Atomic structure of the clamp loader small subunit from Pyrococcus furiosus.

In eukaryotic DNA replication, replication factor-C (RFC) acts as the clamp loader, which correctly installs the sliding clamp onto DNA strands at replication forks. The eukaryotic RFC is a complex consisting of one large and four small subunits. We have determined the crystal structure of the clamp loader small subunit (RFCS) from Pyrococcus furiosus. The six subunits, of which four bind ADP in their canonical nucleotide binding clefts, assemble into a dimer of semicircular trimers. The crescent-like architecture of each subunit formed by the three domains resembles that of the delta' subunit of the E. coli clamp loader. The trimeric architecture of archaeal RFCS, with its mobile N-terminal domains, involves intersubunit interactions that may be conserved in eukaryotic functional complexes.     

Distinct roles for ATP binding and hydrolysis at individual subunits of an archaeal clamp loader

Circular clamps are utilised by replicative polymerases to enhance processivity. The topological problem of loading a toroidal clamp onto DNA is overcome by ATP-dependent clamp loader complexes. Different organisms use related protein machines to load clamps, but the mechanisms by which they utilise ATP are surprisingly different. Using mutant clamp loaders that are deficient in either ATP binding or hydrolysis in different subunits, we show how the different subunits of an archaeal clamp loader use ATP binding and hydrolysis in distinct ways at different steps in the loading process. Binding of nucleotide by the large subunit and three of the four small subunits is sufficient for clamp loading. However, ATP hydrolysis by the small subunits is required for release of PCNA to allow formation of the complex between PCNA and the polymerase, while hydrolysis by the large subunit is required for catalytic clamp loading.     

Mechanism of proliferating cell nuclear antigen clamp opening by replication factor C

The eukaryotic replication factor C (RFC) clamp loader is an AAA+ spiral-shaped heteropentamer that opens and closes the circular proliferating cell nuclear antigen (PCNA) clamp processivity factor on DNA. In this study, we examined the roles of individual RFC subunits in opening the PCNA clamp. Interestingly, Rfc1, which occupies the position analogous to the delta clamp-opening subunit in the Escherichia coli clamp loader, is not required to open PCNA. The Rfc5 subunit is required to open PCNA. Consistent with this result, Rfc2.3.4.5 and Rfc2.5 subassemblies are capable of opening and unloading PCNA from circular DNA. Rfc5 is positioned opposite the PCNA interface from Rfc1, and therefore, its action with Rfc2 in opening PCNA indicates that PCNA is opened from the opposite side of the interface that the E. coli delta wrench acts upon. This marks a significant departure in the mechanism of eukaryotic and prokaryotic clamp loaders. Interestingly, the Rad.RFC DNA damage checkpoint clamp loader unloads PCNA clamps from DNA. We propose that Rad.RFC may clear PCNA from DNA to facilitate shutdown of replication in the face of DNA damage.     

Hypothesis: bacterial clamp loader ATPase activation through DNA-dependent repositioning of the catalytic base and of a trans-acting catalytic threonine

The prokaryotic DNA polymerase III clamp loader complex loads the β clamp onto DNA to link the replication complex to DNA during processive synthesis and unloads it again once synthesis is complete. This minimal complex consists of one δ, one δ′ and three γ subunits, all of which possess an AAA+ module—though only the γ subunit exhibits ATPase activity. Here clues to underlying clamp loader mechanisms are obtained through Bayesian inference of various categories of selective constraints imposed on the γ and δ′ subunits. It is proposed that a conserved histidine is ionized via electron transfer involving structurally adjacent residues within the sensor 1 region of γ's AAA+ module. The resultant positive charge on this histidine inhibits ATPase activity by drawing the negatively charged catalytic base away from the active site. It is also proposed that this arrangement is disrupted upon interaction of DNA with basic residues in γ implicated previously in DNA binding, regarding which a lysine that is near the sensor 1 region and that is highly conserved both in bacterial and in eukaryotic clamp loader ATPases appears to play a critical role. γ ATPases also appear to utilize a trans-acting threonine that is donated by helix 6 of an adjacent γ or δ′ subunit and that assists in the activation of a water molecule for nucleophilic attack on the γ phosphorous atom of ATP. As eukaryotic and archaeal clamp loaders lack most of these key residues, it appears that eubacteria utilize a fundamentally different mechanism for clamp loader activation than do these other organisms.     

Replication protein A-directed unloading of PCNA by the Ctf18 cohesion establishment complex

The replication clamp PCNA is loaded around DNA by replication factor C (RFC) and functions in DNA replication and repair. Regulated unloading of PCNA during the progression and termination of DNA replication may require additional factors. Here we show that a Saccharomyces cerevisiae complex required for the establishment of sister chromatid cohesion functions as an efficient unloader of PCNA. Unloading requires ATP hydrolysis. This seven-subunit Ctf18-RFC complex consists of the four small subunits of RFC, together with Ctf18, Dcc1, and Ctf8. Ctf18-RFC was also a weak loader of PCNA onto naked template-primer DNA. However, when the single-stranded DNA template was coated by the yeast single-stranded DNA binding protein replication protein A (RPA) but not by a mutant form of RPA or a heterologous single-stranded DNA binding protein, both binding of Ctf18-RFC to substrate DNA and loading of PCNA were strongly inhibited, and unloading predominated. Neither yeast RFC itself nor two other related clamp loaders, containing either Rad24 or Elg1, catalyzed significant unloading of PCNA. The Dcc1 and Ctf8 subunits of Ctf18-RFC, while required for establishing sister chromatid cohesion in vivo, did not function specifically in PCNA unloading in vitro, thereby separating the functionality of the Ctf18-RFC complex into two distinct paths.     

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.     

Clamp loaders and replication initiation

Clamp loaders are ATP-driven multiprotein machines that couple ATP hydrolysis to the opening and closing of a circular protein ring around DNA. This ring-shaped clamp slides along DNA, and interacts with numerous proteins involved in DNA replication, DNA repair and cell cycle control. Recently determined structures of clamp loader complexes from prokaryotic and eukaryotic sources have revealed exciting new details of how these complex AAA+ machines perform this essential clamp loading function.     

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.     

Multiple ATP binding is required to stabilize the "activated" (clamp open) clamp loader of the T4 DNA replication complex

Most DNA replication systems include a sliding clamp that encircles the genomic DNA and links the polymerase to the template to control polymerase processivity. A loading complex is required to open the clamp and place it onto the DNA. In phage T4 this complex consists of a trimeric clamp of gp45 subunits and a pentameric loader assembly of four gp44 and one gp62 subunit(s), with clamp loading driven by ATP binding. We measure this binding as a function of input ligand concentration and show that four ATPs bind to the gp44/62 complex with equal affinity. In contrast, the ATPase rate profile of the clamp-clamp loader complex exhibits a marked peak at an input ATP concentration close to the overall Kd (approximately 30 microm), with further increases in bound ATP decreasing the ATPase rate to a much lower level. Thus the progressive binding of the four ATPs triggers a conformational change in the complex that markedly inhibits ATPase activity. This inhibition is related to ring opening by using a clamp that is covalently cross-linked across its subunit interfaces and thus rendered incapable of opening. Binding of this clamp abolishes substrate inhibition of the ATPase but leaves ATP binding unchanged. We show that four ATP ligands must bind to the T4 clamp loader before the loader can be fully "activated" and the clamp opened, and that ATP hydrolysis is required only for release of the loader complex after clamp loading onto the replication fork has been completed.     

Requirement for ATP by the DNA damage checkpoint clamp loader

The DNA damage clamp loader replication factor C (RFC-Rad24) consists of the Rad24 protein and the four small Rfc2-5 subunits of RFC. This complex loads the heterotrimeric DNA damage clamp consisting of Rad17, Mec3, and Ddc1 (Rad17/3/1) onto partial duplex DNA in an ATP-dependent manner. Interactions between the clamp loader and the clamp have been proposed to mirror those of the replication clamp loader RFC and the sliding clamp proliferating cell nuclear antigen (PCNA). In that system, three ATP molecules bound to the Rfc2, Rfc3, and Rfc4 subunits are necessary and sufficient for efficient loading of PCNA, whereas ATP binding to Rfc1 is not required. In contrast, in this study, we show that mutant RFC-Rad24 with a rad24-K115E mutation in the ATP-binding domain of Rad24 shows defects in the ATPase of the complex and is defective for interaction with Rad17/3/1 and for loading of the checkpoint clamp. A similar defect was measured with a mutant RFC-Rad24 clamp loader carrying a rfc4K55R ATP-binding mutation, whereas the rfc4K55E clamp loader showed partial loading activity, in agreement with genetic studies of these mutants. These studies show that ATP utilization by the checkpoint clamp/clamp loader system is effectively different from that by the structurally analogous replication system.     

Three-dimensional electron microscopy of the clamp loader small subunit from Pyrococcus furiosus

An archaeal clamp loader, replication factor C (RFC), consists of two proteins, the small subunit (RFCS) and large subunit (RFCL), whose sequences are both highly homologous to those of the eukaryotic RFC components. We have investigated the oligomeric structure of RFCS from Pyrococcus furiosus by electron microscopy using single-particle analysis. RFCS forms mostly ring-shaped hexamers at pH 9.0, although it tends to form C-shaped tetramers or pentamers at a lower pH (pH 5.5). The three-dimensional (3D) structure of the RFCS hexamer was obtained by random conical tilt reconstruction at 24.0-A resolution. RFCS forms a hexameric ring with outer and inner diameters of 117 and 27 A, respectively, and with a height of about 55 A. The six subunits are arranged in a twisted manner with a sixfold symmetry around the channel. The 3D map revealed that the six subunits are arranged in a head-to-tail configuration. Although the RFC complex consists of RFCS and RFCL in vivo, RFCS alone, together with PCNA, substantially enhanced the DNA synthesizing activity of P. furiosus DNA polymerase I in vitro. The 3D reconstruction of RFCS with catalytic activity provides important insights into the organization mechanism and the functional state of the RFC complex.     

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.     

The Clamp Loader Assembles the �� Clamp onto Either a 3�� or 5�� Primer Terminus: THE UNDERLYING BASIS FAVORING 3�� LOADING*

Clamp loaders assemble sliding clamps onto 3′ primed sites for DNA polymerases. Clamp loaders are thought to be specific for a 3′ primed site, and unable to bind a 5′ site. We demonstrate here that the Escherichia coli γ complex clamp loader can load the β clamp onto a 5′ primed site, although with at least 20-fold reduced efficiency relative to loading at a 3′ primed site. Preferential clamp loading at a 3′ site does not appear to be due to DNA binding, as the clamp loader forms an avid complex with β at a 5′ site. Preferential loading at a 3′ versus a 5′ site occurs at the ATP hydrolysis step, needed to close the ring around DNA. We also address DNA structural features that are recognized for preferential loading at a 3′ site. Although the single-stranded template strand extends in opposite directions from 3′ and 5′ primed sites, thus making it a favorite candidate for distinguishing between 3′ and 5′ sites, the single-strand polarity at a primed template junction does not determine 3′ site selection for clamp loading. Instead, we find that clamp loader recognition of a 3′ site lies in the duplex portion of the primed site, not the single-strand portion. We present evidence that the β clamp facilitates its own loading specificity for a 3′ primed site. Implications to eukaryotic clamp loader complexes are proposed.     

DNA polymerase clamp loaders and DNA recognition

Clamp loaders are heteropentameric ATPase assemblies that load sliding clamps onto DNA and are critical for processive DNA replication. The DNA targets for clamp loading are double-stranded/single-stranded junctions with recessed 3' ends (primer-template junctions). Here, we briefly review the crystal structures of clamp loader complexes and the insights they have provided into the mechanism of the clamp loading process.     

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.     

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.     

Dynamics of loading the Escherichia coli DNA polymerase processivity clamp

Sliding clamps and clamp loaders are processivity factors required for efficient DNA replication. Sliding clamps are ring-shaped complexes that tether DNA polymerases to DNA to increase the processivity of synthesis. Clamp loaders assemble these ring-shaped clamps onto DNA in an ATP-dependent reaction. The overall process of clamp loading is dynamic in that protein-protein and protein-DNA interactions must actively change in a coordinated fashion to complete the mechanical clamp-loading reaction cycle. The clamp loader must initially have a high affinity for both the clamp and DNA to bring these macromolecules together, but then must release the clamp on DNA for synthesis to begin. Evidence is presented for a mechanism in which the clamp-loading reaction comprises a series of binding reactions to ATP, the clamp, DNA, and ADP, each of which promotes some change in the conformation of the clamp loader that alters interactions with the next component of the pathway. These changes in interactions must be rapid enough to allow the clamp loader to keep pace with replication fork movement. This review focuses on the measurement of dynamic and transient interactions required to assemble the Escherichia coli sliding clamp on DNA.     

NMR resonance assignments for the N-terminal domain of the δ subunit of the <Emphasis Type="Italic">E. coli</Emphasis> γ clamp loader complex

The β-clamp protein and the γ clamp loader complex are essential components of bacterial DNA replication machinery. The β-clamp is a ring-shaped homodimer that encircles DNA and increases the efficiency of replication by providing a binding platform for DNA polymerases and other replication-related proteins. The β-clamp is loaded onto DNA by the five-subunit γ clamp loader complex in a multi-step ATP-dependent process. The initial steps of this process involve the cooperative binding of the β-clamp by the five subunits of ATP-bound clamp loader, which induces or traps an open conformation of the clamp. Remarkably, the δ subunit of the E. coli clamp loader, or even its 140 residue N-terminal domain (called mini-δ), alone can shift conformational equilibrium of the β-clamp towards the open state. Here we report nearly complete backbone and side-chain ¹H, ¹³C and ¹⁵N NMR resonance assignments of mini-δ that will facilitate NMR studies of the mechanisms of β-clamp opening and its loading on DNA by the clamp loader.     

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.     

Dynamics of Loading the Escherichia coli DNA Polymerase Processivity Clamp

ABSTRACT

Sliding clamps and clamp loaders are processivity factors required for efficient DNA replication. Sliding clamps are ring-shaped complexes that tether DNA polymerases to DNA to increase the processivity of synthesis. Clamp loaders assemble these ring-shaped clamps onto DNA in an ATP-dependent reaction. The overall process of clamp loading is dynamic in that protein–protein and protein–DNA interactions must actively change in a coordinated fashion to complete the mechanical clamp-loading reaction cycle. The clamp loader must initially have a high affinity for both the clamp and DNA to bring these macromolecules together, but then must release the clamp on DNA for synthesis to begin. Evidence is presented for a mechanism in which the clamp-loading reaction comprises a series of binding reactions to ATP, the clamp, DNA, and ADP, each of which promotes some change in the conformation of the clamp loader that alters interactions with the next component of the pathway. These changes in interactions must be rapid enough to allow the clamp loader to keep pace with replication fork movement. This review focuses on the measurement of dynamic and transient interactions required to assemble the Escherichia coli sliding clamp on DNA.