Epitope tag-induced synthetic lethality between cohesin subunits and Ctf7/Eco1 acetyltransferase

Ctf7/Eco1-dependent acetylation of Smc3 is essential for sister chromatid cohesion. Here, we use epitope tag-induced lethality in cells diminished for Ctf7/Eco1 activity to map cohesin architecture in vivo. Tagging either Smc1 or Mcd1/Scc1, but not Scc3/Irr1, appears to abolish access to Smc3 in ctf7/eco1 mutant cells, suggesting that Smc1 and Smc3 head domains are in direct contact with each other and also with Mcd1/Scc1. Thus, cohesin complexes may be much more compact than commonly portrayed. We further demonstrate that mutation in ELG1 or RFC5 anti-establishment genes suppress tag-induced lethality, consistent with the notion that the replication fork regulates Ctf7/Eco1.     

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     

Topoisomerase II Suppresses the Temperature Sensitivity of Saccharomyces cerevisiae pds5 Mutants,but not the Defect in Sister Chromatid Cohesion

Sister chromatid cohesion enables chromosomes to achieve bipolar attachment to the mitotic spindle and its dissolution is required for chromosome segregation. The cohesin complex serves as the primary molecular glue responsible for cohesion. Pds5p binds to the same chromosomal loci as the cohesin complex but plays a distinct role as a regulator of cohesion maintenance. Catenation between sister chromatids must also be removed by Topoisomerase II (Top2p) enzymatic activity to enable chromosome segregation. We identified TOP2 as a high-copy suppressor of the temperature sensitivity of pds5 mutants. TOP2 suppression is specific for pds5 mutants as it does not suppress mutants in the cohesin complex. TOP2 suppresses mini-chromosome loss in pds5 mutants indicating that it rescues a chromosome segregation defect. Surprisingly, TOP2 over-expression fails to suppress the cohesion defect of pds5 mutants, suggesting that it suppresses an additional and as yet uncharacterized defect in pds5 mutants that is essential for viability. A catalytically dead TOP2 allele suppresses pds5 temperature sensitivity, suggesting that suppression is unrelated to Top2p enzymatic function. Consistent with this idea, when the pds5 mutant is combined with the top2-4 mutant, which accumulates DNA catenanes due to defective enzymatic activity, the double mutants exhibit synthetic sickness indicating that increased catenation is toxic to pds5 cells. Our results suggest that Pds5p and Top2p cooperate to promote proper chromosome segregation by a mechanism unrelated to either cohesion or catenation/decatenation.     

Cohesin rings devoid of scc3 and pds5 maintain their stable association with the DNA

Cohesin is a protein complex that forms a ring around sister chromatids thus holding them together. The ring is composed of three proteins: Smc1, Smc3 and Scc1. The roles of three additional proteins that associate with the ring, Scc3, Pds5 and Wpl1, are not well understood. It has been proposed that these three factors form a complex that stabilizes the ring and prevents it from opening. This activity promotes sister chromatid cohesion but at the same time poses an obstacle for the initial entrapment of sister DNAs. This hindrance to cohesion establishment is overcome during DNA replication via acetylation of the Smc3 subunit by the Eco1 acetyltransferase. However, the full mechanistic consequences of Smc3 acetylation remain unknown. In the current work, we test the requirement of Scc3 and Pds5 for the stable association of cohesin with DNA. We investigated the consequences of Scc3 and Pds5 depletion in vivo using degron tagging in budding yeast. The previously described DHFR-based N-terminal degron as well as a novel Eco1-derived C-terminal degron were employed in our study. Scc3 and Pds5 associate with cohesin complexes independently of each other and require the Scc1 "core" subunit for their association with chromosomes. Contrary to previous data for Scc1 downregulation, depletion of either Scc3 or Pds5 had a strong effect on sister chromatid cohesion but not on cohesin binding to DNA. Quantity, stability and genome-wide distribution of cohesin complexes remained mostly unchanged after the depletion of Scc3 and Pds5. Our findings are inconsistent with a previously proposed model that Scc3 and Pds5 are cohesin maintenance factors required for cohesin ring stability or for maintaining its association with DNA. We propose that Scc3 and Pds5 specifically function during cohesion establishment in S phase.     

Pds5p regulates the maintenance of sister chromatid cohesion and is sumoylated to promote the dissolution of cohesion

Pds5p and the cohesin complex are required for sister chromatid cohesion and localize to the same chromosomal loci over the same cell cycle window. However, Pds5p and the cohesin complex likely have distinct roles in cohesion. We report that pds5 mutants establish cohesion, but during mitosis exhibit precocious sister dissociation. Thus, unlike the cohesin complex, which is required for cohesion establishment and maintenance, Pds5p is required only for maintenance. We identified SMT4, which encodes a SUMO isopeptidase, as a high copy suppressor of both the temperature sensitivity and precocious sister dissociation of pds5 mutants. In contrast, SMT4 does not suppress temperature sensitivity of cohesin complex mutants. Pds5p is SUMO conjugated, with sumoylation peaking during mitosis. SMT4 overexpression reduces Pds5p sumoylation, whereas smt4 mutants have increased Pds5p sumoylation. smt4 mutants were previously shown to be defective in cohesion maintenance during mitosis. These data provide the first link between a protein required for cohesion, Pds5p, and sumoylation, and suggest that Pds5p sumoylation promotes the dissolution of cohesion.     

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.     

A Conserved Domain in the Scc3 Subunit of Cohesin Mediates the Interaction with Both Mcd1 and the Cohesin Loader Complex

The Structural Maintenance of Chromosome (SMC) complex, termed cohesin, is essential for sister chromatid cohesion. Cohesin is also important for chromosome condensation, DNA repair, and gene expression. Cohesin is comprised of Scc3, Mcd1, Smc1, and Smc3. Scc3 also binds Pds5 and Wpl1, cohesin-associated proteins that regulate cohesin function, and to the Scc2/4 cohesin loader. We mutagenized SCC3 to elucidate its role in cohesin function. A 5 amino acid insertion after Scc3 residue I358, or a missense mutation of residue D373 in the adjacent stromalin conservative domain (SCD) induce inviability and defects in both cohesion and cohesin binding to chromosomes. The I358 and D373 mutants abrogate Scc3 binding to Mcd1. These results define an Scc3 region extending from I358 through the SCD required for binding Mcd1, cohesin localization to chromosomes and cohesion. Scc3 binding to the cohesin loader, Pds5 and Wpl1 are unaffected in I358 mutant and the loader still binds the cohesin core trimer (Mcd1, Smc1 and Smc3). Thus, Scc3 plays a critical role in cohesin binding to chromosomes and cohesion at a step distinct from loader binding to the cohesin trimer. We show that residues Y371 and K372 within the SCD are critical for viability and chromosome condensation but dispensable for cohesion. However, scc3 Y371A and scc3 K372A bind normally to Mcd1. These alleles also provide evidence that Scc3 has distinct mechanisms of cohesin loading to different loci. The cohesion-competence, condensation-incompetence of Y371 and K372 mutants suggests that cohesin has at least one activity required specifically for condensation.     

Pds5 cooperates with cohesin in maintaining sister chromatid cohesion

BACKGROUND: Sister chromatid cohesion depends on a complex called cohesin, which contains at least four subunits: Smc1, Smc3, Scc1 and Scc3. Cohesion is established during DNA replication, is partially dismantled in many, but not all, organisms during prophase, and is finally destroyed at the metaphase-to-anaphase transition. A quite separate protein called Spo76 is required for sister chromatid cohesion during meiosis in the ascomycete Sordaria. Spo76-like proteins are highly conserved amongst eukaryotes and a homologue in Aspergillus nidulans, called BimD, is required for the completion of mitosis. The isolation of the cohesin subunit Smc3 as a suppressor of BimD mutations suggests that Spo76/BimD might function in the same process as cohesin. RESULTS: We show here that the yeast homologue of Spo76, called Pds5, is essential for establishing sister chromatid cohesion and maintaining it during metaphase. We also show that Pds5 co-localizes with cohesin on chromosomes, that the chromosomal association of Pds5 and cohesin is interdependent, that Scc1 recruits Pds5 to chromosomes in G1 and that its cleavage causes dissociation of Pds5 from chromosomes at the metaphase-to-anaphase transition. CONCLUSIONS: Our data show that Pds5 functions as part of the same process as cohesin. Sequence similarities and secondary structure predictions indicate that Pds5 consists of tandemly repeated HEAT repeats, and might therefore function as a protein-protein interaction scaffold, possibly in the cohesin-DNA complex assembly.     

Structure and function of cohesin’s Scc3/SA regulatory subunit

Sister chromatid cohesion involves entrapment of sister DNAs by a cohesin ring created through association of a kleisin subunit (Scc1) with ATPase heads of Smc1/Smc3 heterodimers. Cohesin’s association with chromatin involves subunits recruited by Scc1: Wapl, Pds5, and Scc3/SA, in addition to Scc2/4 loading complex. Unlike Pds5, Wapl, and Scc2/4, Scc3s are encoded by all eukaryotic genomes. Here, a crystal structure of Scc3 reveals a hook-shaped protein composed of tandem α helices. Its N-terminal domain contains a conserved and essential surface (CES) present even in organisms lacking Pds5, Wapl, and Scc2/4, while its C-terminal domain binds a section of the kleisin Scc1. Scc3 turns over in G2/M while maintaining cohesin’s association with chromosomes and it promotes de-acetylation of Smc3 upon Scc1 cleavage.     

Ctf18 is required for homologous recombination-mediated double-strand break repair

The efficient repair of double-strand breaks (DSBs) is crucial in maintaining genomic integrity. Sister chromatid cohesion is important for not only faithful chromosome segregation but also for proper DSB repair. During DSB repair, the Smc1–Smc3 cohesin complex is loaded onto chromatin around the DSB to support recombination-mediated DSB repair. In this study, we investigated whether Ctf18, a factor implicated in the establishment of sister chromatid cohesion, is involved in DSB repair in budding yeast. Ctf18 was recruited to HO-endonuclease induced DSB sites in an Mre11-dependent manner and to damaged chromatin in G₂/M phase-arrested cells. The ctf18 mutant cells showed high sensitivity to DSB-inducible genotoxic agents and defects in DSB repair, as well as defects in damage-induced recombination between sister chromatids and between homologous chromosomes. These results suggest that Ctf18 is involved in damage-induced homologous recombination.     

In vivo analysis of cohesin architecture using FRET in the budding yeast Saccharomyces cerevisiae

Cohesion between sister chromatids in eukaryotes is mediated by the evolutionarily conserved cohesin complex. Cohesin forms a proteinaceous ring, large enough to trap pairs of replicated sister chromatids. The circumference consists of the Smc1 and Smc3 subunits, while Scc1 is thought to close the ring by bridging the Smc (structural maintenance of chromosomes) ATPase head domains. Little is known about two additional subunits, Scc3 and Pds5, and about possible conformational changes of the complex during the cell cycle. We have employed fluorescence resonance energy transfer (FRET) to analyse interactions within the cohesin complex in live budding yeast. These experiments reveal an unexpected geometry of Scc1 at the Smc heads, and suggest that Pds5 plays a role at the Smc hinge on the opposite side of the ring. Key subunit interactions, including close proximity of the two ATPase heads, are constitutive throughout the cell cycle. This depicts cohesin as a stable molecular machine undergoing only transient conformational changes during binding and dissociation from chromosomes. Using FRET, we did not observe interactions between more than one cohesin complex in vivo.     

Pds1p Is Required for Meiotic Recombination and Prophase I Progression in Saccharomyces cerevisiae

Sister-chromatid separation at the metaphase–anaphase transition is regulated by a proteolytic cascade. Destruction of the securin Pds1p liberates the Esp1p separase, which ultimately targets the mitotic cohesin Mcd1p/Scc1p for destruction. Pds1p stabilization by the spindle or DNA damage checkpoints prevents sister-chromatid separation while mutants lacking PDS1 (pds1Δ) are temperature sensitive for growth due to elevated chromosome loss. This report examined the role of the budding yeast Pds1p in meiotic progression using genetic, cytological, and biochemical assays. Similar to its mitotic function, Pds1p destruction is required for metaphase I–anaphase I transition. However, even at the permissive temperature for growth, pds1Δ mutants arrest with prophase I spindle and nuclear characteristics. This arrest was partially suppressed by preventing recombination initiation or by inactivating a subset of recombination checkpoint components. Further studies revealed that Pds1p is required for recombination in both double-strand-break formation and synaptonemal complex assembly. Although deleting PDS1 did not affect the degradation of the meiotic cohesin Rec8p, Mcd1p was precociously destroyed as cells entered the meiotic program. This role is meiosis specific as Mcd1p destruction is not altered in vegetative pds1Δ cultures. These results define a previously undescribed role for Pds1p in cohesin maintenance, recombination, and meiotic progression.     

A multi-step pathway for the establishment of sister chromatid cohesion

The cohesion of sister chromatids is mediated by cohesin, a protein complex containing members of the structural maintenance of chromosome (Smc) family. How cohesins tether sister chromatids is not yet understood. Here, we mutate SMC1, the gene encoding a cohesin subunit of budding yeast, by random insertion dominant negative mutagenesis to generate alleles that are highly informative for cohesin assembly and function. Cohesins mutated in the Hinge or Loop1 regions of Smc1 bind chromatin by a mechanism similar to wild-type cohesin, but fail to enrich at cohesin-associated regions (CARs) and pericentric regions. Hence, the Hinge and Loop1 regions of Smc1 are essential for the specific chromatin binding of cohesin. This specific binding and a subsequent Ctf7/Eco1-dependent step are both required for the establishment of cohesion. We propose that a cohesin or cohesin oligomer tethers the sister chromatids through two chromatin-binding events that are regulated spatially by CAR binding and temporally by Ctf7 activation, to ensure cohesins crosslink only sister chromatids.     

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.     

Mutation of the cohesin related gene PDS5 causes cell death with predominant apoptotic features in Saccharomyces cerevisiae during early meiosis

Pds5p is a cohesin related protein. It is required for maintenance of sister chromatid cohesion in mitosis and meiosis. Here we report that pds5-1 causes cell death in yeast Saccharomyces cerevisiae during early meiosis. The pds5-1 caused cell death possesses characteristics of apoptosis and necrosis, including externalization of phosphatidylserine at cytoplasmic membrane, accumulation of DNA breaks, chromatin condensation and fragmentation, nuclei fragmentation, membrane degeneration and cell size enlargement. Our results also suggest that (1) The defect of DNA repair; (2) The production of reactive oxygen species, in pds5-1 mutant are involved in pds5-1 induced cell death.     

S-phase cyclin-dependent kinases promote sister chromatid cohesion in budding yeast

Genome stability depends on faithful chromosome segregation, which relies on maintenance of chromatid cohesion during S phase. In eukaryotes, Pds1/securin is the only known inhibitor that can prevent loss of cohesion. However, pds1Δ yeast cells and securin-null mice are viable. We sought to identify redundant mechanisms that promote cohesion within S phase in the absence of Pds1 and found that cells lacking the S-phase cyclins Clb5 and Clb6 have a cohesion defect under conditions of replication stress. Similar to the phenotype of pds1Δ cells, loss of cohesion in cells lacking Clb5 and Clb6 is dependent on Esp1. However, Pds1 phosphorylation by Cdk-cyclin is not required for cohesion. Moreover, cells lacking Clb5, Clb6, and Pds1 are inviable and lose cohesion during an unperturbed S phase, indicating that Pds1 and specific B-type cyclins promote cohesion independently of one another. Consistent with this, we find that Mcd1/Scc1 is less abundant on chromosomes in cells lacking Clb5 and Clb6 during replication stress. However, clb5Δ clb6Δ cells do accumulate Mcd1/Scc1 at centromeres upon mitotic arrest, suggesting that the cyclin-dependent mechanism is S phase specific. These data indicate that Clb5 and Clb6 promote cohesion which is then protected by Pds1 and that both mechanisms are required during replication stress.     

Pds5B is required for cohesion establishment and Aurora B accumulation at centromeres

Cohesin mediates sister chromatid cohesion and contributes to the organization of interphase chromatin through DNA looping. In vertebrate somatic cells, cohesin consists of Smc1, Smc3, Rad21, and either SA1 or SA2. Three additional factors Pds5, Wapl, and Sororin bind to cohesin and modulate its dynamic association with chromatin. There are two Pds5 proteins in vertebrates, Pds5A and Pds5B, but their functional specificity remains unclear. Here, we demonstrate that Pds5 proteins are essential for cohesion establishment by allowing Smc3 acetylation by the cohesin acetyl transferases (CoATs) Esco1/2 and binding of Sororin. While both proteins contribute to telomere and arm cohesion, Pds5B is specifically required for centromeric cohesion. Furthermore, reduced accumulation of Aurora B at the inner centromere region in cells lacking Pds5B impairs its error correction function, promoting chromosome mis‐segregation and aneuploidy. Our work supports a model in which the composition and function of cohesin complexes differs between different chromosomal regions.     

Ctf19p: A novel kinetochore protein in Saccharomyces cerevisiae and a potential link between the kinetochore and mitotic spindle

A genetic synthetic dosage lethality (SDL) screen using CTF13 encoding a known kinetochore protein as the overexpressed reference gene identified two chromosome transmission fidelity (ctf) mutants, YCTF58 and YCTF26. These mutant strains carry independent alleles of a novel gene, which we have designated CTF19. In light of its potential role in kinetochore function, we have cloned and characterized the CTF19 gene in detail. CTF19 encodes a nonessential 369-amino acid protein. ctf19 mutant strains display a severe chromosome missegregation phenotype, are hypersensitive to benomyl, and accumulate at G2/M in cycling cells. CTF19 genetically interacts with kinetochore structural mutants and mitotic checkpoint mutants. In addition, ctf19 mutants show a defect in the ability of centromeres on minichromosomes to bind microtubules in an in vitro assay. In vivo cross-linking and chromatin immunoprecipitation demonstrates that Ctf19p specifically interacts with CEN DNA. Furthermore, Ctf19-HAp localizes to the nuclear face of the spindle pole body and genetically interacts with a spindle-associated protein. We propose that Ctf19p is part of a macromolecular kinetochore complex, which may function as a link between the kinetochore and the mitotic spindle.