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 replication factor C clamp loader requires arginine finger sensors to drive DNA binding and proliferating cell nuclear antigen loading

Replication factor C (RFC) is an AAA+ heteropentamer that couples the energy of ATP binding and hydrolysis to the loading of the DNA polymerase processivity clamp, proliferating cell nuclear antigen (PCNA), onto DNA. RFC consists of five subunits in a spiral arrangement (RFC-A, -B, -C, -D, and -E, corresponding to subunits RFC1, RFC4, RFC3, RFC2, and RFC5, respectively). The RFC subunits are AAA+ family proteins and the complex contains four ATP sites (sites A, B, C, and D) located at subunit interfaces. In each ATP site, an arginine residue from one subunit is located near the gamma-phosphate of ATP bound in the adjacent subunit. These arginines act as "arginine fingers" that can potentially perform two functions: sensing that ATP is bound and catalyzing ATP hydrolysis. In this study, the arginine fingers in RFC were mutated to examine the steps in the PCNA loading mechanism that occur after RFC binds ATP. This report finds that the ATP sites of RFC function in distinct steps during loading of PCNA onto DNA. ATP binding to RFC powers recruitment and opening of PCNA and activates a gamma-phosphate sensor in ATP site C that promotes DNA association. ATP hydrolysis in site D is uniquely stimulated by PCNA, and we propose that this event is coupled to PCNA closure around DNA, which starts an ordered hydrolysis around the ring. PCNA closure severs contact to RFC subunits D and E (RFC2 and RFC5), and the gamma-phosphate sensor of ATP site C is switched off, resulting in low affinity of RFC for DNA and ejection of RFC from the site of PCNA loading.     

Impact of Individual Proliferating Cell Nuclear Antigen-DNA Contacts on Clamp Loading and Function on DNA

Ring-shaped clamp proteins encircle DNA and affect the work of many proteins, notably processive replication by DNA polymerases. Crystal structures of clamps show several cationic residues inside the ring, and in a co-crystal of Escherichia coli β clamp-DNA, they directly contact the tilted duplex passing through (Georgescu, R. E., Kim, S. S., Yurieva, O., Kuriyan, J., Kong, X. P., and O''Donnell, M. (2008) Structure of a sliding clamp on DNA. Cell 132, 43–54). To investigate the role of these contacts in reactions involving circular clamps, we examined single arginine/lysine mutants of Saccharomyces cerevisiae proliferating cell nuclear antigen (PCNA) in replication factor C (RFC)-catalyzed loading of the clamp onto primer template DNA (ptDNA). Previous kinetic analysis has shown that ptDNA entry inside an ATP-activated RFC-PCNA complex accelerates clamp opening and ATP hydrolysis, which is followed by slow PCNA closure around DNA and product dissociation. Here we directly measured multiple steps in the reaction (PCNA opening, ptDNA binding, PCNA closure, phosphate release, and complex dissociation) to determine whether mutation of PCNA residues Arg-14, Lys-20, Arg-80, Lys-146, Arg-149, or Lys-217 to alanine affects the reaction mechanism. Contrary to earlier steady state analysis of these mutants (McNally, R., Bowman, G. D., Goedken, E. R., O''Donnell, M., and Kuriyan, J. (2010) Analysis of the role of PCNA-DNA contacts during clamp loading. BMC Struct. Biol. 10, 3), our pre-steady state data show that loss of single cationic residues can alter the rates of all DNA-linked steps in the reaction, as well as movement of PCNA on DNA. These results explain an earlier finding that individual arginines and lysines inside human PCNA are essential for polymerase δ processivity (Fukuda, K., Morioka, H., Imajou, S., Ikeda, S., Ohtsuka, E., and Tsurimoto, T. (1995) Structure-function relationship of the eukaryotic DNA replication factor, proliferating cell nuclear antigen. J. Biol. Chem. 270, 22527–22534). Mutations in the N-terminal domain have greater impact than in the C-terminal domain, indicating a positional bias in PCNA-DNA contacts that can influence its functions on DNA.     

Replication factor C is a more effective proliferating cell nuclear antigen (PCNA) opener than the checkpoint clamp loader, Rad24-RFC

Clamp loaders from all domains of life load clamps onto DNA. The clamp tethers DNA polymerases to DNA to increase the processivity of synthesis as well as the efficiency of replication. Here, we investigated proliferating cell nuclear antigen (PCNA) binding and opening by the Saccharomyces cerevisiae clamp loader, replication factor C (RFC), and the DNA damage checkpoint clamp loader, Rad24-RFC, using two separate fluorescence intensity-based assays. Analysis of PCNA opening by RFC revealed a two-step reaction in which RFC binds PCNA before opening PCNA rather than capturing clamps that have transiently and spontaneously opened in solution. The affinity of RFC for PCNA is about an order of magnitude lower in the absence of ATP than in its presence. The affinity of Rad24-RFC for PCNA in the presence of ATP is about an order magnitude weaker than that of RFC for PCNA, similar to the RFC-PCNA interaction in the absence of ATP. Importantly, fewer open clamp loader-clamp complexes are formed when PCNA is bound by Rad24-RFC than when bound by RFC.     

ATP utilization by yeast replication factor C. I. ATP-mediated interaction with DNA and with proliferating cell nuclear antigen.

Eukaryotic replication factor C is the heteropentameric complex that loads the replication clamp proliferating cell nuclear antigen (PCNA) onto primed DNA. In this study we used a derivative, designated RFC, with a N-terminal truncation of the Rfc1 subunit removing a DNA-binding domain not required for clamp loading. Interactions of yeast RFC with PCNA and DNA were studied by surface plasmon resonance. Binding of RFC to PCNA was stimulated by either adenosine (3-thiotriphosphate) (ATPgammaS) or ATP. RFC bound only to primer-template DNA coated with the single-stranded DNA-binding protein RPA if ATPgammaS was also present. Binding occurred without dissociation of RPA. ATP did not stimulate binding of RFC to DNA, suggesting that hydrolysis of ATP dissociated DNA-bound RFC. However, when RFC and PCNA together were flowed across the DNA chip in the presence of ATP, a signal was observed suggesting loading of PCNA by RFC. With ATPgammaS present instead of ATP, long-lived response signals were observed indicative of loading complexes arrested on the DNA. A primer with a 3' single-stranded extension also allowed loading of PCNA; yet turnover of the reaction intermediates was dramatically slowed down. Filter binding experiments and analysis of proteins bound to DNA-magnetic beads confirmed the conclusions drawn from the surface plasmon resonance studies.     

ATP utilization by yeast replication factor C. II. Multiple stepwise ATP binding events are required to load proliferating cell nuclear antigen onto primed DNA.

Binding of adenosine (3-thiotriphosphate) (ATPgammaS), a nonhydrolyzable analog of ATP, to replication factor C with a N-terminal truncation (Delta2-273) of the Rfc1 subunit (RFC) was studied by filter binding. RFC alone bound 1.8 ATPgammaS molecules. However, when either PCNA or primer-template DNA were also present 2.6 or 2.7 ATPgammaS molecules, respectively, were bound. When both PCNA and DNA were present 3.6 ATPgammaS molecules were bound per RFC. Order of addition experiments using surface plasmon resonance indicate that RFC forms an ATP-mediated binary complex with PCNA prior to formation of a ternary DNA.PCNA.RFC complex. An ATP-mediated complex between RFC and DNA was not competent for binding PCNA, and the RFC.DNA complex dissociated with hydrolysis of ATP. Based on these experiments a model is proposed in which: (i) RFC binds two ATPs (RFC.ATP(2)); (ii) this complex binds PCNA (PCNA.RFC.ATP(2)), which goes through a conformational change to reveal a binding site for one additional ATP (PCNA.RFC.ATP(3)); (iii) this complex can bind DNA to yield DNA.PCNA.RFC.ATP(3); (iv) a conformational change in the latter complex reveals a fourth binding site for ATP; and (v) the DNA.PCNA.RFC.ATP(4) complex is finally competent for completion of PCNA loading and release of RFC upon hydrolysis of ATP.     

Cyclin-Dependent Kinase Inhibitor p21 Modulates the DNA Primer-Template Recognition Complex

The p21 protein, a cyclin-dependent kinase (CDK) inhibitor, is capable of binding to both cyclin-CDK and the proliferating cell nuclear antigen (PCNA). Through its binding to PCNA, p21 can regulate the function of PCNA differentially in replication and repair. To gain an understanding of the precise mechanism by which p21 affects PCNA function, we have designed a new assay for replication factor C (RFC)-catalyzed loading of PCNA onto DNA, a method that utilizes a primer-template DNA attached to agarose beads via biotin-streptavidin. Using this assay, we showed that RFC remains transiently associated with PCNA on the DNA after the loading reaction. Addition of p21 did not inhibit RFC-dependent PCNA loading; rather, p21 formed a stable complex with PCNA on the DNA. In contrast, the formation of a p21-PCNA complex on the DNA resulted in the displacement of RFC from the DNA. The nonhydrolyzable analogs of ATP, adenosine-5′-O-(3-thiotriphosphate) (ATPγS) and adenyl-imidodiphosphate, each stabilized the primer recognition complex containing RFC and PCNA in the absence of p21. RFC in the ATPγS-activated complex was no longer displaced from the DNA by p21. We propose that p21 stimulates the dissociation of the RFC from the PCNA-DNA complex in a process that requires ATP hydrolysis and then inhibits subsequent PCNA-dependent events in DNA replication. The data suggest that the conformation of RFC in the primer recognition complex might change on hydrolysis of ATP. We also suggest that the p21-PCNA complex that remains attached to DNA might function to tether cyclin-CDK complexes to specific regions of the genome.     

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.     

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.     

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.     

Replication factor C clamp loader subunit arrangement within the circular pentamer and its attachment points to proliferating cell nuclear antigen

Replication factor C (RFC) is a heteropentameric AAA+ protein clamp loader of the proliferating cell nuclear antigen (PCNA) processivity factor. The prokaryotic homologue, gamma complex, is also a heteropentamer, and structural studies show the subunits are arranged in a circle. In this report, Saccharomyces cerevisiae RFC protomers are examined for their interaction with each other and PCNA. The data lead to a model of subunit order around the circle. A characteristic of AAA+ oligomers is the use of bipartite ATP sites in which one subunit supplies a catalytic arginine residue for hydrolysis of ATP bound to the neighboring subunit. We find that the RFC(3/4) complex is a DNA-dependent ATPase, and we use this activity to determine that RFC3 supplies a catalytic arginine to the ATP site of RFC4. This information, combined with the subunit arrangement, defines the composition of the remaining ATP sites. Furthermore, the RFC(2/3) and RFC(3/4) subassemblies bind stably to PCNA, yet neither RFC2 nor RFC4 bind tightly to PCNA, indicating that RFC3 forms a strong contact point to PCNA. The RFC1 subunit also binds PCNA tightly, and we identify two hydrophobic residues in RFC1 that are important for this interaction. Therefore, at least two subunits in RFC make strong contacts with PCNA, unlike the Escherichia coli gamma complex in which only one subunit makes strong contact with the beta clamp. Multiple strong contact points to PCNA may reflect the extra demands of loading the PCNA trimeric ring onto DNA compared with the dimeric beta ring.     

Division of labor--sequential ATP hydrolysis drives assembly of a DNA polymerase sliding clamp around DNA.

The beta sliding clamp encircles DNA and enables processive replication of the Escherichia coli genome by DNA polymerase III holoenzyme. The clamp loader, gamma complex, assembles beta around DNA in an ATP-fueled reaction. Previous studies have shown that gamma complex opens the beta ring and also interacts with DNA on binding ATP. Here, a rapid kinetic analysis demonstrates that gamma complex hydrolyzes two ATP molecules sequentially when placing beta around DNA. The first ATP is hydrolyzed fast, at 25-30 s(-1), while the second ATP hydrolysis is limited to the steady-state rate of 2 s(-1). This step-wise reaction depends on both primed DNA and beta. DNA alone promotes rapid hydrolysis of two ATP molecules, while beta alone permits hydrolysis of only one ATP. These results suggest that beta inserts a slow step between the two ATP hydrolysis events in clamp assembly, during which the clamp loader may perform work on the clamp. Moreover, one ATP hydrolysis is sufficient for release of beta from the gamma complex. This implies that DNA-dependent hydrolysis of the other ATP is coupled to a separate function, perhaps involving work on DNA. A model is presented in which sequential ATP hydrolysis drives distinct events in the clamp-assembly pathway. We also discuss underlying principles of this step-wise mechanism that may apply to the workings of other ATP-fueled biological machines.     

Communication between subunits within an archaeal clamp-loader complex

We have investigated the communication between subunits in replication factor C (RFC) from Archaeoglobus fulgidus. Mutation of the proposed arginine finger in the small subunits results in a complex that can still bind ATP but has impaired clamp-loading activity, a process that normally only requires binding of nucleotide. The small subunit alone forms a hexameric ring that is six-fold symmetric in the absence of ATP. However, this symmetry is broken when the nucleotide is bound to the complex. A conformational change associated with nucleotide binding may relate to the opening of PCNA rings by RFC during the loading reaction. The structures also reveal the importance of the N-terminal helix of each subunit at the ATP-binding site. Analysis of mutant protein complexes containing subunits lacking this N-terminal helix reveals key distinct regulatory roles during clamp loading that are different for the large and small subunits in the RFC complex.     

ATP utilization by yeast replication factor C. III. The ATP-binding domains of Rfc2, Rfc3, and Rfc4 are essential for DNA recognition and clamp loading.

The conserved lysine in the Walker A motif of the ATP-binding domain encoded by the yeast RFC1, RFC2, RFC3, and RFC4 genes was mutated to glutamic acid. Complexes of replication factor C with a N-terminal truncation (Delta2-273) of the Rfc1 subunit (RFC) containing a single mutant subunit were overproduced in Escherichia coli for biochemical analysis. All of the mutant RFC complexes were capable of interacting with PCNA. Complexes containing a rfc1-K359E mutation were similar to wild type in replication activity and ATPase activity; however, the mutant complex showed increased susceptibility to proteolysis. In contrast, complexes containing either a rfc2-K71E mutation or a rfc3-K59E mutation were severely impaired in ATPase and clamp loading activity. In addition to their defects in ATP hydrolysis, these complexes were defective for DNA binding. A mutant complex containing the rfc4-K55E mutation performed as well as a wild type complex in clamp loading, but only at very high ATP concentrations. Mutant RFC complexes containing rfc2-K71R or rfc3-K59R, carrying a conservative lysine --> arginine mutation, had much milder clamp loading defects that could be partially (rfc2-K71R) or completely (rfc3-K59R) suppressed at high ATP concentrations.     

Reverse-chaperoning activity of an AAA+ protein

Speed and processivity of replicative DNA polymerases can be enhanced via coupling to a sliding clamp. Due to the closed ring shape of the clamp, a clamp loader protein, belonging to the AAA+ class of ATPases, needs to open the ring-shaped clamp before loading it to DNA. Here, we developed real-time fluorescence assays to study the clamp (PCNA) and the clamp loader (RFC) from the mesophilic archaeon Methanosarcina acetivorans. Unexpectedly, we discovered that RFC can assemble a PCNA ring from monomers in solution. A motion-based DNA polymerization assay showed that the PCNA assembled by RFC is functional. This PCNA assembly activity required the ATP-bound conformation of RFC. Our work demonstrates a reverse-chaperoning activity for an AAA+ protein that can act as a template for the assembly of another protein complex.     

The clamp-loading complex for processive DNA replication

DNA polymerase requires two processing factors, sliding clamps and clamp loaders, to direct rapid and accurate duplication of genomic DNA. In eukaryotes, proliferating cell nuclear antigen (PCNA), the ring-shaped sliding clamp, encircles double-stranded DNA within its central hole and tethers the DNA polymerases onto DNA. Replication factor C (RFC) acts as the clamp loader, which correctly installs the sliding clamp onto DNA strands in an ATP-dependent manner. Here we report the three-dimensional structure of an archaeal clamp-loading complex (RFC-PCNA-DNA) determined by single-particle EM. The three-dimensional structure of the complex, reconstituted in vitro using a nonhydrolyzable ATP analog, reveals two components, a closed ring and a horseshoe-shaped element, which correspond to PCNA and RFC, respectively. The atomic structure of PCNA fits well into the closed ring, suggesting that this ternary complex represents a state just after the PCNA ring has closed to encircle the DNA duplex.     

Biochemical characterisation of the clamp/clamp loader proteins from the euryarchaeon Archaeoglobus fulgidus

Replicative polymerases of eukaryotes, prokaryotes and archaea obtain processivity using ring-shaped DNA sliding clamps that are loaded onto DNA by clamp loaders [replication factor C (RFC) in eukaryotes]. In this study, we cloned the two genes for the subunits of the RFC homologue of the euryarchaeon Archaeoglobus fulgidus. The proteins were expressed and purified from Escherichia coli both individually and as a complex. The afRFC subunits form a heteropentameric complex consisting of one copy of the large subunit and four copies of the small subunits. To analyse the functionality of afRFC, we also expressed the A.fulgidus PCNA homologue and a type B polymerase (PolB1) in E.coli. In primer extension assays, afRFC stimulated the processivity of afPolB1 in afPCNA-dependent reactions. Although the afRFC complex showed significant DNA-dependent ATPase activity, which could be further stimulated by afPCNA, neither of the isolated afRFC subunits showed this activity. However, both the large and small afRFC subunits showed interaction with afPCNA. Furthermore, we demonstrate that ATP binding, but not hydrolysis, is needed to stimulate interactions of the afRFC complex with afPCNA and DNA.     

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.     

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.