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.     

Chl1p, a DNA helicase-like protein in budding yeast, functions in sister-chromatid cohesion

From the time of DNA replication until anaphase onset, sister chromatids remain tightly paired along their length. Ctf7p/Eco1p is essential to establish sister-chromatid pairing during S-phase and associates with DNA replication components. DNA helicases precede the DNA replication fork and thus will first encounter chromatin sites destined for cohesion. In this study, I provide the first evidence that a DNA helicase is required for proper sister-chromatid cohesion. Characterizations of chl1 mutant cells reveal that CHL1 interacts genetically with both CTF7/ECO1 and CTF18/CHL12, two genes that function in sister-chromatid cohesion. Consistent with genetic interactions, Chl1p physically associates with Ctf7p/Eco1p both in vivo and in vitro. Finally, a functional assay reveals that Chl1p is critical for sister-chromatid cohesion. Within the budding yeast genome, Chl1p exhibits the highest degree of sequence similarity to human CHL1 isoforms and BACH1. Previous studies revealed that human CHLR1 exhibits DNA helicase-like activities and that BACH1 is a helicase-like protein that associates with the tumor suppressor BRCA1 to maintain genome integrity. Our findings document a novel role for Chl1p in sister-chromatid cohesion and provide new insights into the possible mechanisms through which DNA helicases may contribute to cancer progression when mutated.     

Sister chromatid cohesion establishment occurs in concert with lagging strand synthesis

Cohesion establishment is central to sister chromatid tethering reactions and requires Ctf7/Eco1-dependent acetylation of the cohesin subunit Smc3. Ctf7/Eco1 is essential during S phase, and a number of replication proteins (RFC complexes, PCNA and the DNA helicase Chl1) all play individual roles in sister chromatid cohesion. While the mechanism of cohesion establishment is largely unknown, a popular model is that Ctf7/Eco1 acetylates cohesins encountered by and located in front of the fork. In turn, acetylation is posited both to allow fork passage past cohesin barriers and convert cohesins to a state competent to capture subsequent production of sister chromatids. Here, we report evidence that challenges this pre-replicative cohesion establishment model. Our genetic and biochemical studies link Ctf7/Eco1 to the Okazaki fragment flap endonuclease, Fen1. We further report genetic and biochemical interactions between Fen1 and the cohesion-associated DNA helicase, Chl1. These results raise a new model wherein cohesin deposition and establishment occur in concert with lagging strand-processing events and in the presence of both sister chromatids.     

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     

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.     

Sister chromatid cohesion establishment occurs in concert with lagging strand synthesis

Cohesion establishment is central to sister chromatid tethering reactions and requires Ctf7/Eco1-dependent acetylation of the cohesin subunit Smc3. Ctf7/Eco1 is essential during S phase, and a number of replication proteins (RFC complexes, PCNA and the DNA helicase Chl1) all play individual roles in sister chromatid cohesion. While the mechanism of cohesion establishment is largely unknown, a popular model is that Ctf7/Eco1 acetylates cohesins encountered by and located in front of the fork. In turn, acetylation is posited both to allow fork passage past cohesin barriers and convert cohesins to a state competent to capture subsequent production of sister chromatids. Here, we report evidence that challenges this pre-replicative cohesion establishment model. Our genetic and biochemical studies link Ctf7/Eco1 to the Okazaki fragment flap endonuclease, Fen1. We further report genetic and biochemical interactions between Fen1 and the cohesion-associated DNA helicase, Chl1. These results raise a new model wherein cohesin deposition and establishment occur in concert with lagging strand-processing events and in the presence of both sister chromatids.     

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.     

Human EFO1p exhibits acetyltransferase activity and is a unique combination of linker histone and Ctf7p/Eco1p chromatid cohesion establishment domains

Proper segregation of chromosomes during mitosis requires that the products of chromosome replication are paired together-termed sister chromatid cohesion. In budding yeast, Ctf7p/Eco1p is an essential protein that establishes cohesion between sister chromatids during S phase. In fission yeast, Eso1p also functions in cohesion establishment, but is comprised of a Ctf7p/Eco1p domain fused to a Rad30p domain (a DNA polymerase) both of which are independently expressed in budding yeast. In this report, we identify and characterize the first candidate human ortholog of Ctf7p/Eco1p, which we term hEFO1p (human Establishment Factor Ortholog). As in fission yeast Eso1p, the hEFO1p open reading frame extends well upstream of the C-terminal Ctf7p/Eco1p domain. However, this N-terminal extension in hEFO1p is unlike Rad30p, but instead exhibits significant homology to linker histone proteins. Thus, hEFO1p is a unique fusion of linker histone and cohesion establishment domains. hEFO1p is widely expressed among the tissues tested. Consistent with a role in chromosome segregation, hEFO1p localizes exclusively to the nucleus when expressed in HeLa tissue culture cells. Moreover, biochemical analyses reveal that hEFO1p exhibits acetyltransferase activity. These findings document the first characterization of a novel human acetyltransferase, hEFO1p, that is comprised of both linker histone and Ctf7p/Eco1p domains.     

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.     

Setting the stage for cohesion establishment by the replication fork

Comment on: Rudra S, et al. Cell Cycle 2012; 2114-21The complex process of semi-conservative DNA replication involves a mechanism whereby the leading and lagging strands with opposite polarity serve as templates for concerted synthesis of complementary base pairs.¹ Lagging-strand synthesis creates discontinuous Okazaki fragments that require timely processing of the 5′ flaps, so that adjacent nascent DNA strands are ligated together to insure genomic stability. While the genetic and molecular requirements of Okazaki fragment maturation have been studied in much detail, the precise temporal and spatial relationship of lagging-strand processing to sister chromatid cohesion remains unclear.² The newly replicated daughter duplex DNA molecules (i.e., the sister chromatids) become tethered during DNA replication and remain paired in order to permit proper segregation of the chromosomes to respective poles during mitosis and nuclear division. Elegant genetic studies in yeast have implicated posttranslational modification of cohesins (specialized protein complexes responsible for tethering sister pairs) by Ctf7/Eco1 acetylase as a key regulatory step in the process, enabling cohesins to perform their function in capturing the newly synthesized sister chromatids. Previous work suggested that genetic and physical interactions among the yeast acetyltransferase Ctf7/Eco1, helicase Chl1, Flap Endonuclease (Fen1) and accessory replication factors [e.g., RFC (clamp loader) and PCNA (clamp)] play an integral role in cohesion establishment. Based on these pieces of evidence, several models to explain the relationship between replication fork dynamics and sister chromatid cohesion have been proposed; however, our understanding of the precise timing of cohesin acetylation and the passage of the replication fork machinery has remained murky at best. Given the importance of proper chromosome segregation for chromosomal stability and the suppression of developmental disorders and tumorigenesis, a comprehensive understanding of the molecular acrobatics involved in sister chromatid cohesion is highly important.In a recent study, the temporal relationship between sister chromatid establishment and lagging-strand synthesis was illuminated.³ The authors have elucidated the link between the catalytic functions of DNA unwinding, flap processing and acetylation, which supports a model of cohesion deposition and establishment that occurs after the passage of the replication fork, similar to how genomic DNA becomes chromatinized. This is a significant advance from an earlier and very popular model of sister chromatid cohesion predicted that Ctf7/Eco1 acetylated cohesin proteins before the encounter by the DNA replication fork, which was thought to permit fork progression and the proper cohesion state for sister chromatid tethering (for review, see ref. ²). Instead, the genetic evidence presented by the Skibbens lab supports a model whereby cohesion establishment is temporally coupled to lagging-strand processing.³ In support of the genetic proof, Rudra and Skibbens went on to show that both Ctf7/Eco1 and Chl1 are associated with the lagging-strand processing nuclease Fen1. Altogether, the experimental results implicate a post-fork establishment model that is analogous to how histone protein complexes are deposited onto newly synthesized sister chromatids and become posttranslationally modified to confer epigenetic status.The discovery from the Skibbens lab that cohesion establishment is closely orchestrated with Okazaki fragment processing prompts a new line of inquiry about the control of flap processing by acetylation and its dual purpose for proper sister chromatid cohesion and replication fidelity in eukaryotes (Fig. 1). The catalytic activity of human FEN-1^4,5 and a functionally related endonuclease known as Dna2⁴ have been shown to be modulated by p300 acetylation, which suggested a model for creating long flap intermediates to promote genomic stability and suppress mutagenesis. Given evidence that ChlR1 is implicated in the genetic disorder Warsaw Breakage syndrome and that the human homolog of yeast Chl1⁶ interacts with the RFC complex and Fen1,⁷ it will be informative to determine if acetyltransferases such as the human orthologs Esco1 and Esco2, the latter mutated in the cohesinopathy Roberts syndrome,⁸ and perhaps other acetyltransferases (e.g., p300) are master regulators of lagging-strand synthesis that not only affect replication fidelity and genomic stability, but also sister chromatid cohesion. Coordination of sister chromatid cohesion establishment with lagging strand synthesis may also involve replication fork stabilization by the Timeless-Tipin protein complex implicated in replication checkpoint.⁹ Defects in the efficient coupling of lagging-strand synthesis to sister chromatid cohesion may contribute to the chromosomal instability characteristic of age-related diseases and cancer.Open in a separate windowFigure 1. Interplay between acetylation, replication fork dynamics and cohesion establishment important for chromosomal integrity.     

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.     

An Eco1-independent sister chromatid cohesion establishment pathway in S. cerevisiae

Cohesion between sister chromatids, mediated by the chromosomal cohesin complex, is a prerequisite for their alignment on the spindle apparatus and segregation in mitosis. Budding yeast cohesin first associates with chromosomes in G1. Then, during DNA replication in S-phase, the replication fork-associated acetyltransferase Eco1 acetylates the cohesin subunit Smc3 to make cohesin’s DNA binding resistant to destabilization by the Wapl protein. Whether stabilization of cohesin molecules that happen to link sister chromatids is sufficient to build sister chromatid cohesion, or whether additional reactions are required to establish these links, is not known. In addition to Eco1, several other factors contribute to cohesion establishment, including Ctf4, Ctf18, Tof1, Csm3, Chl1 and Mrc1, but little is known about their roles. Here, we show that each of these factors facilitates cohesin acetylation. Moreover, the absence of Ctf4 and Chl1, but not of the other factors, causes a synthetic growth defect in cells lacking Eco1. Distinct from acetylation defects, sister chromatid cohesion in ctf4Δ and chl1Δ cells is not improved by removing Wapl. Unlike previously thought, we do not find evidence for a role of Ctf4 and Chl1 in Okazaki fragment processing, or of Okazaki fragment processing in sister chromatid cohesion. Thus, Ctf4 and Chl1 delineate an additional acetylation-independent pathway that might hold important clues as to the mechanism of sister chromatid cohesion establishment.     

Cloning and characterization of hCTF18, hCTF8, and hDCC1. Human homologs of a Saccharomyces cerevisiae complex involved in sister chromatid cohesion establishment

A growing body of evidence suggests that establishment of sister chromatid cohesion is dependent on replication fork passage over a precohesion area. In Saccharomyces cerevisiae, this process involves an alternative replication factor C (RFC) complex that contains the four small RFC subunits as well as CTF18, CTF8, and DCC1. Here, we show that an evolutionarily conserved homologous complex exists in the nucleus of human cells. We demonstrate that hCTF18, hCTF8, and hDCC1 interact with each other as well as with the p38 subunit of RFC. This alternative RFC-containing complex interacts with proliferating cell nuclear antigen but not with the Rad9/Rad1/Hus1 complex, a proliferating cell nuclear antigen-like clamp involved in the DNA damage response. hCTF18 preferentially binds chromatin during S phase, suggesting a role during replication. Our data provide evidence for the existence of an alternative RFC complex with a probable role in mammalian sister chromatid cohesion establishment.     

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.     

Replication factor C complexes play unique pro- and anti-establishment roles in sister chromatid cohesion

Recent studies have lead to a rapid expansion of sister chromatid cohesion pathways. Of particular interest is the growth in classifications of anti-establishment factors-now including those that are cohesin-associated (Rad61/WAPL and Pds5) or DNA replication fork-associated (Elg1-RFC). In this study, we show that the two classes of anti-establishment complexes are indistinguishable when challenged both genetically and functionally. These findings suggest that both classes function in a singular pathway that is centered on Ctf7/Eco1 (herein termed Ctf7) regulation. The anti-establishment activity of Elg1-RFC complex is particular intriguing given that an alternate Ctf18-RFC complex exhibits robust pro-establishment activity. Here, we provide several lines of evidence, including the use of Ctf7 bypass suppressors, indicating that these activities are not simply antagonistic. Moreover, the results suggest that Ctf18-RFC is capable of promoting sister chromatid pairing reactions independent of Ctf7. The combination of these studies suggest a new model of sister chromatid pairing regulation.     

RFCCtf18 and the Swi1-Swi3 complex function in separate and redundant pathways required for the stabilization of replication forks to facilitate sister chromatid cohesion in Schizosaccharomyces pombe

Sister chromatid cohesion is established during S phase near the replication fork. However, how DNA replication is coordinated with chromosomal cohesion pathway is largely unknown. Here, we report studies of fission yeast Ctf18, a subunit of the RFC(Ctf18) replication factor C complex, and Chl1, a putative DNA helicase. We show that RFC(Ctf18) is essential in the absence of the Swi1-Swi3 replication fork protection complex required for the S phase stress response. Loss of Ctf18 leads to an increased sensitivity to S phase stressing agents, a decreased level of Cds1 kinase activity, and accumulation of DNA damage during S phase. Ctf18 associates with chromatin during S phase, and it is required for the proper resumption of replication after fork arrest. We also show that chl1Delta is synthetically lethal with ctf18Delta and that a dosage increase of chl1(+) rescues sensitivities of swi1Delta to S phase stressing agents, indicating that Chl1 is involved in the S phase stress response. Finally, we demonstrate that inactivation of Ctf18, Chl1, or Swi1-Swi3 leads to defective centromere cohesion, suggesting the role of these proteins in chromosome segregation. We propose that RFC(Ctf18) and the Swi1-Swi3 complex function in separate and redundant pathways essential for replication fork stabilization to facilitate sister chromatid cohesion in fission yeast.     

Fission yeast Eso1p is required for establishing sister chromatid cohesion during S phase

Sister chromatid cohesion is essential for cell viability. We have isolated a novel temperature-sensitive lethal mutant named eso1-H17 that displays spindle assembly checkpoint-dependent mitotic delay and abnormal chromosome segregation. At the permissive temperature, the eso1-H17 mutant shows mild sensitivity to UV irradiation and DNA-damaging chemicals. At the nonpermissive temperature, the mutant is arrested in M phase with a viability loss due to a failure to establish sister chromatid cohesion during S phase. The lethal M-phase arrest phenotype, however, is suppressed by inactivation of a spindle checkpoint. The eso1(+) gene is not essential for the onset and progression of DNA replication but has remarkable genetic interactions with those genes regulating the G(1)-S transition and DNA replication. The N-terminal two-thirds of Eso1p is highly homologous to DNA polymerase eta of budding yeast and humans, and the C-terminal one-third is homologous to budding yeast Eco1p (also called Ctf7p), which is required for the establishment of sister chromatid cohesion. Deletion analysis and determination of the mutation site reveal that the function of the Eco1p/Ctf7p-homologous domain is necessary and sufficient for sister chromatid cohesion. On the other hand, deletion of the DNA polymerase eta domain in Eso1p increases sensitivity to UV irradiation. These results indicate that Eso1p plays a dual role during DNA replication. The C-terminal region acts to establish sister chromatid cohesion, and the N-terminal region presumably catalyzes translesion DNA synthesis when template DNA contains lesions that block regular DNA replication.     

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.     

PCNA controls establishment of sister chromatid cohesion during S phase

Accurate segregation of the genetic material during cell division requires that sister chromatids are kept together by cohesion proteins until anaphase, when the chromatids become separated and distributed to the two daughter cells. Studies in yeast revealed that chromatid cohesion is essential for viability and is triggered by the conserved protein Eco1 (Ctf7). Cohesion must be established already in S phase in order to tie up sister chromatids instantly after replication, but how this crucial timing is achieved remains enigmatic. Here, we report that in yeast and humans Eco1 is directly physically coupled to the replication protein PCNA, a ring-shaped cofactor of DNA polymerases. Binding to PCNA is crucial, as yeast Eco1 mutants deficient in Eco1-PCNA interaction are defective in cohesion and inviable. Our study thus indicates that PCNA, a central matchmaker for replication-linked functions, is also crucially involved in the establishment of cohesion in S phase.     

The zinc finger of Eco1 enhances its acetyltransferase activity during sister chromatid cohesion

Eco1p/Ctf7p is an essential acetyltransferase required for the establishment of sister chromatid cohesion. Eco1p acetylates Smc3p and Mcd1p (Scc1p or Rad21p) to establish cohesion during S phase and in response to DNA damage, respectively. In addition to its acetyltransferase domain, Eco1p harbors a conserved zinc finger domain. The zinc finger has been implicated in the establishment of sister chromatid cohesion in S phase, yet its function on the molecular level and its contribution to damage-induced cohesion are unknown. Here, we show that the zinc finger is essential for the establishment of cohesion in both S phase and in response to DNA damage. Our results suggest that the zinc finger augments the acetylation of Eco1p itself, Smc3p and likely Mcd1p. We propose that the zinc finger is a general enhancer of substrate recognition, thereby enhances the ability of Eco1p to acetylate its substrates above a threshold needed to generate cohesion during DNA replication and repair. Finally our studies of the zinc finger led to the discovery that Eco1 is a multimer, a property that could be exploited to coordinate acetylation of substrates either spatially or temporally for establishment of sister chromatid cohesion.