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.     

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.     

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.     

The Elg1 Clamp Loader Plays a Role in Sister Chromatid Cohesion

Mutations in the ELG1 gene of yeast lead to genomic instability, manifested in high levels of genetic recombination, chromosome loss, and gross chromosomal rearrangements. Elg1 shows similarity to the large subunit of the Replication Factor C clamp loader, and forms a RFC-like (RLC) complex in conjunction with the 4 small RFC subunits. Two additional RLCs exist in yeast: in one of them the large subunit is Ctf18, and in the other, Rad24. Ctf18 has been characterized as the RLC that functions in sister chromatid cohesion. Here we present evidence that the Elg1 RLC (but not Rad24) also plays an important role in this process. A genetic screen identified the cohesin subunit Mcd1/Scc1 and its loader Scc2 as suppressors of the synthetic lethality between elg1 and ctf4. We describe genetic interactions between ELG1 and genes encoding cohesin subunits and their accessory proteins. We also show that defects in Elg1 lead to higher precocious sister chromatid separation, and that Ctf18 and Elg1 affect cohesion via a joint pathway. Finally, we localize both Ctf18 and Elg1 to chromatin and show that Elg1 plays a role in the recruitment of Ctf18. Our results suggest that Elg1, Ctf4, and Ctf18 may coordinate the relative movement of the replication fork with respect to the cohesin ring.     

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.     

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.     

Identification of RFC(Ctf18p, Ctf8p, Dcc1p): an alternative RFC complex required for sister chromatid cohesion in S. cerevisiae

We have identified and characterized an alternative RFC complex RFC(Ctf18p, Ctf8p, Dcc1p) that is required for sister chromatid cohesion and faithful chromosome transmission. Ctf18p, Ctf8p, and Dcc1p interact physically in a complex with Rfc2p, Rfc3p, Rfc4p, and Rfc5p but not with Rfc1p or Rad24p. Deletion of CTF18, CTF8, or DCC1 singly or in combination (ctf18Deltactf8Deltadcc1Delta) leads to sensitivity to microtubule depolymerizing drugs and a severe sister chromatid cohesion defect. Furthermore, temperature-sensitive mutations in RFC4 result in precocious sister chromatid separation. Our results highlight a novel function of the RFC proteins and support a model in which sister chromatid cohesion is established at the replication fork via a polymerase switching mechanism and a replication-coupled remodeling of chromatin.     

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.     

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.     

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     

Elg1, the major subunit of an alternative RFC complex,interacts with SUMO-processing proteins

PCNA is a homotrimeric ring with important roles in DNA replication and repair. PCNA is loaded and unloaded by the RFC complex, which is composed of five subunits (Rfc1-5). Three additional complexes that share with RFC the small subunits (Rfc2-5) and contain alternative large subunits were found in yeast and other eukaryotes. We have recently reported that one of these, the Elg1-RFC complex, interacts with SUMOylated PCNA and may play a role in its unloading during DNA repair. Here we report that a yeast-two-hybrid screen with the N terminus of Elg1(which interacts with SUMOylated PCNA) uncovered interactions with proteins that belong to the SUMO pathway, including Slx5 and Slx8, which form an E3 ubiquitin ligase that ubiquitinates SUMOylated proteins. Mutations in SLX5 result in a genomic instability phenotype similar to that of elg1 mutants. The physical interaction between the N terminus of Elg1 and Slx5 is mediated by poly-SUMO chains but not by PCNA modifications, and requires Siz2, but not Siz1, activity. Thus our results highlight the many important roles played by Elg1, some of which are PCNA-dependent and some PCNA-independent.     

New functions of Ctf18-RFC in preserving genome stability outside its role in sister chromatid cohesion

Expansion of DNA trinucleotide repeats causes at least 15 hereditary neurological diseases, and these repeats also undergo contraction and fragility. Current models to explain this genetic instability invoke erroneous DNA repair or aberrant replication. Here we show that CAG/CTG tracts are stabilized in Saccharomyces cerevisiae by the alternative clamp loader/unloader Ctf18-Dcc1-Ctf8-RFC complex (Ctf18-RFC). Mutants in Ctf18-RFC increased all three forms of triplet repeat instability--expansions, contractions, and fragility--with effect over a wide range of allele lengths from 20-155 repeats. Ctf18-RFC predominated among the three alternative clamp loaders, with mutants in Elg1-RFC or Rad24-RFC having less effect on trinucleotide repeats. Surprisingly, chl1, scc1-73, or scc2-4 mutants defective in sister chromatid cohesion (SCC) did not increase instability, suggesting that Ctf18-RFC protects triplet repeats independently of SCC. Instead, three results suggest novel roles for Ctf18-RFC in facilitating genomic stability. First, genetic instability in mutants of Ctf18-RFC was exacerbated by simultaneous deletion of the fork stabilizer Mrc1, but suppressed by deletion of the repair protein Rad52. Second, single-cell analysis showed that mutants in Ctf18-RFC had a slowed S phase and a striking G2/M accumulation, often with an abnormal multi-budded morphology. Third, ctf18 cells exhibit increased Rad52 foci in S phase, often persisting into G2, indicative of high levels of DNA damage. The presence of a repeat tract greatly magnified the ctf18 phenotypes. Together these results indicate that Ctf18-RFC has additional important functions in preserving genome stability, besides its role in SCC, which we propose include lesion bypass by replication forks and post-replication repair.     

An SNP-guided microRNA map of fifteen common human disorders identifies a consensus disease phenocode aiming at principal components of the nuclear import pathway

Maintaining genome stability is crucial for all cells. The budding yeast Elg1 protein, the major subunit of a replication factor C-like complex, is important for genome stability, since cells lacking Elg1 exhibit increased recombination and chromosomal rearrangements. This genome maintenance function of Elg1 seems to be conserved in higher eukaryotes, since removal of the human Elg1 homolog, encoded by the ATAD5 gene, also causes genome instability leading to tumorigenesis. The fundamental molecular function of the Elg1/ATAD5-replication factor C-like complex (RLC) was, until recently, elusive, although Elg1/ATAD5-RLC was known to interact with the replication sliding clamp PCNA. Two papers have now reported that following DNA replication, the Elg1/ATAD5-RLC is required to remove PCNA from chromatin in yeast and human cells. In this Review, we summarize the evidence that Elg1/ATAD5-RLC acts as a PCNA unloader and discuss the still enigmatic relationship between the function of Elg1/ATAD5-RLC in PCNA unloading and the role of Elg1/ATAD5 in maintaining genomic stability.     

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.     

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.     

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.     

Role of Elg1 protein in double strand break repair

The inaccurate repair of DNA double-strand breaks (DSBs) can result in genomic instability, and additionally cell death or the development of cancer. Elg1, which forms an alternative RFC-like complex with RFC2-5, is required for the maintenance of genome stability in Saccharomyces cerevisiae, and its function has been linked to DNA replication or damage checkpoint response. Here, we show that Elg1 is involved in homologous recombination (HR)-mediated DSB repair. Mutants of elg1 were partially defective in HR induced by methylmethanesufonate (MMS) and phleomycin. Deletion of ELG1 resulted in less efficient repair of phleomycin-induced DSBs in G₂/M phase-arrested cells. During HR between MAT and HML loci, Elg1 associated with both the MAT locus near the HO endonuclease-induced DSB site, and the HML homologous donor locus. The association of Elg1 with the MAT locus was not dependent on Rad52. However, Elg1 association with the HML locus depended on Rad52. Importantly, we found that two of the later steps in HR-mediated repair of an HO endonuclease-induced DSB, primer extension after strand invasion and ligation, were less efficient in elg1 mutants. Our results suggest that Elg1 is involved in DSB repair by HR.     

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.     

Saccharomyces cerevisiae CTF18 and CTF4 are required for sister chromatid cohesion

CTF4 and CTF18 are required for high-fidelity chromosome segregation. Both exhibit genetic and physical ties to replication fork constituents. We find that absence of either CTF4 or CTF18 causes sister chromatid cohesion failure and leads to a preanaphase accumulation of cells that depends on the spindle assembly checkpoint. The physical and genetic interactions between CTF4, CTF18, and core components of replication fork complexes observed in this study and others suggest that both gene products act in association with the replication fork to facilitate sister chromatid cohesion. We find that Ctf18p, an RFC1-like protein, directly interacts with Rfc2p, Rfc3p, Rfc4p, and Rfc5p. However, Ctf18p is not a component of biochemically purified proliferating cell nuclear antigen loading RF-C, suggesting the presence of a discrete complex containing Ctf18p, Rfc2p, Rfc3p, Rfc4p, and Rfc5p. Recent identification and characterization of the budding yeast polymerase kappa, encoded by TRF4, strongly supports a hypothesis that the DNA replication machinery is required for proper sister chromatid cohesion. Analogous to the polymerase switching role of the bacterial and human RF-C complexes, we propose that budding yeast RF-C(CTF18) may be involved in a polymerase switch event that facilities sister chromatid cohesion. The requirement for CTF4 and CTF18 in robust cohesion identifies novel roles for replication accessory proteins in this process.