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.     

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.     

Mechanism of loading the Escherichia coli DNA polymerase III beta sliding clamp on DNA. Bona fide primer/templates preferentially trigger the gamma complex to hydrolyze ATP and load the clamp

The Escherichia coli DNA polymerase III gamma complex clamp loader assembles the ring-shaped beta sliding clamp onto DNA. The core polymerase is tethered to the template by beta, enabling processive replication of the genome. Here we investigate the DNA substrate specificity of the clamp-loading reaction by measuring the pre-steady-state kinetics of DNA binding and ATP hydrolysis using elongation-proficient and deficient primer/template DNA. The ATP-bound clamp loader binds both elongation-proficient and deficient DNA substrates either in the presence or absence of beta. However, elongation-proficient DNA preferentially triggers gamma complex to release beta onto DNA with concomitant hydrolysis of ATP. Binding to elongation-proficient DNA converts the gamma complex from a high affinity ATP-bound state to an ADP-bound state having a 10(5)-fold lower affinity for DNA. Steady-state binding assays are misleading, suggesting that gamma complex binds much more avidly to non-extendable primer/template DNA because recycling to the high affinity binding state is rate-limiting. Pre-steady-state rotational anisotropy data reveal a dynamic association-dissociation of gamma complex with extendable primer/templates leading to the diametrically opposite conclusion. The strongly favored dynamic recognition of extendable DNA does not require the presence of beta. Thus, the gamma complex uses ATP binding and hydrolysis as a mechanism for modulating its interaction with DNA in which the ATP-bound form binds with high affinity to DNA but elongation-proficient DNA substrates preferentially trigger hydrolysis of ATP and conversion to a low affinity state.     

Mechanism of loading the Escherichia coli DNA polymerase III sliding clamp: I. Two distinct activities for individual ATP sites in the gamma complex

The Escherichia coli DNA polymerase III gamma complex loads the beta clamp onto DNA, and the clamp tethers the core polymerase to DNA to increase the processivity of synthesis. ATP binding and hydrolysis promote conformational changes within the gamma complex that modulate its affinity for the clamp and DNA, allowing it to accomplish the mechanical task of assembling clamps on DNA. This is the first of two reports (Snyder, A. K., Williams, C. R., Johnson, A., O'Donnell, M., and Bloom, L. B. (2004) J. Biol. Chem. 279, 4386-4393) addressing the question of how ATP binding and hydrolysis modulate specific interactions with DNA and beta. Pre-steady-state rates of ATP hydrolysis were slower when reactions were initiated by addition of ATP than when the gamma complex was equilibrated with ATP and were limited by the rate of an intramolecular reaction, possibly ATP-induced conformational changes. Kinetic modeling of assays in which the gamma complex was incubated with ATP for different periods of time prior to adding DNA to trigger hydrolysis suggests a mechanism in which a relatively slow conformational change step (kforward = 6.5 s(-1)) produces a species of the gamma complex that is activated for DNA (and beta) binding. In the absence of beta, 2 of the 3 molecules of ATP are hydrolyzed rapidly prior to releasing DNA, and the 3rd molecule is hydrolyzed slowly. In the presence of beta, all 3 molecules of ATP are hydrolyzed rapidly. These results suggest that hydrolysis of 2 molecules of ATP may be coupled to conformational changes that reduce interactions with DNA, whereas hydrolysis of the 3rd is coupled to changes that result in release of beta.     

Molecular mechanism and energetics of clamp assembly in Escherichia coli. The role of ATP hydrolysis when gamma complex loads beta on DNA

Escherichia coli DNA polymerase III holoenzyme is a multisubunit composite containing the beta sliding clamp and clamp loading gamma complex. The gamma complex requires ATP to load beta onto DNA. A two-color fluorescence spectroscopic approach was utilized to study this system, wherein both assembly (red fluorescence; X-rhodamine labeled DNA anisotropy assay) and ATP hydrolysis (green fluorescence; phosphate binding protein assay) were simultaneously measured with millisecond timing resolution. The two temporally correlated stopped-flow signals revealed that a preassembled beta. gamma complex composite rapidly binds primer/template DNA in an ATP hydrolysis independent step. Once bound, two molecules of ATP are rapidly hydrolyzed (approximately 34 s(-1)). Following hydrolysis, gamma complex dissociates from the DNA ( approximately 22 s(-1)). Once dissociated, the next cycle of loading is severely compromised, resulting in steady-state ATP hydrolysis rates with a maximum of only approximately 3 s(-1). Two single-site beta dimer interface mutants were examined which had impaired steady-state rates of ATP hydrolysis. The pre-steady-state correlated kinetics of these mutants revealed a pattern essentially identical to wild type. The anisotropy data showed that these mutants decrease the steady-state rates of ATP hydrolysis by causing a buildup of "stuck" binary-ternary complexes on the primer/template DNA.     

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.     

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.     

Ordered ATP hydrolysis in the gamma complex clamp loader AAA+ machine

The gamma complex couples ATP hydrolysis to the loading of beta sliding clamps onto DNA for processive replication. The gamma complex structure shows that the clamp loader subunits are arranged as a circular heteropentamer. The three gamma motor subunits bind ATP, the delta wrench opens the beta ring, and the delta' stator modulates the delta-beta interaction. Neither delta nor delta' bind ATP. This report demonstrates that the delta' stator contributes a catalytic arginine for hydrolysis of ATP bound to the adjacent gamma(1) subunit. Thus, the delta' stator contributes to the motor function of the gamma trimer. Mutation of arginine 169 of gamma, which removes the catalytic arginines from only the gamma(2) and gamma(3) ATP sites, abolishes ATPase activity even though ATP site 1 is intact and all three sites are filled. This result implies that hydrolysis of the three ATP molecules occurs in a particular order, the reverse of ATP binding, where ATP in site 1 is not hydrolyzed until ATP in sites 2 and/or 3 is hydrolyzed. Implications of these results to clamp loaders of other systems are discussed.     

A function for the psi subunit in loading the Escherichia coli DNA polymerase sliding clamp

Crystal structures of an Escherichia coli clamp loader have provided insight into the mechanism by which this molecular machine assembles ring-shaped sliding clamps onto DNA. The contributions made to the clamp loading reaction by two subunits, chi and psi, which are not present in the crystal structures, were determined by measuring the activities of three forms of the clamp loader, gamma(3)deltadelta', gamma(3)deltadelta'psi, and gamma(3)deltadelta'psichi. The psi subunit is important for stabilizing an ATP-induced conformational state with high affinity for DNA, whereas the chi subunit does not contribute directly to clamp loading in our assays lacking single-stranded DNA-binding protein. The psi subunit also increases the affinity of the clamp loader for the clamp in assays in which ATPgammaS is substituted for ATP. Interestingly, the affinity of the gamma(3)deltadelta' complex for beta is no greater in the presence than in the absence of ATPgammaS. A role for psi in stabilizing or promoting ATP- and ATPgammaS-induced conformational changes may explain why large conformational differences were not seen in gamma(3)deltadelta' structures with and without bound ATPgammaS. The beta clamp partially compensates for the activity of psi when this subunit is not present and possibly serves as a scaffold on which the clamp loader adopts the appropriate conformation for DNA binding and clamp loading. Results from our work and others suggest that the psi subunit may introduce a temporal order to the clamp loading reaction in which clamp binding precedes DNA binding.     

Mechanism of loading the Escherichia coli DNA polymerase III sliding clamp: II. Uncoupling the beta and DNA binding activities of the gamma complex

Sliding clamps tether DNA polymerases to DNA to increase the processivity of synthesis. The Escherichia coli gamma complex loads the beta sliding clamp onto DNA in an ATP-dependent reaction in which ATP binding and hydrolysis modulate the affinity of the gamma complex for beta and DNA. This is the second of two reports (Williams, C. R., Snyder, A. K., Kuzmic, P., O'Donnell, M., and Bloom, L. B. (2004) J. Biol. Chem. 279, 4376-4385) addressing the question of how ATP binding and hydrolysis regulate specific interactions with DNA and beta. Mutations were made to an Arg residue in a conserved SRC motif in the delta' and gamma subunits that interacts with the ATP site of the neighboring gamma subunit. Mutation of the delta' subunit reduced the ATP-dependent beta binding activity, whereas mutation of the gamma subunits reduced the DNA binding activity of the gamma complex. The gamma complex containing the delta' mutation gave a pre-steady-state burst of ATP hydrolysis, but at a reduced rate and amplitude relative to the wild-type gamma complex. A pre-steady-state burst of ATP hydrolysis was not observed for the complex containing the gamma mutations, consistent with the reduced DNA binding activity of this complex. The differential effects of these mutations suggest that ATP binding at the gamma1 site may be coupled to conformational changes that largely modulate interactions with beta, whereas ATP binding at the gamma2 and/or gamma3 site may be coupled to conformational changes that have a major role in interactions with DNA. Additionally, these results show that the "arginine fingers" play a structural role in facilitating the formation of a conformation that has high affinity for beta and DNA.     

ATP binding and hydrolysis-driven rate-determining events in the RFC-catalyzed PCNA clamp loading reaction

The multi-subunit replication factor C (RFC) complex loads circular proliferating cell nuclear antigen (PCNA) clamps onto DNA where they serve as mobile tethers for polymerases and coordinate the functions of many other DNA metabolic proteins. The clamp loading reaction is complex, involving multiple components (RFC, PCNA, DNA, and ATP) and events (minimally: PCNA opening/closing, DNA binding/release, and ATP binding/hydrolysis) that yield a topologically linked clamp·DNA product in less than a second. Here, we report pre-steady-state measurements of several steps in the reaction catalyzed by Saccharomyces cerevisiae RFC and present a comprehensive kinetic model based on global analysis of the data. Highlights of the reaction mechanism are that ATP binding to RFC initiates slow activation of the clamp loader, enabling it to open PCNA (at ~2 s(-1)) and bind primer-template DNA (ptDNA). Rapid binding of ptDNA leads to formation of the RFC·ATP·PCNA(open)·ptDNA complex, which catalyzes a burst of ATP hydrolysis. Another slow step in the reaction follows ATP hydrolysis and is associated with PCNA closure around ptDNA (8 s(-1)). Dissociation of PCNA·ptDNA from RFC leads to catalytic turnover. We propose that these early and late rate-determining events are intramolecular conformational changes in RFC and PCNA that control clamp opening and closure, and that ATP binding and hydrolysis switch RFC between conformations with high and low affinities, respectively, for open PCNA and ptDNA, and thus bookend the clamp loading reaction.     

DNA structure requirements for the Escherichia coli gamma complex clamp loader and DNA polymerase III holoenzyme

The Escherichia coli chromosomal replicase, DNA polymerase III holoenzyme, is highly processive during DNA synthesis. Underlying high processivity is a ring-shaped protein, the beta clamp, that encircles DNA and slides along it, thereby tethering the enzyme to the template. The beta clamp is assembled onto DNA by the multiprotein gamma complex clamp loader that opens and closes the beta ring around DNA in an ATP-dependent manner. This study examines the DNA structure required for clamp loading action. We found that the gamma complex assembles beta onto supercoiled DNA (replicative form I), but only at very low ionic strength, where regions of unwound DNA may exist in the duplex. Consistent with this, the gamma complex does not assemble beta onto relaxed closed circular DNA even at low ionic strength. Hence, a 3'-end is not required for clamp loading, but a single-stranded DNA (ssDNA)/double-stranded DNA (dsDNA) junction can be utilized as a substrate, a result confirmed using synthetic oligonucleotides that form forked ssDNA/dsDNA junctions on M13 ssDNA. On a flush primed template, the gamma complex exhibits polarity; it acts specifically at the 3'-ssDNA/dsDNA junction to assemble beta onto the DNA. The gamma complex can assemble beta onto a primed site as short as 10 nucleotides, corresponding to the width of the beta ring. However, a protein block placed closer than 14 base pairs (bp) upstream from the primer 3' terminus prevents the clamp loading reaction, indicating that the gamma complex and its associated beta clamp interact with approximately 14-16 bp at a ssDNA/dsDNA junction during the clamp loading operation. A protein block positioned closer than 20-22 bp from the 3' terminus prevents use of the clamp by the polymerase in chain elongation, indicating that the polymerase has an even greater spatial requirement than the gamma complex on the duplex portion of the primed site for function with beta. Interestingly, DNA secondary structure elements placed near the 3' terminus impose similar steric limits on the gamma complex and polymerase action with beta. The possible biological significance of these structural constraints is discussed.     

A central swivel point in the RFC clamp loader controls PCNA opening and loading on DNA

Replication factor C (RFC) is a five-subunit complex that loads proliferating cell nuclear antigen (PCNA) clamps onto primer-template DNA (ptDNA) during replication. RFC subunits belong to the AAA(+) superfamily, and their ATPase activity drives interactions between the clamp loader, the clamp, and the ptDNA, leading to topologically linked PCNA·ptDNA. We report the kinetics of transient events in Saccharomyces cerevisiae RFC-catalyzed PCNA loading, including ATP-induced RFC activation, PCNA opening, ptDNA binding, ATP hydrolysis, PCNA closing, and PCNA·ptDNA release. This detailed perspective enables assessment of individual RFC-A, RFC-B, RFC-C, RFC-D, and RFC-E subunit functions in the reaction mechanism. Functions have been ascribed to RFC subunits previously based on a steady-state analysis of 'arginine-finger' ATPase mutants; however, pre-steady-state analysis provides a different view. The central subunit RFC-C serves as a critical swivel point in the clamp loader. ATP binding to this subunit initiates RFC activation, and the clamp loader adopts a spiral conformation that stabilizes PCNA in a corresponding open spiral. The importance of RFC subunit response to ATP binding decreases as RFC-C>RFC-D>RFC-B, with RFC-A being unnecessary. RFC-C-dependent activation of RFC also enables ptDNA binding, leading to the formation of the RFC·ATP·PCNA(open)·ptDNA complex. Subsequent ATP hydrolysis leads to complex dissociation, with RFC-D activity contributing the most to rapid ptDNA release. The pivotal role of the RFC-B/C/D subunit ATPase core in clamp loading is consistent with the similar central location of all three ATPase active subunits of the Escherichia coli clamp loader.     

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.     

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.     

Dissection of the ATP-driven reaction cycle of the bacteriophage T4 DNA replication processivity clamp loading system

Processive DNA replication requires the loading of a multisubunit ring-shaped protein complex, known as a sliding or processivity clamp, onto the primer-template (p/t) DNA. This clamp then binds to the replication polymerase to form a processive polymerase holoenzyme. The processivity of the holoenzyme derives from the topological properties of the clamp, which encircles the DNA without actually binding to it. Multisubunit complexes known as clamp-loaders utilize ATP to drive the placement of this ring around the DNA. To further understand the role of ATP binding and hydrolysis in driving clamp-loading in the DNA replication system of bacteriophage T4, we report the results of a series of presteady-state and steady-state kinetic ATPase experiments involving the various components of the reconstituted system. The results obtained are consistent with a mechanism in which a slow step, which involves the binary ATP-bound clamp-clamp loader complex, activates this complex and permits p/t DNA to bind and stimulate ATP hydrolysis. ATP hydrolysis itself, as well as the subsequent (after clamp-loading) dissociation of the clamp-loader and the slippage of the loaded clamp from the p/t DNA construct, are shown to be fast steps. A second slow step occurs after ATP hydrolysis. This step involves the dissociated clamp loader complex and may reflect ADP release. Only one molecule of ATP is hydrolyzed per clamp-loading event. Rate constants for each step, and an overall reaction mechanism for the T4 clamp-loading system, are derived from these data and from other results in the literature. The principles that emerge fit into a general framework that can apply to many biological processes involving ATP-driven reaction cycles.     

Clamp loader structure predicts the architecture of DNA polymerase III holoenzyme and RFC.

Recent determinations of the crystal structure of the Escherichia coli gamma complex and delta-beta assembly have shed light on the bacterial clamp loading reaction. In this review, we discuss the structures of delta-beta and the gamma(3)deltadelta' complex and its mechanism of action as a clamp loader of the E. coli beta sliding clamp. We also expand upon the implications of the structural findings to the structure and function of the eukaryotic clamp loader, RFC, and the structure of E. coli DNA polymerase III holoenzyme.     

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.     

Devoted to the lagging strand-the subunit of DNA polymerase III holoenzyme contacts SSB to promote processive elongation and sliding clamp assembly.

Escherichia coli DNA polymerase III holoenzyme contains 10 different subunits which assort into three functional components: a core catalytic unit containing DNA polymerase activity, the beta sliding clamp that encircles DNA for processive replication, and a multisubunit clamp loader apparatus called gamma complex that uses ATP to assemble the beta clamp onto DNA. We examine here the function of the psi subunit of the gamma complex clamp loader. Omission of psi from the holoenzyme prevents contact with single-stranded DNA-binding protein (SSB) and lowers the efficiency of clamp loading and chain elongation under conditions of elevated salt. We also show that the product of a classic point mutant of SSB, SSB-113, lacks strong affinity for psi and is defective in promoting clamp loading and processive replication at elevated ionic strength. SSB-113 carries a single amino acid replacement at the penultimate residue of the C-terminus, indicating the C-terminus as a site of interaction with psi. Indeed, a peptide of the 15 C-terminal residues of SSB is sufficient to bind to psi. These results establish a role for the psi subunit in contacting SSB, thus enhancing the clamp loading and processivity of synthesis of the holoenzyme, presumably by helping to localize the holoenzyme to sites of SSB-coated ssDNA.     

A model for Escherichia coli DNA polymerase III holoenzyme assembly at primer/template ends. DNA triggers a change in binding specificity of the gamma complex clamp loader

The gamma complex of the Escherichia coli DNA polymerase III holoenzyme assembles the beta sliding clamp onto DNA in an ATP hydrolysis-driven reaction. Interactions between gamma complex and primer/template DNA are investigated using fluorescence depolarization to measure binding of gamma complex to different DNA substrates under steady-state and presteady-state conditions. Surprisingly, gamma complex has a much higher affinity for single-stranded DNA (K(d) in the nM range) than for a primed template (K(d) in the microM range) under steady-state conditions. However, when examined on a millisecond time scale, we find that gamma complex initially binds very rapidly and with high affinity to primer/template DNA but is converted subsequently to a much lower affinity DNA binding state. Presteady-state data reveals an effective dissociation constant of 1.5 nM for the initial binding of gamma complex to DNA and a dissociation constant of 5.7 microM for the low affinity DNA binding state. Experiments using nonhydrolyzable ATPgammaS show that ATP binding converts gamma complex from a low affinity "inactive" to high affinity "active" DNA binding state while ATP hydrolysis has the reverse effect, thus allowing cycling between active and inactive DNA binding forms at steady-state. We propose that a DNA-triggered switch between active and inactive states of gamma complex provides a two-tiered mechanism enabling gamma complex to recognize primed template sites and load beta, while preventing gamma complex from competing with DNA polymerase III core for binding a newly loaded beta.DNA complex.