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Cohesin is a conserved, ring-shaped protein complex that topologically entraps DNA. This ability makes this member of the structural maintenance of chromosomes (SMC) complex family a central hub of chromosome dynamics regulation. Besides its essential role in sister chromatid cohesion, cohesin shapes the interphase chromatin domain architecture and plays important roles in transcriptional regulation and DNA repair. Cohesin is loaded onto chromosomes at centromeres, at the promoters of highly expressed genes, as well as at DNA replication forks and sites of DNA damage. However, the features that determine these binding sites are still incompletely understood. We recently described a role of the budding yeast RSC chromatin remodeler in cohesin loading onto chromosomes. RSC has a dual function, both as a physical chromatin receptor of the Scc2/Scc4 cohesin loader complex, as well as by providing a nucleosome-free template for cohesin loading. Here, we show that the role of RSC in sister chromatid cohesion is conserved in fission yeast. We discuss what is known about the broader conservation of the contribution of chromatin remodelers to cohesin loading onto chromatin.

The cohesin complex topologically encircles DNA to promote sister chromatid cohesion. Alternatively cohesin extrudes DNA loops, thought to reflect chromatin domain formation. Here, we propose a structure-based model explaining both activities, supported by biochemical experiments. ATP and DNA binding to cohesin promote conformational changes that guide DNA through a kleisin gate into a DNA gripping state. Two HEAT-repeat DNA binding modules, associated with cohesin’s heads and hinge, are now juxtaposed. ATP hydrolysis disassembles the gripping state, allowing unidirectional hinge module movement to complete topological DNA entry. Without initial kleisin gate passage, biased hinge module motion during gripping state resolution creates a Brownian ratchet that drives loop extrusion. Molecular-mechanical simulations of gripping state formation and resolution cycles recapitulate experimentally observed DNA loop extrusion characteristics. Our model extends to asymmetric and symmetric loop extrusion, as well as z-loop formation. Loop extrusion by biased Brownian fluctuations has important implications for chromosomal cohesin function.

Yeast cohesin complexes cluster onto long DNA into liquid droplets, which they accomplish by ATP-independent DNA bridging., Structural maintenance of chromosome (SMC) protein complexes are able to extrude DNA loops. While loop extrusion constitutes a fundamental building block of chromosomes, other factors may be equally important. Here, we show that yeast cohesin exhibits pronounced clustering on DNA, with all the hallmarks of biomolecular condensation. DNA-cohesin clusters exhibit liquid-like behavior, showing fusion of clusters, rapid fluorescence recovery after photobleaching and exchange of cohesin with the environment. Strikingly, the in vitro clustering is DNA length dependent, as cohesin forms clusters only on DNA exceeding 3 kilo–base pairs. We discuss how bridging-induced phase separation, a previously unobserved type of biological condensation, can explain the DNA-cohesin clustering through DNA-cohesin-DNA bridges. We confirm that, in yeast cells in vivo, a fraction of cohesin associates with chromatin in a manner consistent with bridging-induced phase separation. Biomolecular condensation by SMC proteins constitutes a new basic principle by which SMC complexes direct genome organization.

The ring-like cohesin complex plays an essential role in chromosome segregation, organization, and double-strand break repair through its ability to bring two DNA double helices together. Scc2 (NIPBL in humans) together with Scc4 function as the loader of cohesin onto chromosomes. The RSC complex has been shown to act as a chromatin adapter, facilitating localization of the Scc2-Scc4 cohesin loader. Here we identify a broad range of Scc2- chromatin protein interactions that are evolutionarily conserved, consistent with a multifactorial adaptor landscape. We reveal a role for one complex, Mediator, in recruitment of the cohesin loader. Med14, a subunit of the Mediator complex, is a high copy suppressor of Scc2 mutants. Physical and genetic interactions between Scc2 and Mediator are functionally substantiated in direct recruitment and cohesion assays. Mediator can provide recruitment function for Scc2 based on a chromatin tethering experiment. Depletion of Med14 results in defective sister chromatid cohesion and decreased binding of Scc2 at RNA Pol II transcribed genes. Previous work has suggested that Mediator, Nipbl, and cohesin connect enhancers and promoters of active mammalian genes. Our studies in yeast suggest an evolutionarily conserved fundamental role for Mediator in direct recruitment of Scc2 to RNA pol II transcribed genes.

Summary Despite key roles in sister chromatid cohesion and chromosome organization, the mechanism by which cohesin rings are loaded onto DNA is still unknown. Here we combine biochemical approaches and cryoelectron microscopy (cryo-EM) to visualize a cohesin loading intermediate in which DNA is locked between two gates that lead into the cohesin ring. Building on this structural framework, we design experiments to establish the order of events during cohesin loading. In an initial step, DNA traverses an N-terminal kleisin gate that is first opened upon ATP binding and then closed as the cohesin loader locks the DNA against the ATPase gate. ATP hydrolysis will lead to ATPase gate opening to complete DNA entry. Whether DNA loading is successful or results in loop extrusion might be dictated by a conserved kleisin N-terminal tail that guides the DNA through the kleisin gate. Our results establish the molecular basis for cohesin loading onto DNA., Graphical Abstract, Highlights • 3.9-Å cryo-EM structure of the cohesin complex in a DNA gripping state intermediate • DNA is trapped between two gates that lead into the cohesin ring • The kleisin N-tail guides DNA through a kleisin N-gate into the gripping state • ATP hydrolysis opens the head gate to complete DNA entry, Cohesin is a ring-shaped protein complex that topologically entraps DNA to fulfil key functions in chromosome architecture. In a multidisciplinary approach, Higashi et al. use cryo-EM and biochemical techniques to describe how ATP-fueled structural changes of the cohesin complex drive the DNA entry reaction into the cohesin ring.

Cohesin is a key protein complex that organizes the spatial structure of chromosomes during interphase. Here, we show that yeast cohesin shows pronounced clustering on DNA in an ATP-independent manner, exhibiting all the hallmarks of phase separation. In vitro visualization of cohesin on DNA shows DNA-cohesin clusters that exhibit liquid-like behavior. This includes mutual fusion and reversible dissociation upon depleting the cohesin concentration, increasing the ionic strength, or adding 1,6-hexanediol, conditions that disrupt weak interactions. We discuss how bridging-induced phase separation can explain the DNA-cohesin clustering through DNA-cohesin-DNA bridges. We confirm that, in vivo, a fraction of cohesin associates with chromatin in yeast cells in a manner consistent with phase separation. Our findings establish that SMC proteins can exhibit phase separation, which has potential to clarify previously unexplained aspects of in vivo SMC behavior and constitute an additional principle by which SMC complexes impact genome organization.One sentence summaryYeast cohesin complex is observed to phase separate with DNA into liquid droplets, which it accomplishes by ATP-independent DNA bridging.

Summary Structural maintenance of chromosome (SMC) complexes are proteinaceous rings that embrace DNA to enable vital chromosomal functions. The ring is formed by two SMC subunits, closed at a pair of ATPase heads, whose interaction is reinforced by a kleisin subunit. Using biochemical analysis of fission-yeast cohesin, we find that a similar series of events facilitates both topological entrapment and release of DNA. DNA-sensing lysines trigger ATP hydrolysis to open the SMC head interface, whereas the Wapl subunit disengages kleisin, but only after ATP rebinds. This suggests an interlocking gate mechanism for DNA transport both into and out of the cohesin ring. The entry direction is facilitated by a cohesin loader that appears to fold cohesin to expose the DNA sensor. Our results provide a model for dynamic DNA binding by all members of the SMC family and explain how lysine acetylation of cohesin establishes enduring sister chromatid cohesion., Graphical Abstract, Highlights • Biochemical reconstitution of DNA entry into and exit out of the cohesin ring • DNA-sensing lysines on the Psm3/Smc3 subunit’s ATPase trigger main ring opening • The Wapl subunit disengages an interlocking kleisin subunit “outer gate” • A unified model for DNA entry and exit with implications for all SMC complexes, The biochemical reconstitution of the topological entrapment and release of DNA by the fission-yeast cohesin complex indicates that DNA sensing by lysine residues triggers entry into and exit from nested gates comprised of the cohesin complex proteins in a manner that can be regulated by acetylation.

Separases are crucial cell cycle proteases that control the metaphase-to-anaphase transition by cleaving chromosomal cohesin rings. Two new high-resolution structures of separase bound by its inhibitory chaperone securin illustrate intriguing regulatory mechanisms.

Summary The ring-shaped structural maintenance of chromosome (SMC) complexes are multi-subunit ATPases that topologically encircle DNA. SMC rings make vital contributions to numerous chromosomal functions, including mitotic chromosome condensation, sister chromatid cohesion, DNA repair, and transcriptional regulation. They are thought to do so by establishing interactions between more than one DNA. Here, we demonstrate DNA-DNA tethering by the purified fission yeast cohesin complex. DNA-bound cohesin efficiently and topologically captures a second DNA, but only if that is single-stranded DNA (ssDNA). Like initial double-stranded DNA (dsDNA) embrace, second ssDNA capture is ATP-dependent, and it strictly requires the cohesin loader complex. Second-ssDNA capture is relatively labile but is converted into stable dsDNA-dsDNA cohesion through DNA synthesis. Our study illustrates second-DNA capture by an SMC complex and provides a molecular model for the establishment of sister chromatid cohesion., Graphical Abstract, Highlights • The recombinant cohesin complex mediates DNA-DNA interactions • Second-DNA capture by cohesin requires ssDNA • DNA replication stabilizes second-strand capture • Second-DNA capture at a replication fork could establish sister chromatid cohesion, Cohesin sets up the initial steps of chromosome cohesion by anchoring single-stranded DNA along to a region of duplex DNA.

Sister-chromatid cohesion is established by Eco1-mediated acetylation on two conserved tandem lysines in the cohesin Smc3 subunit. However, the molecular basis of Eco1 substrate recognition and acetylation in cohesion is not fully understood. Here, we discover and rationalize the substrate specificity of Eco1 using mass spectrometry coupled with in-vitro acetylation assays and crystallography. Our structures of the X. laevis Eco2 (xEco2) bound to its primary and secondary Smc3 substrates demonstrate the plasticity of the substrate-binding site, which confers substrate specificity by concerted conformational changes of the central β hairpin and the C-terminal extension.

The cohesin complex is involved in a broad range of chromosomal biology, including DNA repair, gene transcription as well as sister chromatid cohesion. Cohesin is a large, ring-shaped protein complex and is thought to entrap DNA molecules inside of its ring. The unique DNA association is central to cohesin function and requires its ATPase and another heterodimer complex called the cohesin loader. Here we describe the biochemical reconstitution of topological cohesin loading onto DNA using the purified fission yeast cohesin proteins.

The cohesin complex is at the heart of many chromosomal activities, including sister chromatid cohesion and transcriptional regulation. Cohesin loading onto chromosomes depends on the Scc2-Scc4 cohesin loader complex, but the chromatin features that form cohesin loading sites remain poorly understood. Here we show that the RSC chromatin remodeling complex recruits budding yeast Scc2-Scc4 to broad nucleosome-free regions, which the cohesin loader helps to maintain. Consequently, inactivation of either the cohesin loader or the RSC complex has similar effects on nucleosome positioning, gene expression and sister chromatid cohesion. These results show an intimate link between local chromatin structure and higher-order chromosome architecture. Our findings pertain to the similarities between two severe human disorders, Cornelia de Lange syndrome, which is caused by alterations in the human cohesin loader, and Coffin-Siris syndrome, which results from alterations in human RSC complex components. Both syndromes can arise from gene misregulation due to related changes in the nucleosome landscape.

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. Electronic supplementary material The online version of this article (doi:10.1007/s00412-013-0396-y) contains supplementary material, which is available to authorized users.

Sister-chromatid cohesion is established by Eco1-mediated acetylation on two conserved tandem lysines in the cohesin Smc3 subunit. However, the molecular basis of Eco1 substrate recognition and acetylation in cohesion is not fully understood. Here, we discover and rationalize the substrate specificity of Eco1 using mass spectrometry coupled with in-vitro acetylation assays and crystallography. Our structures of the X. laevis Eco2 (xEco2) bound to its primary and secondary Smc3 substrates demonstrate the plasticity of the substrate-binding site, which confers substrate specificity by concerted conformational changes of the central β hairpin and the C-terminal extension.

The functions of cohesin are central to genome integrity, chromosome organization and transcription regulation through its prevention of premature sister-chromatid separation and the formation of DNA loops. The loading of cohesin onto chromatin depends on the Scc2–Scc4 complex; however, little is known about how it stimulates the cohesion-loading activity. Here we determine the large ‘hook' structure of Scc2 responsible for catalysing cohesin loading. We identify key Scc2 surfaces that are crucial for cohesin loading in vivo. With the aid of previously determined structures and homology modelling, we derive a pseudo-atomic structure of the full-length Scc2–Scc4 complex. Finally, using recombinantly purified Scc2–Scc4 and cohesin, we performed crosslinking mass spectrometry and interaction assays that suggest Scc2–Scc4 uses its modular structure to make multiple contacts with cohesin., The cohesin complex maintains genome integrity by ensuring correct sister-chromatid segregation during mitosis and meiosis. Here, Chao et al. present a pseudo-atomic model of the full-length Scc2–Scc4 cohesin loader complex and reveal key Scc2 surfaces crucial for cohesin loading.

DNA replication during S phase is accompanied by establishment of sister chromatid cohesion to ensure faithful chromosome segregation. The Eco1 acetyltransferase, helped by factors including Ctf4 and Chl1, concomitantly acetylates the chromosomal cohesin complex to stabilize its cohesive links. Here we show that Ctf4 recruits the Chl1 helicase to the replisome via a conserved interaction motif that Chl1 shares with GINS and polymerase α. We visualize recruitment by EM analysis of a reconstituted Chl1-Ctf4-GINS assembly. The Chl1 helicase facilitates replication fork progression under conditions of nucleotide depletion, partly independently of Ctf4 interaction. Conversely, Ctf4 interaction, but not helicase activity, is required for Chl1’s role in sister chromatid cohesion. A physical interaction between Chl1 and the cohesin complex during S phase suggests that Chl1 contacts cohesin to facilitate its acetylation. Our results reveal how Ctf4 forms a replisomal interaction hub that coordinates replication fork progression and sister chromatid cohesion establishment.

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.

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:10.1007/s00412-007-0118-4) contains supplementary material, which is available to authorized users.

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.

SummaryThe remarkable accuracy of eukaryotic cell division is partly maintained by the cohesin complex acting as a molecular glue to prevent premature sister chromatid separation. The loading of cohesin onto chromosomes is catalyzed by the Scc2-Scc4 loader complex. Here, we report the crystal structure of Scc4 bound to the N terminus of Scc2 and show that Scc4 is a tetratricopeptide repeat (TPR) superhelix. The Scc2 N terminus adopts an extended conformation and is entrapped by the core of the Scc4 superhelix. Electron microscopy (EM) analysis reveals that the Scc2-Scc4 loader complex comprises three domains: a head, body, and hook. Deletion studies unambiguously assign the Scc2N-Scc4 as the globular head domain, whereas in vitro cohesin loading assays show that the central body and the hook domains are sufficient to catalyze cohesin loading onto circular DNA, but not chromatinized DNA in vivo, suggesting a possible role for Scc4 as a chromatin adaptor.

EMBO J (2013) 32: 666–676 doi:10.1038/emboj.2013.7; published online January292013 EMBO J (2013) 32: 677–687 doi:10.1038/emboj.2013.16; published online February082013 EMBO J (2013) 32: 656–665 doi:10.1038/emboj.2012.346; published online January222013 Chromosome segregation is triggered when the ring-shaped chromosomal cohesin complex is opened by proteolytic cleavage to release pairs of sister chromatids. Even before this dramatic event in anaphase, many cohesin rings lead a dynamic life on chromosomes, and in metazoan cells a good part of them dissociate from chromosome arms during mitotic prophase. Two new papers in The EMBO Journal address how chromatin can get into and out of the cohesin ring without requiring its cleavage.

The final irreversible step in the duplication and dissemination of eukaryotic genomes takes place when sister chromatid pairs split and separate in anaphase. This is triggered by the protease separase that cleaves the Scc1 subunit of 'cohesin', the protein complex responsible for holding sister chromatids together in metaphase. Only part of cellular cohesin is bound to chromosomes in metaphase, and it is unclear whether and how separase specifically targets this fraction for cleavage. We established an assay to compare cleavage of chromatin-bound versus soluble budding yeast cohesin. Scc1 in chromosomal cohesin is significantly preferred by separase over Scc1 in soluble cohesin. The difference is most likely due to preferential phosphorylation of chromatin-bound Scc1 by Polo-like kinase. Site-directed mutagenesis of 10 Polo phosphorylation sites in Scc1 slowed cleavage of chromatin-bound cohesin, and hyperphosphorylation of soluble Scc1 by Polo overexpression accelerated its cleavage to levels of chromosomal cohesin. Polo is bound to chromosomes independently of cohesin's presence, providing a possible explanation for chromosome-specific cohesin modification and targeting of separase cleavage.

At anaphase onset, the protease separase triggers chromosome segregation by cleaving the chromosomal cohesin complex. Here, we show that cohesin destruction in metaphase is sufficient for segregation of much of the budding yeast genome, but not of the long arm of chromosome XII that contains the rDNA repeats. rDNA in metaphase, unlike most other sequences, remains in an undercondensed and topologically entangled state. Separase, concomitantly with cleaving cohesin, activates the phosphatase Cdc14. We find that Cdc14 exerts two effects on rDNA, both mediated by the condensin complex. Lengthwise condensation of rDNA shortens the chromosome XII arm sufficiently for segregation. This condensation depends on the aurora B kinase complex. Independently of condensation, Cdc14 induces condensin-dependent resolution of cohesin-independent rDNA linkage. Cdc14-dependent sister chromatid resolution at the rDNA could introduce a temporal order to chromosome segregation.

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.

Disjunction of maternal and paternal centromeres during meiosis I requires crossing over between homologous chromatids, which creates chiasmata that hold homologs together. It also depends on a mechanism ensuring that maternal and paternal sister kinetochore pairs attach to oppositely oriented microtubules. Proteolytic cleavage of cohesin's Rec8 subunit by separase destroys cohesion between sister chromatid arms at anaphase I and thereby resolves chiasmata. The Spo12 and Slk19 proteins have been implicated in regulating meiosis I kinetochore orientation and/or in preventing cleavage of Rec8 at centromeres. We show here that the role of these proteins is instead to promote nucleolar segregation, including release of the Cdc14 phosphatase required for Cdk1 inactivation and disassembly of the anaphase I spindle. Separase is also required but surprisingly not its protease activity. It has two mechanistically different roles during meiosis I. Loss of the protease-independent function alone results in a second meiotic division occurring on anaphase I spindles in spo12Δ and slk19Δ mutants.

Sister chromatid cohesion and separation are fundamental for accurate genome inheritance over cell generations. Work over recent years has established the existence of a chromosomal protein complex, cohesin, that connects sister chromatids from the time they are generated in S phase onwards, and which is destroyed at the onset of anaphase through cleavage by the protease separase. Over the last year, the function of cohesin has been investigated in higher eukaryotes, including humans, with results that have uncovered important new aspects of this process. The first structural views of cohesin have become available, and significant steps been made towards a mechanistic understanding of chromosome cohesion. Studies on separase have revealed new levels of regulation of chromosome segregation.

Anaphase in budding yeast is triggered by cleavage of the central subunit, Scc1, of the chromosomal cohesin complex by the protease separase. Here we show that separase also cleaves the kinetochore-associated protein Slk19 at anaphase onset. Separase activity is also required for the proper localization of a stable Slk19 cleavage product to the spindle midzone in anaphase. The cleavage and localization of Slk19 are necessary to stabilize the anaphase spindle, and we show that a stable spindle is a prerequisite for timely exit from mitosis. This demonstrates the cleavage of targets other than cohesin by separase in the orchestration of high-fidelity anaphase.

The final irreversible step in the duplication and distribution of genomes to daughter cells takes place at the metaphase to anaphase transition. At this point aligned sister chromatid pairs split and separate. During metaphase, cohesion between sister chromatids is maintained by the chromosomal multi-subunit cohesin complex. Here, I review recent findings as to how anaphase is initiated by proteolytic cleavage of the Scc1 subunit of cohesin. Scc1 is cleaved by a site-specific protease that is conserved in all eukaryotes, and is now called ‘separase’. As a result of this cleavage, the cohesin complex is destroyed, allowing the spindle to pull sister chromatids into opposite halves of the cell. Because of the final and irreversible nature of Scc1 cleavage, this reaction is tightly controlled. Several independent mechanisms seem to impose regulation on Scc1 cleavage, acting on both the activity of separase and the susceptibility of the substrate.

At the onset of anaphase, a caspase-related protease (separase) destroys the link between sister chromatids by cleaving the cohesin subunit Scc1. During most of the cell cycle, separase is kept inactive by binding to an inhibitory protein called securin. Separase activation requires proteolysis of securin, which is mediated by an ubiquitin protein ligase called the anaphase-promoting complex. Cells regulate anaphase entry by delaying securin ubiquitination until all chromosomes have attached to the mitotic spindle. Though no longer regulated by this mitotic surveillance mechanism, sister separation remains tightly cell cycle regulated in yeast mutants lacking securin. We show here that the Polo/Cdc5 kinase phosphorylates serine residues adjacent to Scc1 cleavage sites and strongly enhances their cleavage. Phosphorylation of separase recognition sites may be highly conserved and regulates sister chromatid separation independently of securin.

Cohesion between sister chromatids is established during DNA replication and depends on a protein complex called cohesin. At the metaphase-anaphase transition in the yeast Saccharomyces cerevisiae, the ESP1-encoded protease separin cleaves SCC1, a subunit of cohesin with a relative molecular mass of 63,000 (Mr 63K). The resulting 33K carboxy-terminal fragment of SCC1 bears an amino-terminal arginine-a destabilizing residue in the N-end rule. Here we show that the SCC1 fragment is short-lived (t1/2 approximately 2 min), being degraded by the ubiquitin/proteasome-dependent N-end rule pathway. Overexpression of a long-lived derivative of the SCC1 fragment is lethal. In ubr1Delta cells, which lack the N-end rule pathway, we found a highly increased frequency of chromosome loss. The bulk of increased chromosome loss in ubr1Delta cells is caused by metabolic stabilization of the ESP1-produced SCC1 fragment. This fragment is the first physiological substrate of the N-end rule pathway that is targeted through its N-terminal residue. A number of yeast proteins bear putative cleavage sites for the ESP1 separin, suggesting other physiological substrates and functions of the N-end rule pathway.

Summary Separase is the protease that cleaves the cohesive link between sister chromatids to trigger chromosome segregation in mitosis and meiosis. This enzyme is known to orchestrate additional mitotic events and we now gain new insight into how it promotes cytokinesis in the nematode Caenorhabditis elegans .

It has been proposed but never proven that cohesion between sister chromatids distal to chiasmata is responsible for holding homologous chromosomes together while spindles attempt to pull them toward opposite poles during metaphase of meiosis I. Meanwhile, the mechanism by which disjunction of homologs is triggered at the onset of anaphase I has remained a complete mystery. In yeast, cohesion between sister chromatid arms during meiosis depends on a meiosis-specific cohesin subunit called Rec8, whose mitotic equivalent, Scc1, is cleaved at the metaphase to anaphase transition by an endopeptidase called separin. We show here that cleavage of Rec8 by separin at one of two different sites is necessary for the resolution of chiasmata and the disjunction of homologous chromosomes during meiosis.

Sister chromatid cohesion is crucial for chromosome segregation during mitosis. Loss of cohesion very possibly triggers sister separation at the metaphase φι anaphase transition. This process depends on the destruction of anaphase inhibitory proteins like Pds1p (Cut2p), which is thought to liberate a sister-separating protein Esp1p (Cut1p). By looking for mutants that separate sister centromeres in the presence of Pds1p, this and a previous study have identified six proteins essential for establishing or maintaining sister chromatid cohesion. Four of these proteins, Scc1p, Scc3p, Smc1p, and Smc3p, are subunits of a ‘Cohesin’ complex that binds chromosomes from late G1 until the onset of anaphase. The fifth protein, Scc2p, is not a stoichiometric Cohesin subunit but it is required for Cohesin’s association with chromosomes. The sixth protein, Eco1p(Ctf7p), is not a Cohesin subunit. It is necessary for the establishment of cohesion during DNA replication but not for its maintenance during G2 and M phases.

Background: Cohesion between sister chromatids, which opposes the splitting force exerted by the mitotic spindle during metaphase, is essential for their segregation to opposite poles of the cell during anaphase. In Saccharomyces cerevisiae , cohesion depends on a set of chromosomal proteins called cohesins, which include structural maintenance of chromosomes 1p (Smc1p), Smc3p and sister chromatid cohesion 1p (Scc1p). Strains with mutations in the genes encoding these proteins separate sister chromatids prematurely and fail to align them in metaphase. This leads to missegregation of chromosomes in the following anaphase. Results: In a normal cell cycle, Scc1p was synthesized and recruited to chromosomes at the onset of S phase. Using cells that expressed Scc1p exclusively from a galactose-inducible promoter, we showed that if Scc1p was synthesised only after completion of S phase, it still bound to chromosomes but failed to promote sister chromatid cohesion. Conclusions: Cohesion between sister chromatids must be established during DNA replication, possibly following the passage of a replication fork. Furthermore, Scc1p (and other cohesins) are needed both for maintaining cohesion during mitosis and for establishing it during S phase. Establishment of sister chromatid cohesion is therefore an essential but hitherto neglected aspect of S phase.

Cohesion between sister chromatids, mediated by the chromosomal cohesin complex, is a prerequisite for faithful chromosome segregation in mitosis. Cohesin also has vital roles in DNA repair and transcriptional regulation. The ring-shaped cohesin complex is thought to encircle sister DNA strands, but its molecular mechanism of action is poorly understood and the biochemical reconstitution of cohesin activity in vitro has remained an unattained goal. Here we reconstitute cohesin loading onto DNA using purified fission yeast cohesin and its loader complex, Mis4(Scc2)-Ssl3(Scc4) (Schizosaccharomyces pombe gene names appear throughout with their more commonly known Saccharomyces cerevisiae counterparts added in superscript). Incubation of cohesin with DNA leads to spontaneous topological loading, but this remains inefficient. The loader contacts cohesin at multiple sites around the ring circumference, including the hitherto enigmatic Psc3(Scc3) subunit, and stimulates cohesin's ATPase, resulting in efficient topological loading. The in vitro reconstitution of cohesin loading onto DNA provides mechanistic insight into the initial steps of the establishment of sister chromatid cohesion and other chromosomal processes mediated by cohesin.

Regulated cell growth and proliferation, as well as the execution of carefully controlled cell death, form the basis for the continuation of all life on our planet. Recent decades have seen an explosion of a wealth of genetic information on the molecular components that make up cells and their regulatory circuits. At first sight, it may seem that we understand the principles of cell growth and death. On closer inspection, however, it becomes quickly obvious that our knowledge has just merely begun to scratch the surface of what goes on inside the cell and between cells. Classic genetics in model organism has been extended by high throughput proteomic and phenotypic screens to include human cells. Yet the parts lists provided are just the starting point to begin asking questions. How do the identified molecules work, how do they orchestrate the multitude of reactions that together make cells grow, divide and die, each at the right place and time. Ideas and concepts have emerged from intelligent observations, yet the cell is not the product of intelligent design. As we move forward, our approach must take into account the complex evolved nature of cellular networks that connect cell growth and cell death. Breathtaking advances in many molecular and imaging techniques allow an unprecedented view of important aspects of cell growth and death. Together with a systems level analysis of these observations we hope to increase our understanding of cellular reactions, information processing and decision making. This issue brings together reviews on recent advances on topics of central importance to the processes and regulation of cell growth and death. In preparation for cell division, the cellular content needs to be duplicated. This poses a particularly formidable challenge for the accurate information stored in the genomic DNA. The fluctuating levels of cyclin-dependent kinase (Cdk) activity provide universal control over the cell division cycle, yet how Cdk activity regulates the initiation of chromosomal DNA replication has only recently been revealed. The emerging ideas and their implications are considered by Hiroyuki Araki, `Control of initiation of eukaryotic DNA replication'. Following on from there, Kaitlin Stimpson and Beth Sullivan, `Epigenomics of Centromere Assembly and Function', discuss how a single region on each chromosome is singled out to become the attachment site for the mitotic spindle apparatus that segregates the replicated copies of each chromosome during cell division. This process is made possible by pairwise cohesion between sister chromatids after their synthesis. The ring-shaped cohesin complex is thought to embrace pairs of sister chromatids and Maria Carretero, Silvia Remeseiro and Ana Losada, `Cohesin ties up the genome', describe how these cohesin rings have emerged as versatile organisers of DNA interactions within the chromosome. The body of any metazoan species consists of organs of the right size, containing the correct number of cells. This is an observation that is as obvious as it is puzzling, as Barry Thompson, `Developmental control of cell growth and division in Drosophila', illustrates. Not only will the rate of cell growth impact on cell division, it turns out that the cell cycle status feeds back on the rate and pattern of cell growth, as reported by Alexi Goranov and Angelika Amon, `Growth and Division - not a one-way road'. A multitude of intrinsic and extrinsic parameters are likely to influence cellular behaviour. How information can be processed and robust decisions reached is discussed in examples by Bela Novak, Orsolya Kapuy, Maria Rosa Domingo-Sananes and John Tyson, `Regulated Protein Kinases and Phosphatases in Cell Cycle Decisions'. An important part of biological systems is their continuous optimization and adaptation to changing environments by evolution. Among various drivers of variation that forms the basis of evolution, Norman Pavelka, Giulia Rancati and Rong Li, `Dr. Jekyll and Mr. Hyde: Role of Aneuploidy in Cellular Adaptation and Cancer', set the spotlight on the distinct contribution of whole chromosome number changes. Such changes may not only have contributed to phylogenetic changes in our ancestry, they may also contribute significantly to the evolution of devastating human neoplastic disease. It is in such a disease setting that cell cycle regulators are put into unprecedented context, and the complex but important implications of this are dissected by Vassilis Gorgoulis and Thanos Halazonetis, `Oncogene Induced Senescence: The bright and dark side of the response'. Apoptosis is not the only mechanism of programmed cell death, but it is the best known and most widely investigated. Peter D. Mace and Stefan J. Riedl, `Molecular Cell Death Platforms and Assemblies' describe how scientific interrogation of fundamental apoptotic mechanisms has recently led to the atomic level elucidation of how cellular signals engage different arms of this death response, revealing distinct upstream mechanisms to engage conserved downstream events. Taking advantage of the latent apoptotic potential of cancer cells has led to the development of innovative anti-tumor therapies, as described by Annie Yang, Nicholas S. Wilson and Avi Ashkenazi, `Proapoptotic DR4 and DR5 signaling in cancer cells: toward clinical translation'. Although the role of BCL2 family members in regulating apoptosis has been appreciated for at least 15 years, only recently have scientists been able to define the mechanisms, and Christian Bogner, Brian Leber and David W. Andrews, `Apoptosis: Embedded in membranes' illuminate the interaction of these pro- and anti-apoptotic proteins with each other and with the mitochondrion, a theme that is continued by Stephane G. Rolland and Barbara Conradt, `New role of the BCL2 family of proteins in the regulation of mitochondrial dynamics'. Ultimately, apoptotic death is executed through specific limited proteolysis by caspases, and Francis Impens, Joel Vandekerckhove and Kris Gevaert, `Who gets cut during cell death?' explain how state-of-the-art focused proteomics has revealed the protein victims of proteolytic reprogramming of cells fated to die. In keeping with the theme of this issue, Jeniffer B. Hernandez, Ryan H. Newton and Craig M. Walsh, `Life and death in the thymus – cell death signaling during T cell development' explore the emerging cross-talk between cell proliferation and cell death in education of the adaptive immune system, a mechanism that is also explored by Juanita Lopez and Pascal Meier, `To fight or die - Inhibitor of Apoptosis Proteins at the crossroad of innate immunity and death' in terms of innate immunity. Finally, Kim McCall, `Genetic control of necrosis - another type of programmed cell death' and Hyung Don Ryoo and Eric H. Baehrecke, `Distinct death mechanisms in Drosophila development' reveal how blossoming investigations into non-apoptotic cell death mechanisms, utilizing model organisms, are forcing revisions of how specific organs and tissues deal with their requirements for cell ablation. As we move into the second decade of a new century we face the challenge of understanding how cell growth, proliferation, and death are coordinated. The chapters in this issue are in the hands of individual experts on these forms of cell behavior. But the wave of upcoming scientists is going to have to grapple with the fact that these forms are interconnected, and should probably be considered together, before the next surge of scientific advances can be made to understand how the complex machine that we know as a cell makes its decision to divide or to die.

Cohesion between sister chromatids is mediated by the chromosomal cohesin complex. In budding yeast, cohesin is loaded onto chromosomes during the G1 phase of the cell cycle. During S phase, the replication fork-associated acetyltransferase Eco1 acetylates the cohesin subunit Smc3 to promote the establishment of sister chromatid cohesion. At the time of anaphase, Smc3 loses its acetylation again, but the Smc3 deacetylase and the possible importance of Smc3 deacetylation are unknown. Here, we show that the class I histone deacetylase family member Hos1 is responsible for Smc3 deacetylation. Cohesin is protected from deacetylation while bound to chromosomes but is deacetylated as soon as it dissociates from chromosomes at anaphase onset. Nonacetylated Smc3 is required as a substrate for cohesion establishment in the following cell cycle. Our results complete the description of an Smc3 acetylation cycle and provide unexpected insight into the importance of de novo Smc3 acetylation for cohesion establishment.

In eukaryotic cells, replicated DNA molecules remain physically connected from their synthesis in S phase until they are separated during anaphase. This phenomenon, called sister chromatid cohesion, is essential for the temporal separation of DNA replication and mitosis and for the equal separation of the duplicated genome. Recent work has identified a number of chromosomal proteins required for cohesion. In this review we discuss how these proteins may connect sister chromatids and how they are removed from chromosomes to allow sister chromatid separation at the onset of anaphase.

Replicated chromosomes are held together by the chromosomal cohesin complex from the time of their synthesis in S phase onward. This requires the replication fork–associated acetyl transferase Eco1, but Eco1's mechanism of action is not known. We identified spontaneous suppressors of the thermosensitive eco1-1 allele in budding yeast. An acetylation-mimicking mutation of a conserved lysine in cohesin's Smc3 subunit makes Eco1 dispensable for cell growth, and we show that Smc3 is acetylated in an Eco1-dependent manner during DNA replication to promote sister chromatid cohesion. A second set of eco1-1 suppressors inactivate the budding yeast ortholog of the cohesin destabilizer Wapl. Our results indicate that Eco1 modifies cohesin to stabilize sister chromatid cohesion in parallel with a cohesion establishment reaction that is in principle Eco1-independent.

International audience; Sister chromatid cohesion is mediated by cohesin, but the process of cohesion establishment during S-phase is still enigmatic. In mammalian cells, cohesin binding to chromatin is dynamic in G1, but becomes stabilized during S-phase. Whether the regulation of cohesin stability is integral to the process of cohesion establishment is unknown. Here, we provide evidence that fission yeast cohesin also displays dynamic behavior. Cohesin association with G1 chromosomes requires continued activity of the cohesin loader Mis4/Ssl3, suggesting that repeated loading cycles maintain cohesin binding. Cohesin instability in G1 depends on wpl1, the fission yeast ortholog of mammalian Wapl, suggestive of a conserved mechanism that controls cohesin stability on chromosomes. wpl1 is nonessential, indicating that a change in wpl1-dependent cohesin dynamics is dispensable for cohesion establishment. Instead, we find that cohesin stability increases at the time of S-phase in a reaction that can be uncoupled from DNA replication. Hence, cohesin stabilization might be a pre-requisite for cohesion establishment rather than its consequence.

SMC proteins are key components of large ring-shaped chromosomal protein complexes, such as cohesin and condensin. New evidence supports the idea that these rings topologically encircle DNA. Hints also emerge as to what it may take for DNA to enter the ring.

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 chromatids, the products of eukaryotic DNA replication, are held together by the chromosomal cohesin complex after their synthesis. This allows the spindle in mitosis to recognize pairs of replication products for segregation into opposite directions. Cohesin forms large protein rings that may bind DNA strands by encircling them, but the characterization of cohesin binding to chromosomes in vivo has remained vague. We have performed high resolution analysis of cohesin association along budding yeast chromosomes III-VI. Cohesin localizes almost exclusively between genes that are transcribed in converging directions. We find that active transcription positions cohesin at these sites, not the underlying DNA sequence. Cohesin is initially loaded onto chromosomes at separate places, marked by the Scc2/Scc4 cohesin loading complex, from where it appears to slide to its more permanent locations. But even after sister chromatid cohesion is established, changes in transcription lead to repositioning of cohesin. Thus the sites of cohesin binding and therefore probably sister chromatid cohesion, a key architectural feature of mitotic chromosomes, display surprising flexibility. Cohesin localization to places of convergent transcription is conserved in fission yeast, suggesting that it is a common feature of eukaryotic chromosomes.

Each of our cells inherit their genetic information in the form of chromosomes from a mother cell. In order that we obtain the full genetic complement, cells need to ensure that replicated chromosomes are accurately split and distributed during cell division. Mistakes in this process lead to aneuploidies, cells with supernumerous or missing chromosomes. Most aneuploid human embryos are not viable, and if they are, they develop severe birth defects. Aneuploidies later in human life are frequently found associated with the development of malignant cancer. DNA replication during S-phase is linked to segregation of the sister copies in mitosis by sister chromatid cohesion. A chromosomal protein complex, cohesin, holds replicated sister DNA strands together after their synthesis. This allows pairs of replication products to be recognised by the spindle apparatus in mitosis for segregation into opposite direction. At anaphase onset, cohesin is destroyed by a site-specific protease, separase. Here I review what we have learned about the molecular mechanism of sister chromatid cohesion. Cohesin forms a large proteinaceous ring that may hold sister chromatids by encircling and topological trapping. To understand how cohesin links newly synthesised replication products, biochemical assays to study the enzymology of cohesin will be required.

The final, irreversible step in the duplication and distribution of genomes to daughter cells takes place when chromosomes split at the metaphase-to-anaphase transition. A protease of the CD clan, separase (C50 family), is the key regulator of this transition. During metaphase, cohesion between sister chromatids is maintained by a chromosomal protein complex, cohesin. Anaphase is triggered when separase cleaves the Scc1 subunit of cohesin at two specific recognition sequences. As a result of this cleavage, the cohesin complex is destroyed, allowing the spindle to pull sister chromatids into opposite halves of the cell. Because of the final and irreversible nature of Scc1 cleavage, this reaction is tightly controlled. Several independent mechanisms impose regulation on separase activity, as well as on the susceptibility of the cleavage target Scc1 to cleavage by separase. This chapter provides an overview of these multiple levels of regulation.

Separase is a protease that triggers chromosome segregation at anaphase onset by cleaving cohesin, the chromosomal protein complex responsible for sister chromatid cohesion1,2. After anaphase, cells exit from mitosis; that is, they complete downregulation of cyclin-dependent kinase activity, undergo cytokinesis and enter G1 of the next cell cycle. Here we show that separase activation at the onset of anaphase is sufficient to promote release from the nucleolus and activation of the budding yeast phosphatase, Cdc14, a key step in mitotic exit3,4,5. The ability of separase to activate Cdc14 is independent of its protease function but may involve promoting phosphorylation of the Cdc14 inhibitor Net1. This novel separase function is coregulated with its proteolytic activity by the separase inhibitor securin. This helps to explain the coupling of anaphase and mitotic exit — after securin degradation at anaphase onset, separase cleaves cohesin to trigger chromosome segregation and concurrently uses a non-proteolytic mechanism to initiate mitotic exit.

Rad21L—a new meiotic cohesin described in a recent paper in EMBO reports by the Watanabe group—probably has a role in the establishment of homologous chromosome recognition.

The faithful segregation of the genetic material into daughter cells during cell division is crucial for the production of healthy progeny. Sister chromatid cohesion and separation are fundamental to this process. Progress has been made in our molecular understanding of cohesion and mechanisms for the dissolution of cohesion have been uncovered.