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.     

Eco1 is a novel acetyltransferase that can acetylate proteins involved in cohesion

Cohesion between sister chromatids is established during S phase and maintained through G2 phase until it is resolved in anaphase (for review, see [1-3]). In Saccharomyces cerevisiae, a complex consisting of Scc1, Smc1, Smc3, and Scc3 proteins, called "cohesin," mediates the connection between sister chromatids. The evolutionary conserved yeast protein Eco1 is required for establishment of sister chromatid cohesion during S phase but not for its further maintenance during G2 or M phases or for loading the cohesin complex onto DNA. We address the molecular functions of Eco1 with sensitive sequence analytic techniques, including hidden Markov model domain fragment searches. We found a two-domain architecture with an N-terminal C2H2 Zn finger-like domain and an approximately 150 residue C-terminal domain with an apparent acetyl coenzyme A binding motif (http://mendel.imp.univie.ac.at/SEQUENCES/ECO1/). Biochemical tests confirm that Eco1 has the acetyltransferase activity in vitro. In vitro Eco1 acetylates itself and components of the cohesin complex but not histones. Thus, the establishment of cohesion between sister chromatids appears to be regulated, directly or indirectly, by a specific acetyltransferase.     

Establishment of sister chromatid cohesion at the S. cerevisiae replication fork

Two identical sister copies of eukaryotic chromosomes are synthesized during S phase. To facilitate their recognition as pairs for segregation in mitosis, sister chromatids are held together from their synthesis onward by the chromosomal cohesin complex. Replication fork progression is thought to be coupled to establishment of sister chromatid cohesion, facilitating identification of replication products, but evidence for this has remained circumstantial. Here we show that three proteins required for sister chromatid cohesion, Eco1, Ctf4, and Ctf18, are found at, and Ctf4 travels along chromosomes with, replication forks. The ring-shaped cohesin complex is loaded onto chromosomes before S phase in an ATP hydrolysis-dependent reaction. Cohesion establishment during DNA replication follows without further cohesin recruitment and without need for cohesin to re-engage an ATP hydrolysis motif that is critical for its initial DNA binding. This provides evidence for cohesion establishment in the context of replication forks and imposes constraints on the mechanism involved.     

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.     

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.     

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.     

Cohesin Acetylation Promotes Sister Chromatid Cohesion Only in Association with the Replication Machinery

Acetylation of the Smc3 subunit of cohesin is essential to establish functional cohesion between sister chromatids. Smc3 acetylation is catalyzed by members of the Eco family of acetyltransferases, although the mechanism by which acetylation is regulated and how it promotes cohesion are largely unknown. In vertebrates, the cohesin complex binds to chromatin during mitotic exit and is converted to a functional form during or shortly after DNA replication. The conserved proliferating cell nuclear antigen-interacting protein box motif in yeast Eco1 is required for function, and cohesin is acetylated during the S phase. This has led to the notion that acetylation of cohesin is stimulated by interaction of Eco1 with the replication machinery. Here we show that in vertebrates Smc3 acetylation occurs independently of DNA replication. Smc3 is readily acetylated before replication is initiated and after DNA replication is complete. However, we also show that functional acetylation occurs only in association with the replication machinery: disruption of the interaction between XEco2 and proliferating cell nuclear antigen prevents cohesion establishment while having little impact on the overall levels of Smc3 acetylation. These results demonstrate that Smc3 acetylation can occur throughout interphase but that only acetylation in association with the replication fork promotes sister chromatid cohesion. These data reveal how the generation of cohesion is limited to the appropriate time and place during the cell cycle and provide insight into the mechanism by which acetylation ensures cohesion.     

Sister chromatid cohesion is required for postreplicative double-strand break repair in Saccharomyces cerevisiae.

The repair of DNA double-strand breaks by recombination requires the presence of an undamaged copy that is used as a template during the repair process. Because cells acquire resistance to gamma irradiation during DNA replication and because sister chromatids are the preferred partner for double-strand break repair in mitotic diploid yeast cells, it has long been suspected that cohesion between sister chromatids might be crucial for efficient repair. This hypothesis is consistent with the sensitivity to gamma irradiation of mutants defective in the cohesin complex that holds sister chromatids together from DNA replication until the onset of anaphase (reviewed in) . It is also in accordance with the finding that surveillance mechanisms (checkpoints) that sense DNA damage arrest cell cycle progression in yeast by causing stabilization of the securin Pds1, thereby blocking sister chromatid separation. The hypersensitivity to irradiation of cohesin mutants could, however, be due to a more direct involvement of the cohesin complex in the process of DNA repair. We show here that passage through S phase in the presence of cohesin, and not cohesin per se, is essential for efficient double-strand break repair during G2 in yeast. Proteins needed to load cohesin onto chromosomes (Scc2) and to generate cohesion during S phase (Eco1) are also shown to be required for repair. Our results confirm what has long been suspected but never proven, that cohesion between sister chromatids is essential for efficient double-strand break repair in mitotic cells.     

Establishment and maintenance of sister chromatid cohesion in fission yeast by a unique mechanism

During S phase, chromatid cohesion is established only between nascent sisters and with faithful pairing along their entire region, but how this is ensured is unknown. Here we report that sister chromatid cohesion is formed and maintained by a unique mechanism. In fission yeast, Eso1p, functioning in close coupling to DNA replication, establishes sister chromatid cohesion whereas the newly identified Cohesin-associated protein Pds5p hinders the establishment of cohesion until counteracted by Eso1p, yet stabilizes cohesion once it is established. Eso1p interacts physically with Pds5p via its Ctf7p/Eco1p-homologous domain.     

Tipin/Tim1/And1 protein complex promotes Polα chromatin binding and sister chromatid cohesion

The Tipin/Tim1 complex plays an important role in the S‐phase checkpoint and replication fork stability. However, the biochemical function of this complex is poorly understood. Using Xenopus laevis egg extract we show that Tipin is required for DNA replication in the presence of limiting amount of replication origins. Under these conditions the DNA replication defect correlates with decreased levels of DNA Polα on chromatin. We identified And1, a Polα chromatin‐loading factor, as new Tipin‐binding partner. We found that both Tipin and And1 promote stable binding of Polα to chromatin and that this is required for DNA replication under unchallenged conditions. Strikingly, extracts lacking Tipin and And1 also show reduced sister chromatids cohesion. These data indicate that Tipin/Tim1/And1 form a complex that links stabilization of replication fork and establishment of sister chromatid cohesion.     

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.     

Rtt101‐Mms1‐Mms22 coordinates replication‐coupled sister chromatid cohesion and nucleosome assembly

Two sister chromatids must be held together by a cohesion process from their synthesis during S phase to segregation in anaphase. Despite its pivotal role in accurate chromosome segregation, how cohesion is established remains elusive. Here, we demonstrate that yeast Rtt101‐Mms1, Cul4 family E3 ubiquitin ligases are stronger dosage suppressors of loss‐of‐function eco1 mutants than PCNA. The essential cohesion reaction, Eco1‐catalyzed Smc3 acetylation is reduced in the absence of Rtt101‐Mms1. One of the adaptor subunits, Mms22, associates directly with Eco1. Point mutations (L61D/G63D) in Eco1 that abolish the interaction with Mms22 impair Smc3 acetylation. Importantly, an eco1LGpol30A251V double mutant displays additive Smc3ac reduction. Moreover, Smc3 acetylation and cohesion defects also occur in the mutants of other replication‐coupled nucleosome assembly (RCNA) factors upstream or downstream of Rtt101‐Mms1, indicating unanticipated cross talk between histone modifications and cohesin acetylation. These data suggest that fork‐associated Cul4‐Ddb1 E3s, together with PCNA, coordinate chromatin reassembly and cohesion establishment on the newly replicated sister chromatids, which are crucial for maintaining genome and chromosome stability.     

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.     

A SUMO-Dependent Step during Establishment of Sister Chromatid Cohesion

Cohesin is a protein complex that ties sister DNA molecules from the time of DNA replication until the metaphase to anaphase transition. Current models propose that the association of the Smc1, Smc3, and Scc1/Mcd1 subunits creates a ring-shaped structure that entraps the two sister DNAs [1]. Cohesin is essential for correct chromosome segregation and recombinational repair. Its activity is therefore controlled by several posttranslational modifications, including acetylation, phosphorylation, sumoylation, and site-specific proteolysis. Here we show that cohesin sumoylation occurs at the time of cohesion establishment, after cohesin loading and ATP binding, and independently from Eco1-mediated cohesin acetylation. In order to test the functional relevance of cohesin sumoylation, we have developed a novel approach in budding yeast to deplete SUMO from all subunits in the cohesin complex, based on fusion of the Scc1 subunit to a SUMO peptidase Ulp domain (UD). Downregulation of cohesin sumoylation is lethal, and the Scc1-UD chimeras have a failure in sister chromatid cohesion. Strikingly, the unsumoylated cohesin rings are acetylated. Our findings indicate that SUMO is a novel molecular determinant for the establishment of sister chromatid cohesion, and we propose that SUMO is required for the entrapment of sister chromatids during the acetylation-mediated closure of the cohesin ring.     

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.     

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.     

Acetylation of Smc3 by Eco1 is required for S phase sister chromatid cohesion in both human and yeast

Sister chromatid cohesion is normally established in S phase in a process that depends on the cohesion establishment factor Eco1, a conserved acetyltransferase. However, due to the lack of known in vivo substrates, how Eco1 regulates cohesion is not understood. Here we report that yeast Eco1 and its human ortholog, ESCO1, both acetylate Smc3, a component of the cohesin complex that physically holds the sister chromatid together, at two conserved lysine residues. Mutating these lysine residues to a nonacetylatable form leads to increased loss of sister chromatid cohesion and genome instability in both yeast and human. In addition, we clarified that the acetyltransferase activity of Eco1 is essential for its function. Our study thus identified a molecular target for the acetyltransferase Eco1 and revealed that Smc3 acetylation is a conserved mechanism in regulating sister chromatid cohesion.