Recruitment of Xenopus Scc2 and cohesin to chromatin requires the pre-replication complex

Cohesin is a multi-subunit, ring-shaped protein complex that holds sister chromatids together from the time of their synthesis in S phase until they are segregated in anaphase. In yeast, the loading of cohesin onto chromosomes requires the Scc2 protein. In vertebrates, cohesins first bind to chromosomes as cells exit mitosis, but the mechanism is unknown. Concurrent with cohesin binding, pre-replication complexes (pre-RCs) are assembled at origins of DNA replication through the sequential loading of the initiation factors ORC, Cdc6, Cdt1 and MCM2-7 (the 'licensing' reaction). In S phase, the protein kinase Cdk2 activates pre-RCs, causing origin unwinding and DNA replication. Here, we use Xenopus egg extracts to show that the recruitment of cohesins to chromosomes requires fully licensed chromatin and is dependent on ORC, Cdc6, Cdt1 and MCM2-7, but is independent of Cdk2. We further show that Xenopus Scc2 is required for cohesin loading and that binding of XScc2 to chromatin is MCM2-7 dependent. Our results define a novel pre-RC-dependent pathway for cohesin recruitment to chromosomes in a vertebrate model system.     

Cohesin's binding to chromosomes depends on a separate complex consisting of Scc2 and Scc4 proteins

Cohesion between sister chromatids depends on a multisubunit cohesin complex that binds to chromosomes around DNA replication and dissociates from them at the onset of anaphase. Scc2p, though not a cohesin subunit, is also required for sister chromatid cohesion. We show here that Scc2p forms a complex with a novel protein, Scc4p, which is also necessary for sister cohesion. In scc2 or scc4 mutants, cohesin complexes form normally but fail to bind both to centromeres and to chromosome arms. Our data suggest that a major role for the Scc2p/Scc4p complex is to facilitate the loading of cohesin complexes onto chromosomes.     

A model for ATP hydrolysis-dependent binding of cohesin to DNA

BACKGROUND: Cohesion between sister chromatids is promoted by the chromosomal cohesin complex that forms a proteinaceous ring, large enough in principle to embrace two sister strands. The mechanism by which cohesin binds to DNA, and how sister chromatid cohesion is established, is unknown. RESULTS: Biochemical studies of cohesin have largely been limited to protein isolated from soluble cellular fractions. Here, we characterize cohesin purified from budding yeast chromatin, suggesting that chromosomal cohesin is sufficiently described by its known distinctive ring structure. We present evidence that the two Smc subunits of cohesin by themselves form a ring, closed at interacting ATPase head domains. A motif in the Smc1 subunit implicated in ATP hydrolysis is essential for loading cohesin onto DNA. In addition to functional ATPase heads, an intact cohesin ring structure is indispensable for DNA binding, suggesting that ATP hydrolysis may be coupled to DNA transport into the cohesin ring. DNA is released in anaphase when separase cleaves cohesin's Scc1 subunit. We show that a cleavage fragment of Scc1 disrupts the interaction between the two Smc heads, thereby opening the ring. CONCLUSIONS: We present a model for cohesin binding to chromatin by ATP hydrolysis-dependent transport of DNA into the cohesin ring. After DNA replication, two DNA strands may be trapped to promote sister chromatid cohesion. In anaphase, Scc1 cleavage opens the ring to release sister chromatids.     

Displacement and re-accumulation of centromeric cohesin during transient pre-anaphase centromere splitting

The ring-shaped cohesin complex links sister chromatids until their timely segregation during mitosis. Cohesin is enriched at centromeres where it provides the cohesive counterforce to bipolar tension produced by the mitotic spindle. As a consequence of spindle tension, centromeric sequences transiently split in pre-anaphase cells, in some organisms up to several micrometers. This ‘centromere breathing’ presents a paradox, how sister sequences separate where cohesin is most enriched. We now show that in the budding yeast Saccharomyces cerevisiae, cohesin binding diminishes over centromeric sequences that split during breathing. We see no evidence for cohesin translocation to surrounding sequences, suggesting that cohesin is removed from centromeres during breathing. Two pools of cohesin can be distinguished. Cohesin loaded before DNA replication, which has established sister chromatid cohesion, disappears during breathing. In contrast, cohesin loaded after DNA replication is partly retained. As sister centromeres re-associate after transient separation, cohesin is reloaded in a manner independent of the canonical cohesin loader Scc2/Scc4. Efficient centromere re-association requires the cohesion establishment factor Eco1, suggesting that re-establishment of sister chromatid cohesion contributes to the dynamic behaviour of centromeres in mitosis. These findings provide new insights into cohesin behaviour at centromeres. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

A screen for cohesion mutants uncovers Ssl3, the fission yeast counterpart of the cohesin loading factor Scc4

Sister-chromatid cohesion is mediated by cohesin, a ring-shape complex made of four core subunits called Scc1, Scc3, Smc1, and Smc3 in Saccharomyces cerevisiae (Rad21, Psc3, Psm1, and Psm3 in Schizosaccharomyces pombe). How cohesin ensures cohesion is unknown, although its ring shape suggests that it may tether sister DNA strands by encircling them . Cohesion establishment is a two-step process. Cohesin is loaded on chromosomes before replication and cohesion is subsequently established during S phase. In S. cerevisiae, cohesin loading requires a separate complex containing the Scc2 and Scc4 proteins. Cohesin rings fail to associate with chromatin and cohesion can not establish when Scc2 is impaired . The mechanism of loading is unknown, although some data suggest that hydrolysis of ATP bound to Smc1/3 is required . Scc2 homologs exist in fission yeast (Mis4), Drosophila, Xenopus, and human . By contrast, no homolog of Scc4 has been identified so far. We report here on the identification of fission yeast Ssl3 as a Scc4-like factor. Ssl3 is in complex with Mis4 and, as a bona fide loading factor, Ssl3 is required in G1 for cohesin binding to chromosomes but dispensable in G2 when cohesion is established. The discovery of a functional homolog of Scc4 indicates that the machinery of cohesin loading is conserved among eukaryotes.     

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.     

A matter of choice: the establishment of sister chromatid cohesion

Sister chromatid cohesion is the basis for the recognition of chromosomal DNA replication products for their bipolar segregation in mitosis. Fundamental to sister chromatid cohesion is the ring‐shaped cohesin complex, which is loaded onto chromosomes long before the initiation of DNA replication and is thought to hold replicated sister chromatids together by topological embrace. What happens to cohesin when the replication fork approaches, and how cohesin recognizes newly synthesized sister chromatids, is poorly understood. The characterization of a number of cohesion establishment factors has begun to provide hints as to the reactions involved. Cohesin is a member of the evolutionarily conserved family of Smc subunit‐based protein complexes that contribute to many aspects of chromosome biology by mediating long‐range DNA interactions. I propose that the establishment of cohesion equates to the selective stabilization of those cohesin‐mediated DNA interactions that link sister chromatids in the wake of replication forks.     

C-terminus of Sororin interacts with SA2 and regulates sister chromatid cohesion

Sororin is a conserved protein required for accurate separation of sister chromatids in each cell cycle. Sororin is recruited to chromatin during DNA replication, protects sister chromatid cohesion in S and G2 phase, and regulates the resolution of sister chromatid cohesion in mitosis. Sororin binds to cohesin complex, but how Sororin and cohesin subunits interact remains unclear. Here we report that the C-terminus of Sororin, especially the last 12 amino acid (aa) residues, is important for Sororin to bind cohesin core subunit SA2. Deletion of the last 12aa residues not only inhibits the interactions between Sororin and SA2 but also causes precocious chromosome separation. Our data suggest that the C-terminus of Sororin functions as an anchor binding to SA2, which facilitates other conserved motifs on Sororin to interact with other proteins to regulate sister chromatid cohesion and separation.     

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.     

Chromosomal cohesin forms a ring

The cohesin complex is essential for sister chromatid cohesion during mitosis. Its Smc1 and Smc3 subunits are rod-shaped molecules with globular ABC-like ATPases at one end and dimerization domains at the other connected by long coiled coils. Smc1 and Smc3 associate to form V-shaped heterodimers. Their ATPase heads are thought to be bridged by a third subunit, Scc1, creating a huge triangular ring that could trap sister DNA molecules. We address here whether cohesin forms such rings in vivo. Proteolytic cleavage of Scc1 by separase at the onset of anaphase triggers its dissociation from chromosomes. We show that N- and C-terminal Scc1 cleavage fragments remain connected due to their association with different heads of a single Smc1/Smc3 heterodimer. Cleavage of the Smc3 coiled coil is sufficient to trigger cohesin release from chromosomes and loss of sister cohesion, consistent with a topological association with chromatin.     

Two putative acetyltransferases, san and deco, are required for establishing sister chromatid cohesion in Drosophila

BACKGROUND: Sister chromatid cohesion is needed for proper alignment and segregation of chromosomes during cell division. Chromatids are linked by the multiprotein cohesin complex, which binds to DNA during G(1) and then establishes cohesion during S phase DNA replication. However, many aspects of the mechanisms that establish and maintain cohesion during mitosis remain unclear.RESULTS: We found that mutations in two evolutionarily conserved Drosophila genes, san (separation anxiety) and deco (Drosophila eco1), disrupt centromeric sister chromatid cohesion very early in division. This failure of sister chromatid cohesion does not require separase and is correlated with a failure of the cohesin component Scc1 to accumulate in centromeric regions. It thus appears that these mutations interfere with the establishment of centromeric sister chromatid cohesion. Secondary consequences of these mutations include activation of the spindle checkpoint, causing metaphase delay or arrest. Some cells eventually escape the block but incur many errors in anaphase chromosome segregation. Both san and deco are predicted to encode acetyltransferases, which transfer acetyl groups either to internal lysine residues or to the N terminus of other proteins. The San protein is itself acetylated, and it associates with the Nat1 and Ard1 subunits of the NatA acetyltransferase.CONCLUSIONS: At least two diverse acetyltransferases play vital roles in regulating sister chromatid cohesion during Drosophila mitosis.     

Metazoan Scc4 homologs link sister chromatid cohesion to cell and axon migration guidance

Saccharomyces cerevisiae Scc2 binds Scc4 to form an essential complex that loads cohesin onto chromosomes. The prevalence of Scc2 orthologs in eukaryotes emphasizes a conserved role in regulating sister chromatid cohesion, but homologs of Scc4 have not hitherto been identified outside certain fungi. Some metazoan orthologs of Scc2 were initially identified as developmental gene regulators, such as Drosophila Nipped-B, a regulator of cut and Ultrabithorax, and delangin, a protein mutant in Cornelia de Lange syndrome. We show that delangin and Nipped-B bind previously unstudied human and fly orthologs of Caenorhabditis elegans MAU-2, a non-axis-specific guidance factor for migrating cells and axons. PSI-BLAST shows that Scc4 is evolutionarily related to metazoan MAU-2 sequences, with the greatest homology evident in a short N-terminal domain, and protein–protein interaction studies map the site of interaction between delangin and human MAU-2 to the N-terminal regions of both proteins. Short interfering RNA knockdown of human MAU-2 in HeLa cells resulted in precocious sister chromatid separation and in impaired loading of cohesin onto chromatin, indicating that it is functionally related to Scc4, and RNAi analyses show that MAU-2 regulates chromosome segregation in C. elegans embryos. Using antisense morpholino oligonucleotides to knock down Xenopus tropicalis delangin or MAU-2 in early embryos produced similar patterns of retarded growth and developmental defects. Our data show that sister chromatid cohesion in metazoans involves the formation of a complex similar to the Scc2-Scc4 interaction in the budding yeast. The very high degree of sequence conservation between Scc4 homologs in complex metazoans is consistent with increased selection pressure to conserve additional essential functions, such as regulation of cell and axon migration during development.     

Sororin is required for stable binding of cohesin to chromatin and for sister chromatid cohesion in interphase

Sister chromatid cohesion depends on cohesin [1-3]. Cohesin associates with chromatin dynamically throughout interphase [4]. During DNA replication, cohesin establishes cohesion [5], and this process coincides with the generation of a cohesin subpopulation that is more stably bound to chromatin [4]. In mitosis, cohesin is removed from chromosomes, enabling sister chromatid separation [6]. How cohesin associates with chromatin and establishes cohesion is poorly understood. By searching for proteins that are associated with chromatin-bound cohesin, we have identified sororin, a protein that was known to be required for cohesion [7]. To obtain further insight into sororin's function, we have addressed when during the cell cycle sororin is required for cohesion. We show that sororin is dispensable for the association of cohesin with chromatin but that sororin is essential for proper cohesion during G2 phase. Like cohesin, sororin is also needed for efficient repair of DNA double-strand breaks in G2. Finally, sororin is required for the presence of normal amounts of the stably chromatin-bound cohesin population in G2. Our data indicate that sororin interacts with chromatin-bound cohesin and functions during the establishment or maintenance of cohesion in S or G2 phase, respectively.     

HIV uses autophagy for its own means

Replicated sister chromatids are held together until mitosis by cohesin, a conserved multisubunit complex comprised of Smc1, Smc3, Scc1, and Scc3, which in vertebrate cells exists as two closely related homologues (SA1 and SA2). Here, we show that cohesin^SA1 and cohesin^SA2 are differentially required for telomere and centromere cohesion, respectively. Cells deficient in SA1 are unable to establish or maintain cohesion between sister telomeres after DNA replication in S phase. The same phenotype is observed upon depletion of the telomeric protein TIN2. In contrast, in SA2-depleted cells telomere cohesion is normal, but centromere cohesion is prematurely lost. We demonstrate that loss of telomere cohesion has dramatic consequences on chromosome morphology and function. In the absence of sister telomere cohesion, cells are unable to repair chromatid breaks and suffer sister telomere loss. Our studies elucidate the functional distinction between the Scc3 homologues in human cells and further reveal an essential role for sister telomere cohesion in genomic integrity.     

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.     

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.     

Structure and stability of cohesin's Smc1-kleisin interaction

A multisubunit complex called cohesin forms a huge ring structure that mediates sister chromatid cohesion, possibly by entrapping sister DNAs following replication. Cohesin's kleisin subunit Scc1 completes the ring, connecting the ABC-like ATPase heads of a V-shaped Smc1/3 heterodimer. Proteolytic cleavage of Scc1 by separase triggers sister chromatid disjunction, presumably by breaking the Scc1 bridge. One half of the SMC-kleisin bridge is revealed here by a crystal structure of Smc1's ATPase complexed with Scc1's C-terminal domain. The latter forms a winged helix that binds a pair of beta strands in Smc1's ATPase head. Mutation of conserved residues within the contact interface destroys Scc1's interaction with Smc1/3 heterodimers and eliminates cohesin function. Interaction of Scc1's N terminus with Smc3 depends on prior C terminus connection with Smc1. There is little or no turnover of Smc1-Scc1 interactions within cohesin complexes in vivo because expression of noncleavable Scc1 after DNA replication does not hinder anaphase.     

Sororin, a substrate of the anaphase-promoting complex, is required for sister chromatid cohesion in vertebrates

We have identified a regulator of sister chromatid cohesion in a screen for cell cycle-controlled proteins. This 35 kDa protein is degraded through anaphase-promoting complex (APC)-dependent ubiquitination in G1. The protein is nuclear in interphase cells, dispersed from the chromatin in mitosis, and interacts with the cohesin complex. In Xenopus embryos, overexpression of the protein causes failure to resolve and segregate sister chromatids in mitosis and an increase in the level of cohesin associated with metaphase chromosomes. In cultured cells, depletion of the protein causes mitotic arrest and complete failure of sister chromatid cohesion. This protein is thus an essential, cell cycle-dependent mediator of sister chromatid cohesion. Based on sequence analysis, this protein has no apparent orthologs outside of the vertebrates. We speculate that the protein, which we have named sororin, regulates the ability of the cohesin complex to mediate sister chromatid cohesion, perhaps by altering the nature of the interaction of cohesin with the chromosomes.