The DNA polymerase III holoenzyme: an asymmetric dimeric replicative complex with leading and lagging strand polymerases.

The DNA Polymerase III holoenzyme forms initiation complexes on primed DNA in an ATP-dependent reaction. We demonstrate that the nonhydrolyzable ATP analog, ATP gamma S, supports the formation of an isolable leading strand complex that loads and replicates the lagging strand only in the presence of ATP, beta, and the single-stranded DNA binding protein. The single endogenous DnaX complex within DNA polymerase III holoenzyme assembles beta onto both the leading and lagging strand polymerases by an ordered mechanism. The dimeric replication complex disassembles in the opposite order from which it assembled. Upon ATP gamma S-induced dissociation, the leading strand polymerase is refractory to disassembly allowing cycling to occur exclusively on the lagging strand. These results establish holoenzyme as an intrinsic asymmetric dimer with distinguishable leading and lagging strand polymerases.     

DNA polymerase III holoenzyme from Thermus thermophilus identification, expression, purification of components, and use to reconstitute a processive replicase

DNA replication in bacteria is performed by a specialized multicomponent replicase, the DNA polymerase III holoenzyme, that consist of three essential components: a polymerase, the beta sliding clamp processivity factor, and the DnaX complex clamp-loader. We report here the assembly of the minimal functional holoenzyme from Thermus thermophilus (Tth), an extreme thermophile. The minimal holoenzyme consists of alpha (pol III catalytic subunit), beta (sliding clamp processivity factor), and the essential DnaX (tau/gamma), delta and delta' components of the DnaX complex. We show with purified recombinant proteins that these five components are required for rapid and processive DNA synthesis on long single-stranded DNA templates. Subunit interactions known to occur in DNA polymerase III holoenzyme from mesophilic bacteria including delta-delta' interaction, deltadelta'-tau/gamma complex formation, and alpha-tau interaction, also occur within the Tth enzyme. As in mesophilic holoenzymes, in the presence of a primed DNA template, these subunits assemble into a stable initiation complex in an ATP-dependent manner. However, in contrast to replicative polymerases from mesophilic bacteria, Tth holoenzyme is efficient only at temperatures above 50 degrees C, both with regard to initiation complex formation and processive DNA synthesis. The minimal Tth DNA polymerase III holoenzyme displays an elongation rate of 350 bp/s at 72 degrees C and a processivity of greater than 8.6 kilobases, the length of the template that is fully replicated after a single association event.     

The asymmetric dimeric polymerase hypothesis: a progress report

In 1983, my laboratory first proposed that the DNA polymerase III holoenzyme is an asymmetric dimer with distinguishable leading and lagging strand polymerases. Here, I review progress by my laboratory and others in testing this hypothesis. To date, the hypothesis is supported by our demonstration of (i) an asymmetry in function of two populations of holoenzyme in solution in their ability to use the ATP analog, ATP gamma S, to support initiation complex formation, (ii) the stabilization of a dimeric polymerase structure by the tau subunit, (iii) allosteric communication between polymerase halves and (iv) the coexistence of gamma and the tau, subunits which share common sequences, within the same holoenzyme assemblies. This latter observation may provide a structural basis for holoenzyme asymmetry. I discuss the implications of the asymmetric dimer hypothesis to the solution of problems encountered by polymerases at the replication fork and delineate further tests required before the hypothesis can be firmly established.     

DNA Polymerase III holoenzyme of Escherichia coli. IV. The holoenzyme is an asymmetric dimer with twin active sites

Pol III, a subassembly of Escherichia coli DNA polymerase III holoenzyme lacking only the auxiliary beta subunit, was purified to homogeneity by an improved procedure. This assembly consists of nine different polypeptides, likely in a 1:1 stoichiometry: a catalytic core (pol III) of alpha (132 kDa), epsilon (27 kDa), and theta (10 kDa), and six auxiliary subunits: tau (71 kDa), gamma (52 kDa), delta (35 kDa), delta' (33 kDa), chi (15 kDa), and psi (12 kDa). The assembly behaves on gel filtration as a particle of about 800 kDa, indicating a content of two each of the subunits. A new procedure for purifying the core yielded a novel dimeric form which may provide the foundation for the dimeric nature of the more complex pol III and holoenzyme forms. Pol III readily dissociates into several subassemblies including pol III', likely a dimeric core with two tau subunits. The holoenzyme, purified by a similar procedure with ATP and Mg2+ present throughout, retained the beta subunit (37 kDa) as well as all the subunits present in pol III; the mass of the holoenzyme was estimated to be 900 kDa. The isolated initiation complex of holoenzyme with a primed template DNA and the elongation complex (formed in the presence of three deoxynucleoside triphosphates) had the same composition and stoichiometry as observed for pol III with two beta dimers in addition. An initiation complex assembled from a mixture of monomeric pol III core, gamma 2 delta delta' chi psi complex (gamma complex), beta, and tau retained the core, one beta dimer, and two tau subunits but was deficient in the gamma complex. When tau was omitted from the assembly mixture, the initiation complex contained one or two gamma complexes instead of the tau subunit. Based on these data, pol III holoenzyme is judged to be an asymmetric dimeric particle with twin pol III core active sites and two different sets of auxiliary units designed to achieve essentially concurrent replication of both leading and lagging strand templates.     

Constitution of the twin polymerase of DNA polymerase III holoenzyme

It is speculated that DNA polymerases which duplicate chromosomes are dimeric to provide concurrent replication of both leading and lagging strands. DNA polymerase III holoenzyme (holoenzyme), is the 10-subunit replicase of the Escherichia coli chromosome. A complex of the alpha (DNA polymerase) and epsilon (3'-5' exonuclease) subunits of the holoenzyme contains only one of each protein. Presumably, one of the eight other subunit(s) functions to dimerize the alpha epsilon polymerase within the holoenzyme. Based on dimeric subassemblies of the holoenzyme, two subunits have been elected as possible agents of polymerase dimerization, one of which is the tau subunit (McHenry, C. S. (1982) J. Biol. Chem. 257, 2657-2663). Here, we have used pure alpha, epsilon, and tau subunits in binding studies to determine whether tau can dimerize the polymerase. We find tau binds directly to alpha. Whereas alpha is monomeric, tau is a dimer in its native state and thereby serves as an efficient scaffold to dimerize the polymerase. The epsilon subunit does not associate directly with tau but becomes dimerized in the alpha epsilon tau complex by virtue of its interaction with alpha. We have analyzed the dimeric alpha epsilon tau complex by different physical methods to increase the confidence that this complex truly contains a dimeric polymerase. The tau subunit is comprised of the NH2-terminal two-thirds of tau but does not bind to alpha epsilon, identifying the COOH-terminal region of tau as essential to its polymerase dimerization function. The significance of these results with respect to the organization of subunits within the holoenzyme is discussed.     

Total reconstitution of DNA polymerase III holoenzyme reveals dual accessory protein clamps

DNA polymerase III holoenzyme (holoenzyme) is the 10-subunit replicase of the Escherichia coli chromosome. In this report, pure preparations of delta, delta', and a gamma chi psi complex are resolved from the five protein gamma complex subassembly. Using these subunits and other holoenzyme subunits isolated from overproducing plasmid strains of E. coli, the rapid and highly processive holoenzyme has been reconstituted from only five pure single subunits: alpha, epsilon, gamma, delta, and beta. The preceding report showed that of the three subunits in the core polymerase, only a complex of alpha (DNA polymerase) and epsilon (3'-5' exonuclease) are required to assemble a processive holoenzyme on a template containing a preinitiation complex (Studwell, P.S., and O'Donnell, M. (1990) J. Biol. Chem. 265, 1171-1178). This report shows that of the five proteins in the gamma complex only a heterodimer of gamma and delta is required with the beta subunit to form the ATP-activated preinitiation complex with a primed template. Surprisingly, the delta' subunit does not form an active complex with gamma but forms a fully active heterodimer complex with the tau subunit (as does delta). Hence, the tau delta' and gamma delta heterodimers are fully active in the preinitiation complex reaction with beta and primed DNA. Holoenzymes reconstituted using the alpha epsilon complex, beta subunit, and either gamma delta or tau delta' are fully processive in DNA synthesis, and upon completing the template they rapidly cycle to a new primed template endowed with a preinitiation complex clamp. Since the holoenzyme molecule contains all of these accessory subunits (gamma, delta, tau, delta', and beta) in all likelihood it has the capacity to form two preinitiation complex clamps simultaneously at two primer termini. Two primer binding components within one holoenzyme may mediate its rapid cycling to multiple primers on the lagging strand and also provides functional evidence for the hypothesis of holoenzyme as a dimeric polymerase capable of simultaneous replication of both leading and lagging strands of a replication fork.     

Studies on human DNA polymerase epsilon and GINS complex and their role in DNA replication

In eukaryotic cells, DNA replication is carried out by the coordinated action of three DNA polymerases (Pols), Pol α, δ, and ε. In this report, we describe the reconstitution of the human four-subunit Pol ε and characterization of its catalytic properties in comparison with Pol α and Pol δ. Human Pol ε holoenzyme is a monomeric complex containing stoichiometric subunit levels of p261/Pol 2, p59, p17, and p12. We show that the Pol ε p261 N-terminal catalytic domain is solely responsible for its ability to catalyze DNA synthesis. Importantly, human Pol (hPol) ε was found more processive than hPol δ in supporting proliferating cell nuclear antigen-dependent elongation of DNA chains, which is in keeping with proposed roles for hPol ε and hPol δ in the replication of leading and lagging strands, respectively. Furthermore, GINS, a component of the replicative helicase complex that is composed of Sld5, Psf1, Psf2, and Psf3, was shown to interact weakly with all three replicative DNA Pols (α, δ, and ε) and to markedly stimulate the activities of Pol α and Pol ε. In vivo studies indicated that siRNA-targeted depletion of hPol δ and/or hPol ε reduced cell cycle progression and the rate of fork progression. Under the conditions used, we noted that depletion of Pol ε had a more pronounced inhibitory effect on cellular DNA replication than depletion of Pol δ. We suggest that reduction in the level of Pol δ may be less deleterious because of its collision-and-release role in lagging strand synthesis.     

Balancing eukaryotic replication asymmetry with replication fidelity

Coordinated replication of eukaryotic nuclear genomes is asymmetric, with copying of a leading strand template preceding discontinuous copying of the lagging strand template. Replication is catalyzed by DNA polymerases α, δ and ?, enzymes that are related yet differ in physical and biochemical properties, including fidelity. Recent studies suggest that Pol ? is normally the primary leading strand replicase, whereas most synthesis by Pol δ occurs during lagging strand replication. New studies show that replication asymmetry can generate strand-specific genome instability resulting from biased deoxynucleotide pools and unrepaired ribonucleotides incorporated into DNA during replication, and that the eukaryotic replication machinery has evolved to most efficiently correct those replication errors that are made at the highest rates.     

Reconstitution of a minimal DNA replicase from Pseudomonas aeruginosa and stimulation by non-cognate auxiliary factors

DNA polymerase III holoenzyme is responsible for chromosomal replication in bacteria. The components and functions of Escherichia coli DNA polymerase III holoenzyme have been studied extensively. Here, we report the reconstitution of replicase activity by essential components of DNA polymerase holoenzyme from the pathogen Pseudomonas aeruginosa. We have expressed and purified the processivity factor (beta), single-stranded DNA-binding protein, a complex containing the polymerase (alpha) and exonuclease (epsilon) subunits, and the essential components of the DnaX complex (tau(3)deltadelta'). Efficient primer elongation requires the presence of alphaepsilon, beta, and tau(3)deltadelta'. Pseudomonas aeruginosa alphaepsilon can substitute completely for E. coli polymerase III in E. coli holoenzyme reconstitution assays. Pseudomonas beta and tau(3)deltadelta' exhibit a 10-fold lower activity relative to their E. coli counterparts in E. coli holoenzyme reconstitution assays. Although the Pseudomonas counterpart to the E. coli psi subunit was not apparent in sequence similarity searches, addition of purified E. coli chi and psi (components of the DnaX complex) increases the apparent specific activity of the Pseudomonas tau(3)deltadelta' complex approximately 10-fold and enables the reconstituted enzyme to function better under physiological salt conditions.     

Characterization of a triple DNA polymerase replisome

The replicase of all cells is thought to utilize two DNA polymerases for coordinated synthesis of leading and lagging strands. The DNA polymerases are held to DNA by circular sliding clamps. We demonstrate here that the E. coli DNA polymerase III holoenzyme assembles into a particle that contains three DNA polymerases. The three polymerases appear capable of simultaneous activity. Furthermore, the trimeric replicase is fully functional at a replication fork with helicase, primase, and sliding clamps; it produces slightly shorter Okazaki fragments than replisomes containing two DNA polymerases. We propose that two polymerases can function on the lagging strand and that the third DNA polymerase can act as a reserve enzyme to overcome certain types of obstacles to the replication fork.     

Polymerase dynamics at the eukaryotic DNA replication fork

This review discusses recent insights in the roles of DNA polymerases (Pol) delta and epsilon in eukaryotic DNA replication. A growing body of evidence specifies Pol epsilon as the leading strand DNA polymerase and Pol delta as the lagging strand polymerase during undisturbed DNA replication. New evidence supporting this model comes from the use of polymerase mutants that show an asymmetric mutator phenotype for certain mispairs, allowing an unambiguous strand assignment for these enzymes. On the lagging strand, Pol delta corrects errors made by Pol alpha during Okazaki fragment initiation. During Okazaki fragment maturation, the extent of strand displacement synthesis by Pol delta determines whether maturation proceeds by the short or long flap processing pathway. In the more common short flap pathway, Pol delta coordinates with the flap endonuclease FEN1 to degrade initiator RNA, whereas in the long flap pathway, RNA removal is initiated by the Dna2 nuclease/helicase.     

A novel assembly mechanism for the DNA polymerase III holoenzyme DnaX complex: association of deltadelta' with DnaX(4) forms DnaX(3)deltadelta'

We have constructed a plasmid-borne artificial operon that expresses the six subunits of the DnaX complex of Escherichia coli DNA polymerase III holoenzyme: tau, gamma, delta, delta', chi and psi. Induction of this operon followed by assembly in vivo produced two taugamma mixed DnaX complexes with stoichiometries of tau(1)gamma(2)deltadelta'chipsi and tau(2)gamma(1)deltadelta'chipsi rather than the expected gamma(2)tau(2)deltadelta'chipsi. We observed the same heterogeneity when taugamma mixed DnaX complexes were reconstituted in vitro. Re-examination of homomeric DnaX tau and gamma complexes assembled either in vitro or in vivo also revealed a stoichiometry of DnaX(3)deltadelta'chipsi. Equilibrium sedimentation analysis showed that free DnaX is a tetramer in equilibrium with a free monomer. An assembly mechanism, in which the association of heterologous subunits with a homomeric complex alters the stoichiometry of the homomeric assembly, is without precedent. The significance of our findings to the architecture of the holoenzyme and the clamp-assembly apparatus of all other organisms is discussed.     

tau binds and organizes Escherichia coli replication proteins through distinct domains: domain III, shared by gamma and tau, oligomerizes DnaX.

The tau and gamma proteins of the DNA polymerase III holoenzyme DnaX complex are products of the dnaX gene with gamma being a truncated version of tau arising from ribosomal frameshifting. tau is comprised of five structural domains, the first three of which are shared by gamma (Gao, D., and McHenry, C. (2001) J. Biol. Chem. 276, 4433-4453). In the absence of the other holoenzyme subunits, DnaX exists as a tetramer. Association of delta, delta', chi, and psi with domain III of DnaX(4) results in a DnaX complex with a stoichiometry of DnaX(3)deltadelta'chipsi. To identify which domain facilitates DnaX self-association, we examined the properties of purified biotin-tagged DnaX fusion proteins containing domains I-II or III-V. Unlike domain I-II, treatment of domain III-V, gamma, and tau with the chemical cross-linking reagent BS3 resulted in the appearance of high molecular weight intramolecular cross-linked protein. Gel filtration of domains I-II and III-V demonstrated that domain I-II was monomeric, and domain III-V was an oligomer. Biotin-tagged domain III-V, and not domain I-II, was able to form a mixed DnaX complex by recruiting tau, delta, delta', chi, and psi onto streptavidin-agarose beads. Thus, domain III not only contains the delta, delta', chi, and psi binding interface, but also the region that enables DnaX to oligomerize.     

Coordinated leading- and lagging-strand synthesis at the Escherichia coli DNA replication fork. V. Primase action regulates the cycle of Okazaki fragment synthesis.

Replication forks formed during rolling-circle DNA synthesis supported by a tailed form II DNA substrate in the presence of the primosome, the single-stranded DNA binding protein, and the DNA polymerase III holoenzyme (Pol III HE) that had been reconstituted from the purified subunits, beta, tau, and the gamma.delta complex, at limiting (with respect to nucleotide incorporation) concentrations of the Pol III core (alpha, epsilon, and theta) produced aberrantly small Okazaki fragments, while the synthesis of the leading strand was unperturbed. These small Okazaki fragments were not arrayed in tandem along the lagging-strand DNA template, but were separated by large gaps. Similarly structured synthetic products were not manufactured by replication forks reconstituted with higher, saturating concentrations of the Pol III core. Replication forks producing these small fragments could respond, by modulating the size of the Okazaki fragments produced, to variations in the concentration of NTPs or the primase, conditions that affect the frequency of priming on the lagging strand, but not to variation in the concentration of dNTPs, conditions that affect the frequency of utilization of the primers. Significantly longer Okazaki fragments (greater than 7 kilobases) could be produced in the presence of a limiting amount of Pol III core at low concentrations of the primase. These observations indicated that the production of small Okazaki fragments was not a result of a debilitated lagging-strand Pol III core, but rather a function of the time available for nascent strand synthesis during the cycle of events that are required for the manufacture of an Okazaki fragment and that it was the association of primase with the replication fork that keyed this cycle.     

Protein-protein interactions in the bacteriophage T4 replisome. The leading strand holoenzyme is physically linked to the lagging strand holoenzyme and the primosome

The bacteriophage T4 replication complex is composed of eight proteins that function together to replicate DNA. This replisome can be broken down into four basic units: a primosome composed of gp41, gp61, and gp59; a leading strand holoenzyme composed of gp43, gp44/62, and gp45; a lagging strand holoenzyme; and a single strand binding protein polymer. These units interact further to form the complete replisome. The leading and lagging strand polymerases are physically linked in the presence of DNA or an active replisome. The region of interaction was mapped to an extension of the finger domain, such that Cys-507 of one subunit is in close proximity to Cys-507 of a second subunit. The leading strand polymerase and the primosome also associate, such that gp59 mediates the contact between the two complexes. Binding of gp43 to the primosome complex causes displacement of gp32 from the gp59.gp61.gp41 primosome complex. The resultant species is a complex of proteins that may allow coordinated leading and lagging strand synthesis, helicase DNA unwinding activity, and polymerase nucleotide incorporation.     

Bacterial replicases and related polymerases

Bacterial replicases are complex, tripartite replicative machines. They contain a polymerase, Pol III, a β(2) processivity factor and a DnaX complex ATPase that loads β(2) onto DNA and chaperones Pol III onto the newly loaded β(2). Many bacteria encode both a full length τ and a shorter γ form of DnaX by a variety of mechanisms. The polymerase catalytic subunit of Pol III, α, contains a PHP domain that not only binds to prototypical ? Mg(2+)-dependent exonuclease, but also contains a second Zn(2+)-dependent proofreading exonuclease, at least in some bacteria. Replication of the chromosomes of low GC Gram-positive bacteria require two Pol IIIs, one of which, DnaE, appears to extend RNA primers a only short distance before handing the product off to the major replicase, PolC. Other bacteria encode a second Pol III (ImuC) that apparently replaces Pol V, required for induced mutagenesis in E. coli. Approaches that permit simultaneous biochemical screening of all components of complex bacterial replicases promise inhibitors of specific protein targets and reaction stages.     

The eukaryotic replisome tolerates leading‐strand base damage by replicase switching

The high‐fidelity replicative DNA polymerases, Pol ε and Pol δ, are generally thought to be poorly equipped to replicate damaged DNA. Direct and complete replication of a damaged template therefore typically requires the activity of low‐fidelity translesion synthesis (TLS) polymerases. Here we show that a yeast replisome, reconstituted with purified proteins, is inherently tolerant of the common oxidative lesion thymine glycol (Tg). Surprisingly, leading‐strand Tg was bypassed efficiently in the presence and absence of the TLS machinery. Our data reveal that following helicase–polymerase uncoupling a switch from Pol ε, the canonical leading‐strand replicase, to the lagging‐strand replicase Pol δ, facilitates rapid, efficient and error‐free lesion bypass at physiological nucleotide levels. This replicase switch mechanism also promotes bypass of the unrelated oxidative lesion, 8‐oxoguanine. We propose that replicase switching may promote continued leading‐strand synthesis whenever the replisome encounters leading‐strand damage that is bypassed more efficiently by Pol δ than by Pol ε.     

DNA polymerase III holoenzyme of Escherichia coli. III. Distinctive processive polymerases reconstituted from purified subunits

The 10 distinctive polypeptides of DNA polymerase III holoenzyme, purified as individual subunits or complexes, could be reconstituted to generate a polymerase with the high catalytic rate of the isolated intact holoenzyme. Functions and interactions of the subunits can be inferred from partial assemblies of the pol III core (alpha, epsilon, and theta subunits) with auxiliary subunits. The core possesses the polymerase and proofreading activities; the auxiliary subunits provide the core with processivity, the capacity to replicate long stretches of DNA without dissociating from the template. In a sequence of reconstruction steps, the beta subunit binds the primed template in an ATP-dependent manner through the catalytic action of a complex made up of the gamma, delta, delta', chi, and psi polypeptides. With the beta subunit in place, a processive polymerase is produced upon addition of the core. When the tau subunit is lacking, binding of polymerase to the primed template is less efficient and stable. The tau-less reconstituted polymerase is more prone to dissociation upon encountering secondary structures in the template in its path, such as a hairpin region in the single strand or a duplex region formed by a strand annealed to the template. With the tau subunit present, the interaction of the core.beta complex (the basic unit of a processive polymerase) with the primed template is strengthened. The tau-containing reconstituted polymerase can replicate DNA continuously through secondary structures in the template. The two distinctive kinds of processivity demonstrated by the tau-less and tau-containing reconstituted polymerases fit nicely into a scheme in which, organized as an asymmetric dimeric holoenzyme, the tau half is responsible for continuous synthesis of one strand, and the less stable half for discontinuous synthesis of the other.     

The asymmetric dimeric polymerase hypothesis: A progress report

In 1983, my laboratory first proposed that the DNA polymerase III holoenzyme is an asymmetric dimer with distinguishable leading and lagging strand polymerases. Here, I review progress by my laboratory and others in testing this hypothesis. To date, the hypothesis is supported by our demonstration of (i) an asymmetry in function of two populations of holoenzyme in solution in their ability to use the ATP analog, ATPγS, to support initiation complex formation, (ii) the stabilization of a dimeric polymerase structure by the τ subunit, (iii) allosteric communication between polymerase halves and (iv) the coexistence of γ and the τ, subunits which share common sequences, within the same holoenzyme assemblies. This latter observation may provide a structural basis for holoenzyme asymmetry. I discuss the implications of the asymmetric dimer hypothesis to the solution of problems encountered by polymerases at the replication fork and delineate further tests required before the hypothesis can be firmly established.