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.     

Physical links between the nuclear envelope protein Mps3, three alternate replication factor C complexes, and a variant histone in Saccharomyces cerevisiae

Viability of cell progeny upon cell division require that genomes are replicated, repaired, and maintained with high fidelity. Central to both DNA replication and repair are Replication Factor C (RFC) complexes which catalyze the unloading/loading of sliding clamps such as PCNA or 9-1-1 complexes on DNA. Budding yeast contain four alternate RFC complexes which play partially redundant roles. Rfc1, Ctf18, Rad24, and Elg1 are all large subunits that bind, in a mutually exclusive fashion to RFC 2-5 small subunits. Ctf18, Rad24, and Elg1 are of particular interest because, in addition to their roles in maintaining genome integrity, all three play critical roles in sister chromatid tethering reactions that appear coupled to their roles in DNA replication/repair. Intriguingly, the nuclear envelope protein Mps3 similarly exhibits roles in repair and cohesion, leading us to hypothesize that Mps3 and RFCs function through a singular mechanism. Here we report that the nuclear envelope protein Mps3 physically associates with all three of these large RFC complex subunits (Ctf18, Elg1, and Rad24). In addition we report a physical interaction between Mps3 and the histone variant Htz1, a factor previously shown to promote DNA repair. In combination, these findings reveal a direct link between the nuclear envelope and chromatin and provide support for a model that telomeres and chromatin interact with the nuclear envelope during both DNA repair and sister chromatid pairing reactions.     

The N- and C-termini of Elg1 contribute to the maintenance of genome stability

ELG1 (enhanced level of genome instability) encodes a Replication Factor C (RFC) homolog that is important for the maintenance of genome stability. Elg1 interacts with Rfc2-5, forming the third alternative RFC complex identified to date. We found that Elg1 plays a role in the suppression of spontaneous DNA damage in addition to its previously identified roles in the resistance to DNA damage. Using mutational analysis we examined the function of conserved and unique regions of Elg1 in these roles. We found that the Walker A motif in the conserved RFC region is dispensable for Elg1 function in vivo. The RFC region is important for association with chromatin although residues predicted to mediate interactions with DNA are dispensable for Elg1 function. The unique C-terminus of Elg1 mediates oligomerization with Rfc2-5, nuclear import, and chromatin association, and is critical for the function of Elg1. Finally, we demonstrated that the N-terminus of Elg1 contributes to the maintenance of genome stability, and that one function of this N-terminus is to promote the nuclear localization of Elg1. Together, these studies delineate the regions of Elg1 important for its function in damage resistance and in the suppression of spontaneous DNA damage.     

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 forms an alternative PCNA-interacting RFC complex required to maintain genome stability

BACKGROUND: Genome instability is a hallmark of cancer and plays a critical role in generating the myriad of phenotypes selected for during tumor progression. However, the mechanisms that prevent genome rearrangements remain poorly understood. RESULTS: To elucidate the mechanisms that ensure genome stability, we screened a collection of candidate genes for suppressors of gross chromosomal rearrangements (GCRs) in budding yeast. One potent suppressor gene encodes Elg1, a conserved but uncharacterized homolog of the large RFC subunit Rfc1 and the alternative RFC subunits Ctf18/Chl12 and Rad24. Our results are consistent with the hypothesis that Elg1 forms a novel and distinct RFC-like complex in both yeast and human cells. We find that Elg1 is required for efficient S phase progression and telomere homeostasis in yeast. Elg1 interacts physically with the PCNA homolog Pol30 and the FEN-1 homolog Rad27. The physical and genetic interactions suggest a role for Elg1 in Okazaki fragment maturation. Furthermore, Elg1 acts in concert with the alternative Rfc1-like proteins Rad24 and Ctf18 to enable Rad53 checkpoint kinase activation in response to replication stress. CONCLUSIONS: Collectively, these results reveal that Elg1 forms a novel and conserved alternative RFC complex. Furthermore, we propose that genome instability arises at high frequency in elg1 mutants due to a defect in Okazaki fragment maturation.     

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.     

The Elg1 replication factor C-like complex: a novel guardian of genome stability

The remarkable stability of the eukaryotic genome is achieved by the activity of many overlapping surveillance and repair mechanism. Two protein complexes with resemblance to replication factor C (RFC) have been recently described, that play important roles in maintaining the stability of the genome. These RFC-like complexes (RLCs) share four common subunits (Rfc2-5) and each carry a unique large subunit (Rad24 or Ctf18) replacing the Rfc1 subunit of the replication complex. Work in several laboratories has recently uncovered a novel yeast gene, ELG1, which seems to play a central role in keeping the genome stable. elg1 mutants exhibit increased rates of spontaneous recombination and gross chromosomal rearrangements during vegetative growth. In addition, they lose chromosomes at an enhanced rate, show hyper-transposition of natural repeated elements and exhibit elongated telomeres. The Elg1 protein also associates with the Rfc2-5 subunits of replication factor C (RFC) to form a third RFC-like complex (RLC). Genetic and biochemical data indicate that the Elg1, Ctf18 and Rad24 RLCs work in three separate pathways important for maintaining the integrity of the genome and for coping with various genomic stresses. ELG1 is evolutionarily conserved and may play an important role in preventing the onset of cancer in humans. The Elg1 function is thus clearly required for maintaining genome stability during normal growth, and its absence has severe genetic consequences.     

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.Key words: sister chromatid cohesion, ctf7/eco1, ELG1 RFC complexes, CTF18 RFC complexes, PDS5     

Mechanical link between cohesion establishment and DNA replication: Ctf7p/Eco1p,a cohesion establishment factor,associates with three different replication factor C complexes

CTF7/ECO1 is an essential yeast gene required for the establishment of sister chromatid cohesion. The findings that CTF7/ECO1, POL30 (PCNA), and CHL12/CTF18 (a replication factor C [RFC] homolog) genetically interact provided the first evidence that the processes of cohesion establishment and DNA replication are intimately coupled-a link now confirmed by other studies. To date, however, it is unknown how Ctf7p/Eco1p function is coupled to DNA replication or whether Ctf7p/Eco1p physically associates with any components of the DNA replication machinery. Here, we report that Ctf7p/Eco1p associates with proteins that perform partially redundant functions in DNA replication. Chl12p/Ctf18p combines with Rfc2p to Rfc5p to form one of three independent RFC complexes. By chromatographic methods, Ctf7p/Eco1p was found to associate with Chl12/Ctf18p and with Rfc2p, Rfc3p, Rfc4p, and Rfc5p. The association between Ctf7p/Eco1p and this RFC complex is biologically relevant in that (i) Ctf7p/Eco1p cosediments with Chl12p/Ctf18p in vivo and (ii) rfc5-1 mutant cells exhibit precocious sister separation. Previous studies revealed that Rfc1p or Rad24p associates with Rfc2p to Rfc5p to form two other RFC complexes independent of Ctf18p-RFC complexes. These Rfc1p-RFC and Rad24p-RFC complexes function in DNA replication or repair and DNA damage checkpoint pathways. Importantly, Ctf7p/Eco1p also associates with Rfc1p and Rad24p, suggesting that these RFC complexes also play critical roles in cohesion establishment. The associations between Ctf7p/Eco1p and RFC subunits provide novel evidence regarding the physical linkage between cohesion establishment and DNA replication. Furthermore, the association of Ctf7p/Eco1p with each of three RFC complexes supplies new insights into the functional redundancy of RFC complexes in cohesion establishment.     

DNA polymerase delta, RFC and PCNA are required for repair synthesis of large looped heteroduplexes in Saccharomyces cerevisiae

Small looped mispairs are corrected by DNA mismatch repair (MMR). In addition, a distinct process called large loop repair (LLR) corrects loops up to several hundred nucleotides in extracts of bacteria, yeast or human cells. Although LLR activity can be readily demonstrated, there has been little progress in identifying its protein components. This study identified some of the yeast proteins responsible for DNA repair synthesis during LLR. Polyclonal antisera to either Pol31 or Pol32 subunits of polymerase δ efficiently inhibited LLR in extracts by blocking repair just prior to gap filling. Gap filling was inhibited regardless of whether the loop was retained or removed. These experiments suggest polymerase δ is uniquely required in yeast extracts for LLR-associated synthesis. Similar results were obtained with antisera to the clamp loader proteins Rfc3 and Rfc4, and to PCNA, i.e. LLR was inhibited just prior to gap filling for both loop removal and loop retention. Thus PCNA and RFC seem to act in LLR only during repair synthesis, in contrast to their roles at both pre- and post-excision steps of MMR. These biochemical experiments support the idea that yeast polymerase δ, RFC and PCNA are required for large loop DNA repair synthesis.     

Regulation of Elg1 activity by phosphorylation

ELG1 is a conserved gene with important roles in the maintenance of genome stability. Elg1''s activity prevents gross chromosomal rearrangements, maintains proper telomere length regulation, helps repairing DNA damage created by a number of genotoxins and participates in sister chromatid cohesion. Elg1 is evolutionarily conserved, and its Fanconi Anemia-related mammalian ortholog (also known as ATAD5) is embryonic lethal when lost in mice and acts as a tumor suppressor in mice and humans. Elg1 encodes a protein that forms an RFC-like complex that unloads the replicative clamp, PCNA, from DNA, mainly in its SUMOylated form. We have identified 2 different regions in yeast Elg1 that undergo phosphorylation. Phosphorylation of one of them, S112, is dependent on the ATR yeast ortholog, Mec1, and probably is a direct target of this kinase. We show that phosphorylation of Elg1 is important for its role at telomeres. Mutants unable to undergo phosphorylation suppress the DNA damage sensitivity of Δrad5 mutants, defective for an error-free post-replicational bypass pathway. This indicates a role of phosphorylation in the regulation of DNA repair. Our results open the way to investigate the mechanisms by which the activity of Elg1 is regulated during DNA replication and in response to DNA damage.     

Functional and Physical Interaction between Rad24 and Rfc5 in the Yeast Checkpoint Pathways

The RFC5 gene encodes a small subunit of replication factor C (RFC) complex in Saccharomyces cerevisiae and has been shown to be required for the checkpoints which respond to replication block and DNA damage. Here we describe the isolation of RAD24, known to play a role in the DNA damage checkpoint, as a dosage-dependent suppressor of rfc5-1. RAD24 overexpression suppresses the sensitivity of rfc5-1 cells to DNA-damaging agents and the defect in DNA damage-induced Rad53 phosphorylation. Rad24, like Rfc5, is required for the regulation of Rad53 phosphorylation in response to DNA damage. The Rad24 protein, which is structurally related to the RFC subunits, interacts physically with RFC subunits Rfc2 and Rfc5 and cosediments with Rfc5. Although the rad24Δ mutation alone does not cause a defect in the replication block checkpoint, it does enhance the defect in rfc5-1 mutants. Furthermore, overexpression of RAD24 suppresses the rfc5-1 defect in the replication block checkpoint. Taken together, our results demonstrate a physical and functional interaction between Rad24 and Rfc5 in the checkpoint pathways.     

Elg1 forms an alternative RFC complex important for DNA replication and genome integrity

Genome-wide synthetic genetic interaction screens with mutants in the mus81 and mms4 replication fork-processing genes identified a novel replication factor C (RFC) homolog, Elg1, which forms an alternative RFC complex with Rfc2-5. This complex is distinct from the DNA replication RFC, the DNA damage checkpoint RFC and the sister chromatid cohesion RFC. As expected from its genetic interactions, elg1 mutants are sensitive to DNA damage. Elg1 is redundant with Rad24 in the DNA damage response and contributes to activation of the checkpoint kinase Rad53. We find that elg1 mutants display DNA replication defects and genome instability, including increased recombination and mutation frequencies, and minichromosome maintenance defects. Mutants in elg1 show genetic interactions with pathways required for processing of stalled replication forks, and are defective in recovery from DNA damage during S phase. We propose that Elg1-RFC functions both in normal DNA replication and in the DNA damage response.     

Rfc5, in cooperation with rad24, controls DNA damage checkpoints throughout the cell cycle in Saccharomyces cerevisiae

RAD24 and RFC5 are required for DNA damage checkpoint control in the budding yeast Saccharomyces cerevisiae. Rad24 is structurally related to replication factor C (RFC) subunits and associates with RFC subunits Rfc2, Rfc3, Rfc4, and Rfc5. rad24Delta mutants are defective in all the G(1)-, S-, and G(2)/M-phase DNA damage checkpoints, whereas the rfc5-1 mutant is impaired only in the S-phase DNA damage checkpoint. Both the RFC subunits and Rad24 contain a consensus sequence for nucleoside triphosphate (NTP) binding. To determine whether the NTP-binding motif is important for Rad24 function, we mutated the conserved lysine(115) residue in this motif. The rad24-K115E mutation, which changes lysine to glutamate, confers a complete loss-of-function phenotype, while the rad24-K115R mutation, which changes lysine to arginine, shows no apparent phenotype. Although neither rfc5-1 nor rad24-K115R single mutants are defective in the G(1)- and G(2)/M-phase DNA damage checkpoints, rfc5-1 rad24-K115R double mutants become defective in these checkpoints. Coimmunoprecipitation experiments revealed that Rad24(K115R) fails to interact with the RFC proteins in rfc5-1 mutants. Together, these results indicate that RFC5, like RAD24, functions in all the G(1)-, S- and G(2)/M-phase DNA damage checkpoints and suggest that the interaction of Rad24 with the RFC proteins is essential for DNA damage checkpoint control.     

Chl12 (Ctf18) forms a novel replication factor C-related complex and functions redundantly with Rad24 in the DNA replication checkpoint pathway

RAD24 has been identified as a gene essential for the DNA damage checkpoint in budding yeast. Rad24 is structurally related to subunits of the replication factor C (RFC) complex, and forms an RFC-related complex with Rfc2, Rfc3, Rfc4, and Rfc5. The rad24Delta mutation enhances the defect of rfc5-1 in the DNA replication block checkpoint, implicating RAD24 in this checkpoint. CHL12 (also called CTF18) encodes a protein that is structurally related to the Rad24 and RFC proteins. We show here that although neither chl12Delta nor rad24Delta single mutants are defective, chl12Delta rad24Delta double mutants become defective in the replication block checkpoint. We also show that Chl12 interacts physically with Rfc2, Rfc3, Rfc4, and Rfc5 and forms an RFC-related complex which is distinct from the RFC and RAD24 complexes. Our results suggest that Chl12 forms a novel RFC-related complex and functions redundantly with Rad24 in the DNA replication block checkpoint.     

A novel Rad24 checkpoint protein complex closely related to replication factor C

Rad24 functions in the DNA damage checkpoint pathway of Saccharomyces cerevisiae. Here, analysis of Rad24 in whole cell extracts demonstrated that its mass was considerably greater than its predicted molecular weight, suggesting that Rad24 is a component of a protein complex. The Rad24 complex was purified to homogeneity. In addition to Rad24, the complex included polypeptides of 40 kDa and 35 kDa. The 40 kDa species was found by mass spectrometry to contain Rfc2 and Rfc3, subunits of replication factor C (RFC), a five subunit protein that is required for the loading of polymerases onto DNA during replication and repair [3]. We hypothesised that other RFC subunits, all of which share sequence homologles with Rad24, might also be components of the Rad24 complex. Reciprocal co-immunoprecipitation studies were performed using extracts prepared from strains containing epitope-tagged RFC proteins. These experiments showed that the small RFC proteins, Rfc2, Rfc3, Rfc4 and Rfc5, interacted with Rad24, whereas the Rfc1 subunit did not. We suggest that this RFC-like Rad24 complex may function as a structure-specific sensor in the DNA damage checkpoint pathway.     

A SUMO-targeted ubiquitin ligase is involved in the degradation of the nuclear pool of the SUMO E3 ligase Siz1

The Slx5/Slx8 heterodimer constitutes a SUMO-targeted ubiquitin ligase (STUbL) with an important role in SUMO-targeted degradation and SUMO-dependent signaling. This STUbL relies on SUMO-interacting motifs in Slx5 to aid in substrate targeting and carboxy-terminal RING domains in both Slx5 and Slx8 for substrate ubiquitylation. In budding yeast cells, Slx5 resides in the nucleus, forms distinct foci, and can associate with double-stranded DNA breaks. However, it remains unclear how STUbLs interact with other proteins and their substrates. To examine the targeting and functions of the Slx5/Slx8 STUbL, we constructed and analyzed truncations of the Slx5 protein. Our structure–function analysis reveals a domain of Slx5 involved in nuclear localization and in the interaction with Slx5, SUMO, Slx8, and a novel interactor, the SUMO E3 ligase Siz1. We further analyzed the functional interaction of Slx5 and Siz1 in vitro and in vivo. We found that a recombinant Siz1 fragment is an in vitro ubiquitylation target of the Slx5/Slx8 STUbL. Furthermore, slx5∆ cells accumulate phosphorylated and sumoylated adducts of Siz1 in vivo. Specifically, we show that Siz1 can be ubiquitylated in vivo and is degraded in an Slx5-dependent manner when its nuclear egress is prevented in mitosis. In conclusion, our data provide a first look into the STUbL-mediated regulation of a SUMO E3 ligase.     

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.     

Biochemical Characterization of DNA Damage Checkpoint Complexes: Clamp Loader and Clamp Complexes with Specificity for 5′ Recessed DNA

The cellular pathways involved in maintaining genome stability halt cell cycle progression in the presence of DNA damage or incomplete replication. Proteins required for this pathway include Rad17, Rad9, Hus1, Rad1, and Rfc-2, Rfc-3, Rfc-4, and Rfc-5. The heteropentamer replication factor C (RFC) loads during DNA replication the homotrimer proliferating cell nuclear antigen (PCNA) polymerase clamp onto DNA. Sequence similarities suggest the biochemical functions of an RSR (Rad17–Rfc2–Rfc3–Rfc4–Rfc5) complex and an RHR heterotrimer (Rad1–Hus1–Rad9) may be similar to that of RFC and PCNA, respectively. RSR purified from human cells loads RHR onto DNA in an ATP-, replication protein A-, and DNA structure-dependent manner. Interestingly, RSR and RFC differed in their ATPase activities and displayed distinct DNA substrate specificities. RSR preferred DNA substrates possessing 5′ recessed ends whereas RFC preferred 3′ recessed end DNA substrates. Characterization of the biochemical loading reaction executed by the checkpoint clamp loader RSR suggests new insights into the mechanisms underlying recognition of damage-induced DNA structures and signaling to cell cycle controls. The observation that RSR loads its clamp onto a 5′ recessed end supports a potential role for RHR and RSR in diverse DNA metabolism, such as stalled DNA replication forks, recombination-linked DNA repair, and telomere maintenance, among other processes.     

Biochemical characterization of DNA damage checkpoint complexes: clamp loader and clamp complexes with specificity for 5' recessed DNA

The cellular pathways involved in maintaining genome stability halt cell cycle progression in the presence of DNA damage or incomplete replication. Proteins required for this pathway include Rad17, Rad9, Hus1, Rad1, and Rfc-2, Rfc-3, Rfc-4, and Rfc-5. The heteropentamer replication factor C (RFC) loads during DNA replication the homotrimer proliferating cell nuclear antigen (PCNA) polymerase clamp onto DNA. Sequence similarities suggest the biochemical functions of an RSR (Rad17–Rfc2–Rfc3–Rfc4–Rfc5) complex and an RHR heterotrimer (Rad1–Hus1–Rad9) may be similar to that of RFC and PCNA, respectively. RSR purified from human cells loads RHR onto DNA in an ATP-, replication protein A-, and DNA structure-dependent manner. Interestingly, RSR and RFC differed in their ATPase activities and displayed distinct DNA substrate specificities. RSR preferred DNA substrates possessing 5′ recessed ends whereas RFC preferred 3′ recessed end DNA substrates. Characterization of the biochemical loading reaction executed by the checkpoint clamp loader RSR suggests new insights into the mechanisms underlying recognition of damage-induced DNA structures and signaling to cell cycle controls. The observation that RSR loads its clamp onto a 5′ recessed end supports a potential role for RHR and RSR in diverse DNA metabolism, such as stalled DNA replication forks, recombination-linked DNA repair, and telomere maintenance, among other processes.