Replication Factor C Subunits Research Articles

Interactional similarities and differences in the protein complex of PCNA and DNA replication factor C between rice and Arabidopsis

BackgroundProliferating cell nuclear antigen (PCNA), a conserved trimeric ring complex, is loaded onto replication fork through a hetero-pentameric AAA+ ATPase complex termed replication factor C (RFC) to maintain genome stability. Although architectures of PCNA-RFC complex in yeast have been revealed, the functions of PCNA and protein-protein interactions of PCNA-RFC complex in higher plants are not very clear. Here, essential regions mediating interactions between PCNA and RFC subunits in Arabidopsis and rice were investigated via yeast-two-hybrid method and bimolecular fluorescence complementation techniques.ResultsWe observed that OsPCNA could interact with all OsRFC subunits, while protein-protein interactions only exist between Arabidopsis RFC2/3/4/5 and AtPCNA1/2. The truncated analyses indicated that the C-terminal of Arabidopsis RFC2/3/4/5 and rice RFC1/2 is essential for binding PCNA while the region of rice RFC3/4/5 mediating interaction with PCNA distributed both at the N- and C-terminal. On the other hand, we found that the C- and N-terminal of Arabidopsis and rice PCNA contribute equally to PCNA-PCNA interaction, and the interdomain connecting loop (IDCL) domain and C-terminal of PCNAs are indispensable for interacting RFC subunits.ConclusionsThese results indicated that Arabidopsis and rice PCNAs are highly conserved in sequence, structure and pattern of interacting with other PCNA monomer. Nevertheless, there are also significant differences between the Arabidopsis and rice RFC subunits in binding PCNA. Taken together, our results could be helpful for revealing the biological functions of plant RFC-PCNA complex.

Subunit Interaction Differences Between the Replication Factor C Complexes in Arabidopsis and Rice.

Replication factor C (RFC) is a multisubunit complex that opens the sliding clamp and loads it onto the DNA chain in an ATP-dependent manner and is thus critical for high-speed DNA synthesis. In yeast (Saccharomyces cerevisiae) and humans, biochemical studies and structural analysis revealed interaction patterns between the subunits and architectures of the clamp loaders. Mutations of ScRFC1/2/3/4/5 lead to loss of cell viability and defective replication. However, the functions of RFC subunits in higher plants are unclear, except for AtRFC1/3/4, and the interaction and arrangement of the subunits have not been studied. Here, we identified rfc2-1/+, rfc3-2/+, and rfc5-1/+ mutants in Arabidopsis, and found that embryos and endosperm arrested at the 2/4-celled embryo proper stage and 6-8 nuclei stages, respectively. Subcellular localization analysis revealed that AtRFC1 and OsRFC1/4/5 proteins were localized in the nucleus, while AtRFC2/3/4/5 and OsRFC2/3 proteins were present both in the nucleus and cytoplasm. By using yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) techniques, we demonstrated the interactions of Arabidopsis and rice (Oryza sativa) RFC subunits, and proposed arrangements of the five subunits within the RFC complex, which were AtRFC5-AtRFC4-AtRFC3/2-AtRFC2/3-AtRFC1 and OsRFC5-OsRFC2-OsRFC3-OsRFC4-OsRFC1, respectively. In addition, AtRFC1 could interact with AtRFC2/3/4/5 in the presence of other subunits, while OsRFC1 directly interacted with the other four subunits. To further characterize the regions required for complex formation, truncated RFC proteins of the subunits were created. The results showed that C-termini of the RFC subunits are required for complex formation. Our studies indicate that the localization and interactions of RFCs in Arabidopsis and rice are distinctly discrepant.

Arabidopsis replication factor C4 is critical for DNA replication during the mitotic cell cycle.

Replication factor C (RFC) is a conserved eukaryotic complex consisting of RFC1/2/3/4/5. It plays important roles in DNA replication and the cell cycle in yeast and fruit fly. However, it is not very clear how RFC subunits function in higher plants, except for the Arabidopsis (At) subunits AtRFC1 and AtRFC3. In this study, we investigated the functions of AtRFC4 and found that loss of function of AtRFC4 led to an early sporophyte lethality that initiated as early as the elongated zygote stage, all defective embryos arrested at the two- to four-cell embryo proper stage, and the endosperm possessed six to eight free nuclei. Complementation of rfc4-1/+ with AtRFC4 expression driven through the embryo-specific DD45pro and ABI3pro or the endosperm-specific FIS2pro could not completely restore the defective embryo or endosperm, whereas a combination of these three promoters in rfc4-1/+ enabled the aborted ovules to develop into viable seeds. This suggests that AtRFC4 functions simultaneously in endosperm and embryo and that the proliferation of endosperm is critical for embryo maturation. Assays of DNA content in rfc4-1/+ verified that DNA replication was disrupted in endosperm and embryo, resulting in blocked mitosis. Moreover, we observed a decreased proportion of late S-phase and M-phase cells in the rfc4-1/-FIS2;DD45;ABI3pro::AtRFC4 seedlings, suggesting that incomplete DNA replication triggered cell cycle arrest in cells of the root apical meristem. Therefore, we conclude that AtRFC4 is a crucial gene for DNA replication.

Structural and Functional Analysis of the interaction between PCNA and ATAD5

Proliferating cell nuclear antigen (PCNA) is a DNA sliding clamp that coordinates DNA replication and repair. PCNA is loaded on and unloaded off of DNA during replication. It is loaded onto DNA by the replication factor C (RFC) complex. The mechanism of PCNA unloading is not fully understood however. ATPase family AAA domain containing protein 5 (ATAD5) interacts with subunits of RFC to form a RFC-like complex that functions in unloading PCNA. Reduced expression of ATAD5 extends the life of replication factories by retaining PCNA and other repliosome proteins on chromatin. ATAD5 also plays a role in maintaining genomic stability, as mutations to ATAD5 cause genomic instability in mice and human cells. The region of ATAD5 that interacts with PCNA had yet to be determined. Proteins interacting with PCNA usually have a PCNA-interaction protein (PIP) motif termed the PIP-box. Through sequence analysis, we identified a putative PIP-box in ATAD5. Using GST pull-down experiments, we found that the putative PIP-box bound to PCNA. Mutation of the key residues in the PIP-box abolished the binding. The interaction was also confirmed by isothermal titration calorimetry (ITC) analysis of PCNA and a synthetic peptide containing the PIP-box. The binding constant (Kd) of the PCNA-peptide interaction is 6.2μM, determined by the ITC assays. To further characterize the interaction, we co-crystallized PCNA and the ATAD5 peptide. The complex crystals diffracted to a resolution of 3.5Å. The crystals belong to the space group H3, with unit-cell parameters a = 83.6Å and c = 211.4Å. Our molecular replacement solution indicates that the peptide binds on PCNA at the conserved PIP-box binding pocket. Taken together, our work demonstrates that ATAD5 does contain a PIP motif that directly interacts with PCNA. The structure-function studies on the interaction will provide insights into the molecular mechanism of PCNA unloading and the role of ATAD5 in DNA replication and repair.

Abscisic Acid Suppresses the Highly Occurred Somatic Homologous Recombination in Arabidopsis rfc1 Mutant

The phytohormone abscisic acid (ABA) regulates many aspects of plant growth, including seed germination, root growth and cell division. Previous study indicates that ABA treatment increases DNA damage and somatic homologous recombination (HR) in Arabidopsis abo4/pol ɛ (aba overly-sensitive 4 /DNA polymerase ɛ) mutants. DNA replication factor C (RFC) complex is required for loading PCNA (Proliferating Cell Nuclear Antigen) during DNA replication. The defect in RFC1, the largest subunit of RFC, causes the high HR and DNA damage sensitivity in Arabidopsis. Here we found that like pol ε/abo4, rfc1 is sensitive to ABA in both ABA-inhibiting seed germination and root growth. However, ABA treatment greatly reduces HR and also reduces the expression of the DNA-damaged marker genes in rfc1. These results suggest that RFC1 plays critical roles in ABA-mediated HR in Arabidopsis.

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·PCNAopen·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.

Rfc5p regulates alternate RFC complex functions in sister chromatid pairing reactions in budding yeast

Sister chromatid pairing reactions, termed cohesion establishment, occur during S-phase and appear to be regulated by Replication Factor C (RFC) complexes. For instance, RFCs that contain Ctf18p exhibit pro-establishment activities while those that contain Elg1p exhibit anti-establishment activities. It remains unknown whether Ctf18p-RFC and Elg1p-RFC functions are simply opposing or instead reveal complicated and non-parallel regulatory mechanisms. To better understand the nature of these novel pathways, we analyzed the small RFC subunit Rfc5p that is common to both Ctf18p-RFC and Elg1p-RFC. Despite this commonality, the data show that diminished Rfc5p function rescues ctf7/eco1 mutant cell phenotypes, revealing that Rfc5p promotes anti-establishment activities. This rescue is specific to establishment pathways in that rfc5-1 greatly accentuates growth defects when expressed in scc2 (deposition), mcd1/scc1 or smc3 (cohesion maintenance) mutated cells. Our results reveal for the first time a role for small RFC subunits in directing RFC complex functions - in this case towards anti-establishment pathways. We further report that Pds5p exhibits both establishment and anti-establishment functions in cohesion. This duality suggests that categorizations of establishment and anti-establishment activities require further examination.

Elg1, an alternative subunit of the RFC clamp loader, preferentially interacts with SUMOylated PCNA

Replication-factor C (RFC) is a protein complex that loads the processivity clamp PCNA onto DNA. Elg1 is a conserved protein with homology to the largest subunit of RFC, but its function remained enigmatic. Here, we show that yeast Elg1 interacts physically and genetically with PCNA, in a manner that depends on PCNA modification, and exhibits preferential affinity for SUMOylated PCNA. This interaction is mediated by three small ubiquitin-like modifier (SUMO)-interacting motifs and a PCNA-interacting protein box close to the N-terminus of Elg1. These motifs are important for the ability of Elg1 to maintain genomic stability. SUMOylated PCNA is known to recruit the helicase Srs2, and in the absence of Elg1, Srs2 and SUMOylated PCNA accumulate on chromatin. Strains carrying mutations in both ELG1 and SRS2 exhibit a synthetic fitness defect that depends on PCNA modification. Our results underscore the importance of Elg1, Srs2 and SUMOylated PCNA in the maintenance of genomic stability.

Recognition of the Ring-Opened State of Proliferating Cell Nuclear Antigen by Replication Factor C Promotes Eukaryotic Clamp-Loading

Proliferating cell nuclear antigen (PCNA, sliding clamp) is a toroidal-shaped protein that encircles DNA and plays a pivotal role in DNA replication, modification and repair. To perform its vital functions, the clamp has to be opened and resealed at primer−template junctions by a clamp loader molecular machine, replication factor C (RFC). The mechanism of this process constitutes a significant piece in the puzzle of processive DNA replication. We show that upon clamp opening the RFC/PCNA complex undergoes a large conformational rearrangement, leading to the formation of an extended interface between the clamp and RFC. Binding of ring-open PCNA to all five RFC subunits transforms the free-energy landscape underlying the closed- to open state transition, trapping PCNA in an open conformation. Careful comparison of free-energy profiles for clamp opening in the presence and absence of RFC allowed us to substantiate the role of RFC in the initial stage of the clamp-loading cycle. RFC does not appreciably destabilize the closed state of PCNA. Instead, the function of the clamp loader is dependent on the selective stabilization of the open conformation of the clamp.

Human ELG1 Regulates the Level of Ubiquitinated Proliferating Cell Nuclear Antigen (PCNA) through Its Interactions with PCNA and USP1

The level of monoubiquitinated proliferating cell nuclear antigen (PCNA) is closely linked with DNA damage bypass to protect cells from a high level of mutagenesis. However, it remains unclear how the level of monoubiquitinated PCNA is regulated. Here, we demonstrate that human ELG1 protein, which comprises an alternative replication factor C (RFC) complex and plays an important role in preserving genomic stability, as an interacting partner for the USP1 (ubiquitin-specific protease 1)-UAF1 (USP1-associated factor 1) complex, a deubiquitinating enzyme complex for PCNA and FANCD2. ELG1 protein interacts with PCNAs that are localized at stalled replication forks. ELG1 knockdown specifically resulted in an increase in the level of PCNA monoubiquitination without affecting the level of FANCD2 ubiquitination. It is a novel function of ELG1 distinct from its role as an alternative RFC complex because knockdowns of any other RFC subunits or other alternative RFCs did not affect PCNA monoubiquitination. Lastly, we identified a highly conserved N-terminal domain in ELG1 that was responsible for the USP1-UAF1 interaction as well as the activity to down-regulate PCNA monoubiquitination. Taken together, ELG1 specifically directs USP1-UAF1 complex for PCNA deubiquitination.

Inactivating pentapeptide insertions in the fission yeast replication factor C subunit Rfc2 cluster near the ATP‐binding site and arginine finger motif

Replication factor C (RFC) plays a key role in eukaryotic chromosome replication by acting as a loading factor for the essential sliding clamp and polymerase processivity factor, proliferating cell nuclear antigen (PCNA). RFC is a pentamer comprising a large subunit, Rfc1, and four small subunits, Rfc2-Rfc5. Each RFC subunit is a member of the AAA+ family of ATPase and ATPase-like proteins, and the loading of PCNA onto double-stranded DNA is an ATP-dependent process. Here, we describe the properties of a collection of 38 mutant forms of the Rfc2 protein generated by pentapeptide-scanning mutagenesis of the fission yeast rfc2 gene. Each insertion was tested for its ability to support growth in fission yeast rfc2Delta cells lacking endogenous Rfc2 protein and the location of each insertion was mapped onto the 3D structure of budding yeast Rfc2. This analysis revealed that the majority of the inactivating mutations mapped in or adjacent to ATP sites C and D in Rfc2 (arginine finger and P-loop, respectively) or to the five-stranded beta sheet at the heart of the Rfc2 protein. By contrast, nonlethal mutations map predominantly to loop regions or to the outer surface of the RFC complex, often in highly conserved regions of the protein. Possible explanations for the effects of the various insertions are discussed.

Mechanism of ATP-Driven PCNA Clamp Loading by S. cerevisiae RFC

Circular clamps tether polymerases to DNA, serving as essential processivity factors in genome replication, and function in other critical cellular processes as well. Clamp loaders catalyze clamp assembly onto DNA, and the question of how these proteins construct a topological link between a clamp and DNA, especially the mechanism by which ATP is utilized for the task, remains open. Here we describe pre-steady-state analysis of ATP hydrolysis, proliferating cell nuclear antigen (PCNA) clamp opening, and DNA binding by Saccharomyces cerevisiae replication factor C (RFC), and present the first kinetic model of a eukaryotic clamp-loading reaction validated by global data analysis. ATP binding to multiple RFC subunits initiates a slow conformational change in the clamp loader, enabling it to bind and open PCNA and to bind DNA as well. PCNA opening locks RFC into an active state, and the resulting RFC.ATP.PCNA((open)) intermediate is ready for the entry of DNA into the clamp. DNA binding commits RFC to ATP hydrolysis, which is followed by PCNA closure and PCNA.DNA release. This model enables quantitative understanding of the multistep mechanism of a eukaryotic clamp loader and furthermore facilitates comparative analysis of loaders from diverse organisms.

Kinetic Analysis of Arginine-Finger Motif Function in S. cerevisiae Clamp Loader

Replication factor C (RFC) loads a circular clamp, proliferating cell nuclear antigen (PCNA), onto a primer-template junction at the replication fork during DNA replication. RFC consists of five subunits, A-E, each of which belongs to the AAA+ family member of ATPases. The subunits are arranged in an open ring form with four ATPase sites located at the interfaces of subunits A-B, B-C, C-D and D-E. In each ATPase site, an Arginine-finger motif (SRC), which is provided by the adjacent subunit, is implicated in sensing ATP binding and regulating ATP hydrolysis. According to a recent study of SRC→SAC point mutations, it appears that RFC subunits play distinct roles during clamp assembly (Johnson, et al. 2006). We analyzed these mutants by measuring their pre-steady state functions of DNA binding and ATPase activities. We found that CSAC (Binactive) and DSAC (Cinactive) mutants have severe defects in DNA binding, while BSAC (Ainactive) and ESAC (Dinactive) mutants have severe defects in DNA dissociation. These disruptions in RFC interactions with DNA, and corresponding effects of RFC ATPase activities, elucidate further the distinct functions of individual RFC subunits in the clamp assembly reaction.Reference: Johnson A. et al., (2006) J. Biol. Chem. 281, 35531-35543.

DNA Damage-induced Ubiquitylation of RFC2 Subunit of Replication Factor C Complex

Many proteins involved in DNA replication and repair undergo post-translational modifications such as phosphorylation and ubiquitylation. Proliferating cell nuclear antigen (PCNA; a homotrimeric protein that encircles double-stranded DNA to function as a sliding clamp for DNA polymerases) is monoubiquitylated by the RAD6-RAD18 complex and further polyubiquitylated by the RAD5-MMS2-UBC13 complex in response to various DNA-damaging agents. PCNA mono- and polyubiquitylation activate an error-prone translesion synthesis pathway and an error-free pathway of damage avoidance, respectively. Here we show that replication factor C (RFC; a heteropentameric protein complex that loads PCNA onto DNA) was also ubiquitylated in a RAD18-dependent manner in cells treated with alkylating agents or H(2)O(2). A mutant form of RFC2 with a D228A substitution (corresponding to a yeast Rfc4 mutation that reduces an interaction with replication protein A (RPA), a single-stranded DNA-binding protein) was heavily ubiquitylated in cells even in the absence of DNA damage. Furthermore RFC2 was ubiquitylated by the RAD6-RAD18 complex in vitro, and its modification was inhibited in the presence of RPA. The inhibitory effect of RPA on RFC2 ubiquitylation was relatively specific because RAD6-RAD18-mediated ubiquitylation of PCNA was RPA-insensitive. Our findings suggest that RPA plays a regulatory role in DNA damage responses via repression of RFC2 ubiquitylation in human cells.

A Second Proliferating Cell Nuclear Antigen Loader Complex, Ctf18-Replication Factor C, Stimulates DNA Polymerase η Activity

Replication factor C (RFC) loads the clamp protein PCNA onto DNA structures. Ctf18-RFC, which consists of the chromosome cohesion factors Ctf18, Dcc1, and Ctf8 and four small RFC subunits, functions as a second proliferating cell nuclear antigen (PCNA) loader. To identify potential targets of Ctf18-RFC, human cell extracts were assayed for DNA polymerase activity specifically stimulated by Ctf18-RFC in conjunction with PCNA. After several chromatography steps, an activity stimulated by Ctf18-RFC but not by RFC was identified. Liquid chromatography/tandem mass spectrometry (LC/MS/MS) analysis revealed the presence of two DNA polymerases, eta and lambda, in the most purified fraction, but experiments with purified recombinant proteins demonstrated that only polymerase (pol) eta was responsible for activity. Ctf18-RFC alone stimulated pol eta, and the addition of PCNA cooperatively increased stimulation. Furthermore, Ctf18-RFC interacted physically with pol eta, as indicated by co-precipitation in human cells. We propose that this novel loader-DNA polymerase interaction allows DNA replication forks to overcome interference by various template structures, including damaged DNA and DNA-protein complexes that maintain chromosome cohesion.

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.

Interplay between primase and replication factor C in the hyperthermophilic archaeonSulfolobus solfataricus

The heterodimeric primase from the hyperthermophilic archaeon Sulfolobus solfataricus synthesizes long RNA and DNA products in vitro. How primer synthesis by primase is coupled to primer extension by DNA polymerase in this organism is unclear. Here we show that the small subunit of the clamp loader replication factor C (RFC) of S. solfataricus interacted with both the catalytic and non-catalytic subunits of the primase by yeast two-hybrid and co-immunoprecipitation assays. Further, the primase-RFC interaction was also identified in the cell extract of S. solfataricus. Deletion analysis indicated that the small subunit of RFC interacted strongly with the N-terminal domain of the catalytic subunit of the primase. RFC stimulated dinucleotide formation but decreased the amount of primers synthesized by the primase. The inhibition of primer synthesis is consistent with the observation that RFC reduced the affinity of the primase for DNA templates. On the other hand, primase stimulated the ATPase activity of RFC. These findings suggest that the primase-RFC interaction modulates the activities of both enzymes and therefore may be involved in the regulation of primer synthesis and the transfer of primers to DNA polymerase in Archaea.

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.

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.

Contrasting effects of Elg1-RFC and Ctf18-RFC inactivation in the absence of fully functional RFC in fission yeast.

Proliferating cell nuclear antigen loading onto DNA by replication factor C (RFC) is a key step in eukaryotic DNA replication and repair processes. In this study, the C-terminal domain (CTD) of the large subunit of fission yeast RFC is shown to be essential for its function in vivo. Cells carrying a temperature-sensitive mutation in the CTD, rfc1-44, arrest with incompletely replicated chromosomes, are sensitive to DNA damaging agents, are synthetically lethal with other DNA replication mutants, and can be suppressed by mutations in rfc5. To assess the contribution of the RFC-like complexes Elg1–RFC and Ctf18–RFC to the viability of rfc1-44, genes encoding the large subunits of these complexes have been deleted and overexpressed. Inactivation of Ctf18–RFC by the deletion of ctf18+, dcc1+ or ctf8+ is lethal in an rfc1-44 background showing that full Ctf18–RFC function is required in the absence of fully functional RFC. In contrast, rfc1-44 elg1Δ cells are viable and overproduction of Elg1 in rfc1-44 is lethal, suggesting that Elg1–RFC plays a negative role when RFC function is inhibited. Consistent with this, the deletion of elg1+ is shown to restore viability to rfc1-44 ctf18Δ cells.