Conserved interactions in the Staphylococcus aureus DNA PolC chromosome replication machine

The PolC holoenzyme replicase of the Gram-positive Staphylococcus aureus pathogen has been reconstituted from pure subunits. We compared individual S. aureus replicase subunits with subunits from the Gram-negative Escherichia coli polymerase III holoenzyme for activity and interchangeability. The central organizing subunit, tau, is smaller than its Gram-negative homolog, yet retains the ability to bind single-stranded DNA and contains DNA-stimulated ATPase activity comparable with E. coli tau. S. aureus tau also stimulates PolC, although they do not form as stabile a complex as E. coli polymerase III.tau. We demonstrate that the extreme C-terminal residues of PolC bind to and function with beta clamps from different bacteria. Hence, this polymerase-clamp interaction is highly conserved. Additionally, the S. aureus delta wrench of the clamp loader binds to E. coli beta. The S. aureus clamp loader is even capable of loading E. coli and Streptococcus pyogenes beta clamps onto DNA. Interestingly, S. aureus PolC lacks functionality with heterologous beta clamps when they are loaded onto DNA by the S. aureus clamp loader, suggesting that the S. aureus clamp loader may have difficulty ejecting from heterologous clamps. Nevertheless, these overall findings underscore the conservation in structure and function of Gram-positive and Gram-negative replicases despite >1 billion years of evolutionary distance between them.     

Analysis of a multicomponent thermostable DNA polymerase III replicase from an extreme thermophile

This report takes a proteomic/genomic approach to characterize the DNA polymerase III replication apparatus of the extreme thermophile, Aquifex aeolicus. Genes (dnaX, holA, and holB) encoding the subunits required for clamp loading activity (tau, delta, and delta') were identified. The dnaX gene produces only the full-length product, tau, and therefore differs from Escherichia coli dnaX that produces two proteins (gamma and tau). Nonetheless, the A. aeolicus proteins form a taudeltadelta' complex. The dnaN gene encoding the beta clamp was identified, and the taudeltadelta' complex is active in loading beta onto DNA. A. aeolicus contains one dnaE homologue, encoding the alpha subunit of DNA polymerase III. Like E. coli, A. aeolicus alpha and tau interact, although the interaction is not as tight as the alpha-tau contact in E. coli. In addition, the A. aeolicus homologue to dnaQ, encoding the epsilon proofreading 3'-5'-exonuclease, interacts with alpha but does not form a stable alpha.epsilon complex, suggesting a need for a brace or bridging protein to tightly couple the polymerase and exonuclease in this system. Despite these differences to the E. coli system, the A. aeolicus proteins function to yield a robust replicase that retains significant activity at 90 degrees C. Similarities and differences between the A. aeolicus and E. coli pol III systems are discussed, as is application of thermostable pol III to biotechnology.     

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.     

Cross-utilization of the beta sliding clamp by replicative polymerases of evolutionary divergent organisms

Chromosomal replicases are multiprotein machines comprised of a DNA polymerase, a sliding clamp, and a clamp loader. This study examines replicase components for their ability to be switched between Gram-positive and Gram-negative organisms. These two cell types diverged over 1 billion years ago, and their sequences have diverged widely. Yet the Escherichia coli beta clamp binds directly to Staphylococcus aureus PolC and makes it highly processive, confirming and extending earlier results (Low, R. L., Rashbaum, S. A. , and Cozzarelli, N. R. (1976) J. Biol. Chem. 251, 1311-1325). We have also examined the S. aureus beta clamp. The results show that it functions with S. aureus PolC, but not with E. coli polymerase III core. PolC is a rather potent polymerase by itself and can extend a primer with an intrinsic speed of 80-120 nucleotides per s. Both E. coli beta and S. aureus beta converted PolC to a highly processive polymerase, but surprisingly, beta also increased the intrinsic rate of DNA synthesis to 240-580 nucleotides per s. This finding expands the scope of beta function beyond a simple mechanical tether for processivity to include that of an effector that increases the intrinsic rate of nucleotide incorporation by the polymerase.     

Analysis of the ATPase subassembly which initiates processive DNA synthesis by DNA polymerase III holoenzyme.

The gamma complex (gamma delta delta' chi psi) subassembly of DNA polymerase III holoenzyme transfers the beta subunit onto primed DNA in a reaction which requires ATP hydrolysis. Once on DNA, beta is a "sliding clamp" which tethers the polymerase to DNA for highly processive synthesis. We have examined beta and the gamma complex to identify which subunit(s) hydrolyzes ATP. We find the gamma complex is a DNA dependent ATPase. The beta subunit, which lacks ATPase activity, enhances the gamma complex ATPase when primed DNA is used as an effector. Hence, the gamma complex recognizes DNA and couples ATP hydrolysis to clamp beta onto primed DNA. Study of gamma complex subunits showed no single subunit contained significant ATPase activity. However, the heterodimers, gamma delta and gamma delta', were both DNA-dependent ATPases. Only the gamma delta ATPase was stimulated by beta and was functional in transferring the beta from solution to primed DNA. Similarity in ATPase activity of DNA polymerase III holoenzyme accessory proteins to accessory proteins of phage T4 DNA polymerase and mammalian DNA polymerase delta suggests the basic strategy of chromosome duplication has been conserved throughout evolution.     

The delta subunit of DNA polymerase III holoenzyme serves as a sliding clamp unloader in Escherichia coli

In Escherichia coli, the circular beta sliding clamp facilitates processive DNA replication by tethering the polymerase to primer-template DNA. When synthesis is complete, polymerase dissociates from beta and DNA and cycles to a new start site, a primed template loaded with beta. DNA polymerase cycles frequently during lagging strand replication while synthesizing 1-2-kilobase Okazaki fragments. The clamps left behind remain stable on DNA (t(12) approximately 115 min) and must be removed rapidly for reuse at numerous primed sites on the lagging strand. Here we show that delta, a single subunit of DNA polymerase III holoenzyme, opens beta and slips it off DNA (k(unloading) = 0.011 s(-)(1)) at a rate similar to that of the multisubunit gamma complex clamp loader by itself (0.015 s(-)(1)) or within polymerase (pol) III* (0.0065 s(-)(1)). Moreover, unlike gamma complex and pol III*, delta does not require ATP to catalyze clamp unloading. Quantitation of gamma complex subunits (gamma, delta, delta', chi, psi) in E. coli cells reveals an excess of delta, free from gamma complex and pol III*. Since pol III* and gamma complex occur in much lower quantities and perform several DNA metabolic functions in replication and repair, the delta subunit probably aids beta clamp recycling during DNA replication.     

Interplay of clamp loader subunits in opening the beta sliding clamp of Escherichia coli DNA polymerase III holoenzyme

The Escherichia coli beta dimer is a ring-shaped protein that encircles DNA and acts as a sliding clamp to tether the replicase, DNA polymerase III holoenzyme, to DNA. The gamma complex (gammadeltadelta'chipsi) clamp loader couples ATP to the opening and closing of beta in assembly of the ring onto DNA. These proteins are functionally and structurally conserved in all cells. The eukaryotic equivalents are the replication factor C (RFC) clamp loader and the proliferating cell nuclear antigen (PCNA) clamp. The delta subunit of the E. coli gamma complex clamp loader is known to bind beta and open it by parting one of the dimer interfaces. This study demonstrates that other subunits of gamma complex also bind beta, although weaker than delta. The gamma subunit like delta, affects the opening of beta, but with a lower efficiency than delta. The delta' subunit regulates both gamma and delta ring opening activities in a fashion that is modulated by ATP interaction with gamma. The implications of these actions for the workings of the E. coli clamp loading machinery and for eukaryotic RFC and PCNA are discussed.     

Mechanism of processivity clamp opening by the delta subunit wrench of the clamp loader complex of E. coli DNA polymerase III

The dimeric ring-shaped sliding clamp of E. coli DNA polymerase III (beta subunit, homolog of eukaryotic PCNA) is loaded onto DNA by the clamp loader gamma complex (homolog of eukaryotic Replication Factor C, RFC). The delta subunit of the gamma complex binds to the beta ring and opens it. The crystal structure of a beta:delta complex shows that delta, which is structurally related to the delta' and gamma subunits of the gamma complex, is a molecular wrench that induces or traps a conformational change in beta such that one of its dimer interfaces is destabilized. Structural comparisons and molecular dynamics simulations suggest a spring-loaded mechanism in which the beta ring opens spontaneously once a dimer interface is perturbed by the delta wrench.     

Fluorescence measurements on the E.coli DNA polymerase clamp loader: implications for conformational changes during ATP and clamp binding

Sliding clamps are ring-shaped proteins that tether DNA polymerases to their templates during processive DNA replication. The action of ATP-dependent clamp loader complexes is required to open the circular clamps and to load them onto DNA. The crystal structure of the pentameric clamp loader complex from Escherichia coli (the gamma complex), determined in the absence of nucleotides, revealed a highly asymmetric and extended form of the clamp loader. Consideration of this structure suggested that a compact and more symmetrical inactive form may predominate in solution in the absence of crystal packing forces. This model has the N-terminal domains of the delta and delta' subunits of the clamp loader close to each other in the inactive state, with the clamp loader opening in a crab-claw-like fashion upon ATP-binding. We have used fluorescence resonance energy transfer (FRET) to investigate the structural changes in the E.coli clamp loader complex that result from ATP-binding and interactions between the clamp loader and the beta clamp. FRET measurements using fluorophores placed in the N-terminal domains of the delta and delta' subunits indicate that the distances between these subunits in solution are consistent with the previously crystallized extended form of the clamp loader. Furthermore, the addition of nucleotide and clamp to the labeled clamp loader does not appreciably alter these FRET distances. Our results suggest that the changes that occur in the relative positioning of the delta and delta' subunits when ATP binds to and activates the complex are subtle, and that crab-claw-like movements are not a significant component of the clamp loader mechanism.     

The internal workings of a DNA polymerase clamp-loading machine.

Replicative DNA polymerases are multiprotein machines that are tethered to DNA during chain extension by sliding clamp proteins. The clamps are designed to encircle DNA completely, and they are manipulated rapidly onto DNA by the ATP-dependent activity of a clamp loader. We outline the detailed mechanism of gamma complex, a five-protein clamp loader that is part of the Escherichia coli replicase, DNA polymerase III holoenzyme. The gamma complex uses ATP to open the beta clamp and assemble it onto DNA. Surprisingly, ATP is not needed for gamma complex to crack open the beta clamp. The function of ATP is to regulate the activity of one subunit, delta, which opens the clamp simply by binding to it. The delta' subunit acts as a modulator of the interaction between delta and beta. On binding ATP, the gamma complex is activated such that the delta' subunit permits delta to bind beta and crack open the ring at one interface. The clamp loader-open clamp protein complex is now ready for an encounter with primed DNA to complete assembly of the clamp around DNA. Interaction with DNA stimulates ATP hydrolysis which ejects the gamma complex from DNA, leaving the ring to close around the duplex.     

Tau binds and organizes Escherichia coli replication proteins through distinct domains. Domain III, shared by gamma and tau, binds delta delta ' and chi psi

The DnaX complex of the DNA polymerase holoenzyme assembles the beta(2) processivity factor onto the primed template enabling highly processive replication. The key ATPases within this complex are tau and gamma, alternative frameshift products of the dnaX gene. Of the five domains of tau, I-III are shared with gamma In vivo, gamma binds the auxiliary subunits deltadelta' and chipsi (Glover, B. P., and McHenry, C. S. (2000) J. Biol. Chem. 275, 3017-3020). To localize deltadelta' and chipsi binding domains within gamma domains I-III, we measured the binding of purified biotin-tagged DnaX proteins lacking specific domains to deltadelta' and chipsi by surface plasmon resonance. Fusion proteins containing either DnaX domains I-III or domains III-V bound deltadelta' and chipsi subunits. A DnaX protein only containing domains I and II did not bind deltadelta' or chipsi. The binding affinity of chipsi for DnaX domains I-III and domains III-V was the same as that of chipsi for full-length tau, indicating that domain III contained all structural elements required for chipsi binding. Domain III of tau also contained deltadelta' binding sites, although the interaction between deltadelta' and domains III-V of tau was 10-fold weaker than the interaction between deltadelta' and full length tau. The presence of both delta and chipsi strengthened the delta'-C(0)tau interaction by at least 15-fold. Domain III was the only domain common to all of tau fusion proteins whose interaction with delta' was enhanced in the presence of delta and chipsi. Thus, domain III of the DnaX proteins not only contains the deltadelta' and chipsi binding sites but also contains the elements required for the positive cooperative assembly of the DnaX complex.     

Assembly of DNA polymerase III holoenzyme: co-assembly of gamma and tau is inhibited by DnaX complex accessory proteins but stimulated by DNA polymerase III core.

Although the two alternative Escherichia coli dnaX gene products, tau and gamma, are found co-assembled in purified DNA polymerase III holoenzyme, the pathway of assembly is not well understood. When the 10 subunits of holoenzyme are simultaneously mixed, they rapidly form a nine-subunit assembly containing tau but not gamma. We developed a new assay based on the binding of complexes containing biotin-tagged tau to streptavidin-coated agarose beads to investigate the effects of various DNA polymerase III holoenzyme subunits on the kinetics of co-assembly of gamma and tau into the same complex. Auxiliary proteins in combination with delta' almost completely blocked co-assembly, whereas chipsi or delta' alone slowed the association only moderately compared with the interaction of tau with gamma alone. In contrast, DNA polymerase III core, in the absence of deltadelta' and chipsi, accelerated the co-assembly of tau and gamma, suggesting a role for DNA polymerase III' [tau(2)(pol III core)(2)] in the assembly pathway of holoenzyme.     

The delta and delta ' subunits of the DNA polymerase III holoenzyme are essential for initiation complex formation and processive elongation.

delta and delta' are required for assembly of the processivity factor beta(2) onto primed DNA in the DNA polymerase III holoenzyme-catalyzed reaction. We developed protocols for generating highly purified preparations of delta and delta'. In holoenzyme reconstitution assays, delta' could not be replaced by delta, tau, or gamma, even when either of the latter were present at a 10,000-fold molar excess. Likewise, delta could not be replaced by delta', tau, or gamma. Bacterial strains bearing chromosomal knockouts of either the holA(delta) or holB(delta') genes were not viable, demonstrating that both delta and delta' are essential. Western blots of isolated initiation complexes demonstrated the presence of both delta and delta'. However, in the absence of chipsi and single-stranded DNA-binding protein, a stable initiation complex lacking deltadelta' was isolated by gel filtration. Lack of delta-delta' decreased the rate of elongation about 3-fold, and the extent of processive replication was significantly decreased. Adding back delta-delta' but not chipsi, delta, or delta' alone restored the diminished activity, indicating that in addition to being key components required for the beta loading activity of the DnaX complex, deltadelta' is present in initiation complex and is required for processive elongation.     

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.     

Mechanism of the delta wrench in opening the beta sliding clamp

The beta sliding clamp encircles DNA and tethers DNA polymerase III holoenzyme to the template for high processivity. The clamp loader, gamma complex (gamma 3 delta delta'chi psi), assembles beta around DNA in an ATP-fueled reaction. The delta subunit of the clamp loader opens the beta ring and is referred to as the wrench; ATP modulates contact between beta and delta among other functions. Crystal structures of delta.beta and the gamma 3 delta delta' minimal clamp loader make predictions of the clamp loader mechanism, which are tested in this report by mutagenesis. The delta wrench contacts beta at two sites. One site is at the beta dimer interface, where delta appears to distort the interface by via a steric clash between a helix on delta and a loop near the beta interface. The energy for this steric clash is thought to derive from the other site of interaction, in which delta binds to a hydrophobic pocket in beta. The current study demonstrates that rather than a simple steric clash with beta, delta specifically contacts beta at this site, but not through amino acid side chains, and thus is presumably mediated by peptide backbone atoms. The results also imply that the interaction of delta at the hydrophobic site on beta contributes to destabilization of the beta dimer interface rather than acting solely as a grip of delta on beta. Within the gamma complex, delta' is proposed to prevent delta from binding to beta in the absence of ATP. This report demonstrates that one or more gamma subunits also contribute to this role. The results also indicate that delta' acts as a backboard upon which the gamma subunits push to attain the ATP induced change needed for the delta wrench to bind and open the beta ring.     

Dual functions, clamp opening and primer-template recognition, define a key clamp loader subunit

Clamp loader proteins catalyze assembly of circular sliding clamps on DNA to enable processive DNA replication. During the reaction, the clamp loader binds primer-template DNA and positions it in the center of a clamp to form a topological link between the two. Clamp loaders are multi-protein complexes, such as the five protein Escherichia coli, Saccharomyces cerevisiae, and human clamp loaders, and the two protein Pyrococcus furiosus and Methanobacterium thermoautotrophicum clamp loaders, and thus far the site(s) responsible for binding and selecting primer-template DNA as the target for clamp assembly remain unknown. To address this issue, we analyzed the interaction between the E.coli gamma complex clamp loader and DNA using UV-induced protein-DNA cross-linking and mass spectrometry. The results show that the delta subunit in the gamma complex makes close contact with the primer-template junction. Tryptophan 279 in the delta C-terminal domain lies near the 3'-OH primer end and may play a key role in primer-template recognition. Previous studies have shown that delta also binds and opens the beta clamp (hydrophobic residues in the N-terminal domain of delta contact beta. The clamp-binding and DNA-binding sites on delta appear positioned for facile entry of primer-template into the center of the clamp and exit of the template strand from the complex. A similar analysis of the S.cerevisiae RFC complex suggests that the dual functionality observed for delta in the gamma complex may be true also for clamp loaders from other organisms.     

A three-domain structure for the delta subunit of the DNA polymerase III holoenzyme delta domain III binds delta' and assembles into the DnaX complex.

Using psi-BLAST, we have developed a method for identifying the poorly conserved delta subunit of the DNA polymerase III holoenzyme from all sequenced bacteria. This approach, starting with Escherichia coli delta, leads not only to the identification of delta but also to the DnaX and delta' subunits of the DnaX complex and other AAA(+)-class ATPases. This suggests that, although not an ATPase, delta is related structurally to the other subunits of the DnaX complex that loads the beta sliding clamp processivity factor onto DNA. To test this prediction, we aligned delta sequences with those of delta' and, using the start of delta' Domain III established from its x-ray crystal structure, predicted the juncture between Domains II and III of delta. This putative delta Domain III could be expressed to high levels, consistent with the prediction that it folds independently. delta Domain III, like Domain III of DnaX and delta', assembles by itself into a complex with the other DnaX complex components. Cross-linking studies indicated a contact of delta with the DnaX subunits. These observations are consistent with a model where two tau subunits and one each of the gamma, delta', and delta subunits mutually interact to form a pentameric functional core for the DnaX complex.     

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.     

Mechanism of beta clamp opening by the delta subunit of Escherichia coli DNA polymerase III holoenzyme

The beta sliding clamp encircles the primer-template and tethers DNA polymerase III holoenzyme to DNA for processive replication of the Escherichia coli genome. The clamp is formed via hydrophobic and ionic interactions between two semicircular beta monomers. This report demonstrates that the beta dimer is a stable closed ring and is not monomerized when the gamma complex clamp loader (gamma(3)delta(1)delta(1)chi(1)psi(1)) assembles the beta ring around DNA. delta is the subunit of the gamma complex that binds beta and opens the ring; it also does not appear to monomerize beta. Point mutations were introduced at the beta dimer interface to test its structural integrity and gain insight into its interaction with delta. Mutation of two residues at the dimer interface of beta, I272A/L273A, yields a stable beta monomer. We find that delta binds the beta monomer mutant at least 50-fold tighter than the beta dimer. These findings suggest that when delta interacts with the beta clamp, it binds one beta subunit with high affinity and utilizes some of that binding energy to perform work on the dimeric clamp, probably cracking one dimer interface open.     

Mechanism of the E. coli tau processivity switch during lagging-strand synthesis

The E. coli replication machinery employs a beta clamp that tethers the polymerase to DNA, thus ensuring high processivity. The replicase also contains a processivity switch that dissociates the polymerase from its beta clamp. The switch requires the tau subunit of the clamp loader and is regulated by different DNA structures. At a primed site, the switch is "off." When the replicase reaches the downstream primer to form a nick, the switch is flipped, and tau ejects the polymerase from beta. This switch has high fidelity for completed synthesis, remaining "off" until just prior to incorporation of the last nucleotide and turning "on" only after addition of the last dNTP. These actions of tau are confined to its C-terminal region, which is located outside the clamp loading apparatus. Thus, this highly processive replication machine has evolved a mechanism to specifically counteract processivity at a defined time in the lagging-strand cycle.