C-termini of each β monomer. As a consequence of this overlap of binding sites, pol Three core and the γ complex cannot be bound to the β clamp at the same fourth dimension. During processive DNA synthesis, the stable association of β with pol III core prevents access past the γ complex and thus effectively prevents premature unloading of the clamp. It is only after completion of an Okazaki fragment, when the pol III cadre dissociates from β, that the γ complex can access the clench and mediate unloading.
It is credible from the foregoing word that the smooth performance of the political leader III replication machine depends upon a number of specific protein–poly peptide and poly peptide–Deoxyribonucleic acid interactions. These interactions ensure that the events required for synthesis of each Okazaki fragment accept place in the proper social club and are completed quickly. One reason for the speed of the motorcar is that all of the reacting components are held in close proximity by protein–protein interactions. Thus, even though the lagging strand polymerase must constantly dissociate from the termini of completed Okazaki fragments, the pol Three holoenzyme is held at the fork past the leading strand polymerase, which remains tethered to the Dna via a β clamp throughout chromosomal DNA replication. The physical proximity of the polymerase agile site to a newly synthesized RNA primer facilitates the cycling of the polymerase. Similarly, the presence of a γ complex within the holoenzyme ensures the rapid associates of β clamps on newly synthesized primer, as well equally their rapid disassembly from completed Dna strands. Although it has not yet been possible to mensurate the fourth dimension required for the intramolecular cycling of pol III core from completed strand to nascent primer, it has been estimated from in vitro studies of intermolecular transfer that the cycle time is considerably less than 1 southward (
). Given that information technology requires one–2 south to complete the synthesis of an Okazaki fragment, it is clear that the performance of the automobile is not express past the time required for polymerase cycling.
Much of what has been learned of the machinery of politico 3 action may be generally applicative to other replicases and fifty-fifty to processes unrelated to DNA replication. Both T4 and Due east. coli employ dimeric polymerases for coordinated synthesis of leading and lagging strand DNA (
,
). Further characterization of the eukaryotic replication mechanisms may also reveal physical coupling of the leading and lagging strand polymerases. As already noted, both the T4 and eukaryotic replication machines make use of ring-shaped homotrimeric sliding clamps (gp45 and PCNA) similar in overall construction to the β dimer, and both accept clamp loaders that role similar the γ circuitous (
,
,
). Interestingly, work in the past several years has uncovered cases in which components of the replicase, particularly the sliding clamps, interact with proteins not involved in Dna synthesis. In the example of bacteriophage T4, for example, information technology has been demonstrated that the gp45 sliding clamps abandoned during Deoxyribonucleic acid replication can serve as mobile enhancer proteins to activate the RNA polymerase responsible for transcribing the tardily genes of the virus (
). This mechanism explains how the switch from early on to late gene expression during T4 infection is coupled to the onset of Deoxyribonucleic acid replication. In eukaryotes it has been shown that a significant fraction of PCNA in the cell is present in complexes with cyclin-dependent kinases and the p21 kinase inhibitor, suggesting that the protein may play a function in linking DNA replication to other processes in the cell cycle (
). Thus, it appears that cells take plant additional uses for the complex mechanism that originally evolved to duplicate the genome. It would not be surprising if other examples are uncovered in the form of future studies of pol III office.
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DOI: https://doi.org/x.1016/S0092-8674(00)80069-0
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