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.     

Replication factor C from the hyperthermophilic archaeon Pyrococcus abyssi does not need ATP hydrolysis for clamp-loading and contains a functionally conserved RFC PCNA-binding domain

The molecular organization of the replication complex in archaea is similar to that in eukaryotes. Only two proteins homologous to subunits of eukaryotic replication factor C (RFC) have been detected in Pyrococcus abyssi (Pab). The genes encoding these two proteins are arranged in tandem. We cloned these two genes and co-expressed the corresponding recombinant proteins in Escherichia coli. Two inteins present in the gene encoding the small subunit (PabRFC-small) were removed during cloning. The recombinant protein complex was purified by anion-exchange and hydroxyapatite chromatography. Also, the PabRFC-small subunit could be purified, while the large subunit (PabRFC-large) alone was completely insoluble. The highly purified PabRFC complex possessed an ATPase activity, which was not enhanced by DNA. The Pab proliferating cell nuclear antigen (PCNA) activated the PabRFC complex in a DNA-dependent manner, but the PabRFC-small ATPase activity was neither DNA-dependent nor PCNA-dependent. The PabRFC complex was able to stimulate PabPCNA-dependent DNA synthesis by the Pabfamily D heterodimeric DNA polymerase. Finally, (i) the PabRFC-large fraction cross-reacted with anti-human-RFC PCNA-binding domain antibody, corroborating the conservation of the protein sequence, (ii) the human PCNA stimulated the PabRFC complex ATPase activity in a DNA-dependent way and (iii) the PabRFC complex could load human PCNA onto primed single-stranded circular DNA, suggesting that the PCNA-binding domain of RFC has been functionally conserved during evolution. In addition, ATP hydrolysis was not required either for DNA polymerase stimulation or PCNA-loading in vitro.     

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.     

Molecular modeling-based analysis of interactions in the RFC-dependent clamp-loading process

Replication and related processes in eukaryotic cells require replication factor C (RFC) to load a molecular clamp for DNA polymerase in an ATP-driven process, involving multiple molecular interactions. The detailed understanding of this mechanism is hindered by the lack of data regarding structure, mutual arrangement, and dynamics of the players involved. In this study, we analyzed interactions that take place during loading onto DNA of either the PCNA clamp or the Rad9-Rad1-Hus1 checkpoint complex, using computationally derived molecular models. Combining the modeled structures for each RFC subunit with known structural, biochemical, and genetic data, we propose detailed models of how two of the RFC subunits, RFC1 and RFC3, interact with the C-terminal regions of PCNA. RFC1 is predicted to bind PCNA similarly to the p21-PCNA interaction, while the RFC3-PCNA binding is proposed to be similar to the E. coli delta-beta interaction. Additional sequence and structure analysis, supported by experimental data, suggests that RFC5 might be the third clamp loader subunit to bind the equivalent PCNA region. We discuss functional implications stemming from the proposed model of the RFC1-PCNA interaction and compare putative clamp-interacting regions in RFC1 and its paralogs, Rad17 and Ctf18. Based on the individual intermolecular interactions, we propose RFC and PCNA arrangement that places three RFC subunits in association with each of the three C-terminal regions in PCNA. The two other RFC subunits are positioned at the two PCNA interfaces, with the third PCNA interface left unobstructed. In addition, we map interactions at the level of individual subunits between the alternative clamp loader/clamp system, Rad17-RFC(2-5)/Rad9-Rad1-Hus1. The proposed models of interaction between two clamp/clamp loader pairs provide both structural framework for interpretation of existing experimental data and a number of specific findings that can be subjected to direct experimental testing.     

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.     

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.     

Biochemical analysis of replication factor C from the hyperthermophilic archaeon Pyrococcus furiosus

Replication factor C (RFC) and proliferating cell nuclear antigen (PCNA) are accessory proteins essential for processive DNA synthesis in the domain Eucarya. The function of RFC is to load PCNA, a processivity factor of eukaryotic DNA polymerases delta and epsilon, onto primed DNA templates. RFC-like genes, arranged in tandem in the Pyrococcus furiosus genome, were cloned and expressed individually in Escherichia coli cells to determine their roles in DNA synthesis. The P. furiosus RFC (PfuRFC) consists of a small subunit (RFCS) and a large subunit (RFCL). Highly purified RFCS possesses an ATPase activity, which was stimulated up to twofold in the presence of both single-stranded DNA (ssDNA) and P. furiosus PCNA (PfuPCNA). The ATPase activity of PfuRFC itself was as strong as that of RFCS. However, in the presence of PfuPCNA and ssDNA, PfuRFC exhibited a 10-fold increase in ATPase activity under the same conditions. RFCL formed very large complexes by itself and had an extremely weak ATPase activity, which was not stimulated by PfuPCNA and DNA. The PfuRFC stimulated PfuPCNA-dependent DNA synthesis by both polymerase I and polymerase II from P. furiosus. We propose that PfuRFC is required for efficient loading of PfuPCNA and that the role of RFC in processive DNA synthesis is conserved in Archaea and Eucarya.     

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.     

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.     

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.     

Isolation and characterization of a split B-type DNA polymerase from the archaeon Methanobacterium thermoautotrophicum deltaH.

We describe here the isolation and characterization of a B-type DNA polymerase (PolB) from the archaeon Methanobacterium thermoautotrophicum DeltaH. Uniquely, the catalytic domains of M. thermoautotrophicum PolB are encoded from two different genes, a feature that has not been observed as yet in other polymerases. The two genes were cloned, and the proteins were overexpressed in Escherichia coli and purified individually and as a complex. We demonstrate that both polypeptides are needed to form the active polymerase. Similar to other polymerases constituting the B-type family, PolB possesses both polymerase and 3'-5' exonuclease activities. We found that a homolog of replication protein A from M. thermoautotrophicum inhibits the PolB activity. The inhibition of DNA synthesis by replication protein A from M. thermoautotrophicum can be relieved by the addition of M. thermoautotrophicum homologs of replication factor C and proliferating cell nuclear antigen. The possible roles of PolB in M. thermoautotrophicum replication are discussed.     

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.     

Functional Interactions of a Homolog of Proliferating Cell Nuclear Antigen with DNA Polymerases in Archaea

Proliferating cell nuclear antigen (PCNA) is an essential component of the DNA replication and repair machinery in the domain Eucarya. We cloned the gene encoding a PCNA homolog (PfuPCNA) from an euryarchaeote, Pyrococcus furiosus, expressed it in Escherichia coli, and characterized the biochemical properties of the gene product. The protein PfuPCNA stimulated the in vitro primer extension abilities of polymerase (Pol) I and Pol II, which are the two DNA polymerases identified in this organism to date. An immunological experiment showed that PfuPCNA interacts with both Pol I and Pol II. Pol I is a single polypeptide with a sequence similar to that of family B (alpha-like) DNA polymerases, while Pol II is a heterodimer. PfuPCNA interacted with DP2, the catalytic subunit of the heterodimeric complex. These results strongly support the idea that the PCNA homolog works as a sliding clamp of DNA polymerases in P. furiosus, and the basic mechanism for the processive DNA synthesis is conserved in the domains Bacteria, Eucarya, and Archaea. The stimulatory effect of PfuPCNA on the DNA synthesis was observed by using a circular DNA template without the clamp loader (replication factor C [RFC]) in both Pol I and Pol II reactions in contrast to the case of eukaryotic organisms, which are known to require the RFC to open the ring structure of PCNA prior to loading onto a circular DNA. Because RFC homologs have been found in the archaeal genomes, they may permit more efficient stimulation of DNA synthesis by archaeal DNA polymerases in the presence of PCNA. This is the first stage in elucidating the archaeal DNA replication mechanism.     

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.     

Recruitment of DNA repair synthesis machinery to sites of DNA damage/repair in living human cells

The eukaryotic sliding DNA clamp, proliferating cell nuclear antigen (PCNA), is essential for DNA replication and repair synthesis. In order to load the ring-shaped, homotrimeric PCNA onto the DNA double helix, the ATPase activity of the replication factor C (RFC) clamp loader complex is required. Although the recruitment of PCNA by RFC to DNA replication sites has well been documented, our understanding of its recruitment during DNA repair synthesis is limited. In this study, we analyzed the accumulation of endogenous and fluorescent-tagged proteins for DNA repair synthesis at the sites of DNA damage produced locally by UVA-laser micro-irradiation in HeLa cells. Accumulation kinetics and in vitro pull-down assays of the large subunit of RFC (RFC140) revealed that there are two distinct modes of recruitment of RFC to DNA damage, a simultaneous accumulation of RFC140 and PCNA caused by interaction between PCNA and the extreme N-terminus of RFC140 and a much faster accumulation of RFC140 than PCNA at the damaged site. Furthermore, RFC140 knock-down experiments showed that PCNA can accumulate at DNA damage independently of RFC. These results suggest that immediate accumulation of RFC and PCNA at DNA damage is only partly interdependent.     

Gene cloning and function analysis of replication factor C from Thermococcus kodakaraensis KOD1

Replication factor C (RFC) catalyzes the assembly of circular proliferating cell nuclear antigen (PCNA) clamps around primed DNA, enabling processive synthesis by DNA polymerase. The RFC-like genes, arranged in tandem in the Thermococcus kodakaraensis KOD1 genome, were cloned individually and co-expressed in Escherichia coli cells. T. kodakaraensis KOD1 RFC homologue (Tk-RFC) consists of the small subunit (Tk-RFCS: MW=37.2 kDa) and the large subunit (Tk-RFCL: MW=57.2 kDa). The DNA elongation rate of the family B DNA polymerase from T. kodakaraensis KOD1 (KOD DNA polymerase), which has the highest elongation rate in all thermostable DNA polymerases, was increased about 1.7 times, when T. kodakaraensis KOD1 PCNA (Tk-PCNA) and the Tk-RFC at the equal molar ratio of KOD DNA polymerase were reacted with primed DNA.     

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.     

Fidelity of eucaryotic DNA polymerase delta holoenzyme from Schizosaccharomyces pombe

The fidelity of Schizosaccharomyces pombe DNA polymerase delta was measured in the presence or absence of its processivity subunits, proliferating cell nuclear antigen (PCNA) sliding clamp and replication factor C (RFC) clamp-loading complex, using a synthetic 30-mer primer/100-mer template. Synthesis by pol delta alone was distributive. Processive synthesis occurred in the presence of PCNA, RFC, and Escherichia coli single strand DNA-binding protein (SSB) and required the presence of ATP. "Passive" self-loading of PCNA onto DNA takes place in the absence of RFC, in an ATP-independent reaction, which was strongly inhibited by SSB. The nucleotide substitution error rate for pol delta holoenzyme (HE) (pol delta + PCNA + RFC) was 4.6 x 10(-4) for T.G mispairs, 5.3 x 10(-5) for G.G mispairs, and 4.5 x 10(-6) for A.G mispairs. The T.G misincorporation frequency for pol delta without the accessory proteins was unchanged. The fidelity of pol delta HE was between 1 and 2 orders of magnitude lower than that measured for the E. coli pol III HE at the same template position. This relatively low fidelity was caused by inefficient proofreading by the S. pombe polymerase-associated proofreading exonuclease. The S. pombe 3'-exonuclease activity was also extremely inefficient in excising primer-3'-terminal mismatches in the absence of dNTP substrates and in hydrolyzing single-stranded DNA. A comparison of pol delta HE with E. coli pol IIIalpha HE (lacking the proofreading exonuclease subunit) showed that both holoenzymes exhibit similar error rates for each mispair.     

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.