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The Cdc28 protein kinase subunits, Cks1 and Cks2, play dual roles in Cdk-substrate specificity and Cdk-independent protein degradation, in concert with the E3 ubiquitin ligase complexes SCFSkp2 and APCCdc20. Notable targets controlled by Cks include p27 and Cyclin A. Here, we demonstrate that Cks1 and Cks2 proteins interact with both the MllN and MllC subunits of Mll1 (Mixed-lineage leukaemia 1), and together, the Cks proteins define Mll1 levels throughout the cell cycle. Overexpression of CKS1B and CKS2 is observed in multiple human cancers, including various MLL-rearranged (MLLr) AML subtypes. To explore the importance of MLL-Fusion Protein regulation by CKS1/2, we used small molecule inhibitors (MLN4924 and C1) to modulate their protein degradation functions. These inhibitors specifically reduced the proliferation of MLLr cell lines compared to primary controls. Altogether, this study uncovers a novel regulatory pathway for MLL1, which may open a new therapeutic approach to MLLr leukaemia., Highlights • Mll1 is controlled by the Cks1/Cks2 axis. • Opposing effects: Cks1 promotes Mll1 degradation, while Cks2 stabilises Mll1. • Cks1/Cks2 impact on Wnt signalling through Mll1. • CKS1 and CKS2 are overexpressed in MLL-rearranged AML. • Pharmacological inhibition of SCFSKP2-CKS1 is cytotoxic for MLL-rearranged AML.

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.

The condensin complex is a key determinant of mitotic chromosome architecture. In addition, condensin promotes resolution of sister chromatids during anaphase, a function that is conserved from prokaryotes to human. Anaphase bridges observed in cells lacking condensin are reminiscent of chromosome segregation failure after inactivation of topoisomerase II (topo II), the enzyme that removes catenanes persisting between sister chromatids following DNA replication. Circumstantial evidence has linked condensin to sister chromatid decatenation but, because of the difficulty of observing chromosome catenation, this link has remained indirect. Alternative models for how condensin facilitates chromosome resolution have been put forward. Here, we follow the catenation status of circular minichromosomes of three sizes during the Saccharomyeces cerevisiae cell cycle. Catenanes are produced during DNA replication and are for the most part swiftly resolved during and following S-phase, aided by sister chromatid separation. Complete resolution, however, requires the condensin complex, a dependency that becomes more pronounced with increasing chromosome size. Our results provide evidence that condensin prevents deleterious anaphase bridges during chromosome segregation by promoting sister chromatid decatenation.

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.

The processes underlying the large-scale reorganisation of chromatin in mitosis that form compact mitotic chromosomes and ensure the fidelity of chromosome segregation during cell division still remain obscure. The chromosomal condensin complex is a major molecular effector of chromosome condensation and segregation in diverse organisms ranging from bacteria to humans. Condensin is a large, evolutionarily conserved, multisubunit protein assembly composed of dimers of the structural maintenance of chromosomes (SMC) family of ATPases, clasped into topologically closed rings by accessory subunits. Condensin binds to DNA dynamically, in a poorly understood cycle of ATP-modulated conformational changes, and exhibits the ability to positively supercoil DNA. During mitosis, condensin is phosphorylated by the cyclin-dependent kinase (CDK), Polo and Aurora B kinases in a manner that correlates with changes in its localisation, dynamics and supercoiling activity. Here we review the reported architecture, biochemical activities and regulators of condensin. We compare models of bacterial and eukaryotic condensins in order to uncover conserved mechanistic principles of condensin action and to propose a model for mitotic chromosome condensation.

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.

Metaphase of mitosis is brought about in all eukaryotes by activation of cylin-dependent kinase (Cdk1), whereas exit from mitosis requires down-regulation of Cdk1 activity and dephosphorylation of its target proteins. In budding yeast, the completion of mitotic exit requires the release and activation of the Cdc14 protein-phosphatase, which is kept inactive in the nucleolus during most of the cell cycle. Activation of Cdc14 is controlled by two regulatory networks called FEAR (Cdc fourteen early anaphase release) and MEN (mitotic exit network). We have shown recently that the anaphase promoting protease (separase) is essential for Cdc14 activation, thereby it makes mitotic exit dependent on execution of anaphase. Based on this finding, we have proposed a new model for mitotic exit in budding yeast. Here we explain the essence of the model by phaseplane analysis, which reveals two underlying bistable switches in the regulatory network. One bistable switch is caused by mutual activation (positive feedback) between Cdc14 activating MEN and Cdc14 itself. The mitosis-inducing Cdk1 activity inhibits the activation of this positive feedback loop and thereby controlling this switch. The other irreversible switch is generated by a double-negative feedback (mutual antagonism) between mitosis inducing Cdk1 activity and its degradation machinery (APC(Cdh1)). The Cdc14 phosphatase helps turning this switch in favor of APC(Cdh1) side. Both of these bistable switches have characteristic thresholds, the first one for Cdk1 activity, while the second for Cdc14 activity. We show that the physiological behaviors of certain cell cycle mutants are suggestive for those Cdk1 and Cdc14 thresholds. The two bistable switches turn on in a well-defined order. In this paper, we explain how the activation of Cdc20 (which causes the activation of separase and a decrease of Cdk1 kinase activity) provides an initial trigger for the activation of the MEN-Cdc14 positive feedback loops, which in turn, flips the second irreversible Cdk-APC(Cdh1) switch on the APC(Cdh1) side).

The mitotic checkpoint prevents a eukaryotic cell from commencing to separate its replicated genome into two daughter cells (anaphase) until all of its chromosomes are properly aligned on the metaphase plate, with the two copies of each chromosome attached to opposite poles of the mitotic spindle. The mitotic checkpoint is exquisitely sensitive in that a single unaligned chromosome, 1 of a total of ∼50, is sufficient to delay progression into anaphase; however, when the last chromosome comes into alignment on the metaphase plate, the mitotic checkpoint is quickly satisfied, and the replicated chromosomes are rapidly partitioned to opposite poles of the dividing cell. The mitotic checkpoint is also curious in the sense that, before metaphase alignment, chromosomes that are not being pulled in opposite directions by the mitotic spindle activate the checkpoint, but during anaphase, these same tensionless chromosomes can no longer activate the checkpoint. These and other puzzles associated with the mitotic checkpoint are addressed by a proposed molecular mechanism, which involves two positive feedback loops that create a bistable response of the checkpoint to chromosomal tension.

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.

The ease with which populations of the budding yeast Saccharomyces cerevisiae can be synchronized using the mating pheromone α-factor has been invaluable for studies of the cell cycle. The α-factor response pathway has also remained an important model to study the molecular mechanism of G-protein coupled receptor signalling. α-Factor is a 13 amino acids long peptide that is readily available by automated peptide synthesis. However, only cells of the a mating type respond to α-factor. Cells of the opposite α mating type respond to a-factor, a farnesylated and C-terminally methylated 12 amino acids peptide. Because of its more difficult chemical synthesis, a-factor is not readily available and consequently the a-factor response is less well understood. Here we describe an improved strategy for producing a-factor, based on solid-phase peptide synthesis, followed by two simple steps in solution that show favourable characteristics and good yield. We demonstrate the successful use of the resulting a-factor to synchronize cell cycle progression of α cells. Notably, the a-factor concentrations required for cell synchronization are an order of magnitude lower than typically used α-factor concentrations. Despite a similar cell cycle response, shmoo formation was less pronounced compared to α-factor-treated a cells. Our protocol makes a-factor widely accessible, extending the ease of cell cycle synchronization to budding yeast cells of both mating types and facilitating the study of a-factor signalling. Copyright © 2012 John Wiley & Sons, Ltd.

Two closely connected mechanisms safeguard the fidelity of chromosome segregation in eukaryotic cells. The mitotic checkpoint monitors the attachment of kinetochores to microtubules and delays anaphase onset until all sister kinetochores have become attached to opposite poles. In addition, an error correction mechanism destabilizes erroneous attachments that do not lead to tension at sister kinetochores. Aurora B kinase, the catalytic subunit of the CPC (chromosomal passenger complex), acts as a sensor and effector in both pathways. In this review we focus on a poorly understood but important aspect of mitotic control: what prevents the mitotic checkpoint from springing into action when sister centromeres are split and tension is suddenly lost at anaphase onset? Recent work has shown that disjunction of sister chromatids, in principle, engages the mitotic checkpoint, and probably also the error correction mechanism, with potentially catastrophic consequences for cell division. Eukaryotic cells have solved this ‘anaphase problem’ by disabling the mitotic checkpoint at the metaphase-to-anaphase transition. Checkpoint inactivation is in part due to the reversal of Cdk1 (cyclin-dependent kinase 1) phosphorylation of the CPC component INCENP (inner centromere protein; Sli15 in budding yeast), which causes the relocation of the CPC from centromeres to the spindle midzone. These findings highlight principles of mitotic checkpoint control: when bipolar chromosome attachment is reached in mitosis, the checkpoint is satisfied, but still active and responsive to loss of tension. Mitotic checkpoint inactivation at anaphase onset is required to prevent checkpoint re-engagement when sister chromatids split.

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.

Completion of mitotic exit and cytokinesis requires the inactivation of mitotic cyclin-dependent kinase (Cdk) activity. A key enzyme that counteracts Cdk during budding yeast mitotic exit is the Cdc14 phosphatase. Cdc14 is inactive for much of the cell cycle, sequestered by its inhibitor Net1 in the nucleolus. At anaphase onset, separase-dependent down-regulation of PP2ACdc55 allows phosphorylation of Net1 and consequent Cdc14 release. How separase causes PP2ACdc55 down-regulation is not known. Here, we show that two Cdc55-interacting proteins, Zds1 and Zds2, contribute to timely Cdc14 activation during mitotic exit. Zds1 and Zds2 are required downstream of separase to facilitate nucleolar Cdc14 release. Ectopic Zds1 expression in turn is sufficient to down-regulate PP2ACdc55 and promote Net1 phosphorylation. These findings identify Zds1 and Zds2 as new components of the mitotic exit machinery, involved in activation of the Cdc14 phosphatase at anaphase onset. Our results suggest that these proteins may act as separase-regulated PP2ACdc55 inhibitors.

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.

Mitotic chromosomes were one of the first cell biological structures to be described, yet their molecular architecture remains poorly understood. We have devised a simple biophysical model of a 300 kb-long nucleosome chain, the size of a budding yeast chromosome, constrained by interactions between binding sites of the chromosomal condensin complex, a key component of interphase and mitotic chromosomes. Comparisons of computational and experimental (4C) interaction maps, and other biophysical features, allow us to predict a mode of condensin action. Stochastic condensin-mediated pairwise interactions along the nucleosome chain generate native-like chromosome features and recapitulate chromosome compaction and individualization during mitotic condensation. Higher order interactions between condensin binding sites explain the data less well. Our results suggest that basic assumptions about chromatin behavior go a long way to explain chromosome architecture and are able to generate a molecular model of what the inside of a chromosome is likely to look like. DOI: http://dx.doi.org/10.7554/eLife.05565.001, eLife digest The genetic material of living things is made up of long strands of DNA. Human cells contain about two meters of DNA split between 46 chromosomes. These chromosomes carry all the instructions to build a human body. To fit all of this information inside each human cell, the DNA is wrapped around hundreds of thousands of proteins such that the chromosomes each resemble a string of beads. Most of the time the chromosomes in a cell are only loosely arranged. But, when a cell prepares to divide into two new cells, its chromosomes become more compacted. This allows the DNA to withstand the physical forces involved when the copies of the chromosomes are pulled into the two daughter cells, and it makes it easier for the cell to handle its genetic material. If a chromosome breaks during cell division, it can result in diseases such as cancer. Several proteins—collectively called condensins—work to compact (or condense) the chromosomes. These proteins are found in a wide range of species, but it remains poorly understood how they cause chromosomes to become more compact. Due to the technical limitations of current imaging methods, it has not been possible to directly visualize the path of the DNA strand within a compacted chromosome. However, Cheng et al. have now overcome this limitation by combining experimental analyses and computational simulations. Cheng et al. used computer modeling to simulate a piece of chromosome that was about the same size as a chromosome from a single-celled microorganism called budding yeast. This model could accurately recreate the behavior of chromosomes as observed in non-dividing cells—and revealed that these chromosomes are in a relaxed state. Cheng et al. then modeled what happens when condensins are introduced. As expected, the chromosomes became more compacted and the model's behavior was then validated using further experiments. This predicted that condensin complexes, bound to regions along the chromosome's length, interact to form pairs that continually separate and form new pairs with other condensins; and that these ‘dynamic pairwise’ interactions compact the chromosome. The current model describes a relatively small chromosome and, in the future, extending the model to larger chromosomes could shed insight. DOI: http://dx.doi.org/10.7554/eLife.05565.002

SummaryAfter anaphase, the high mitotic cyclin-dependent kinase (Cdk) activity is downregulated to promote exit from mitosis. To this end, in the budding yeast S. cerevisiae, the Cdk counteracting phosphatase Cdc14 is activated. In metaphase, Cdc14 is kept inactive in the nucleolus by its inhibitor Net1. During anaphase, Cdk- and Polo-dependent phosphorylation of Net1 is thought to release active Cdc14. How Net1 is phosphorylated specifically in anaphase, when mitotic kinase activity starts to decline, has remained unexplained. Here, we show that PP2ACdc55 phosphatase keeps Net1 underphosphorylated in metaphase. The sister chromatid-separating protease separase, activated at anaphase onset, interacts with and downregulates PP2ACdc55, thereby facilitating Cdk-dependent Net1 phosphorylation. PP2ACdc55 downregulation also promotes phosphorylation of Bfa1, contributing to activation of the “mitotic exit network” that sustains Cdc14 as Cdk activity declines. These findings allow us to present a new quantitative model for mitotic exit in budding yeast.

The final event of the eukaryotic cell cycle is cytokinesis, when two new daughter cells are born. How the timing and execution of cytokinesis is controlled is poorly understood. Here, we show that downregulation of cyclin-dependent kinase (Cdk) activity, together with upregulation of its counteracting phosphatase Cdc14, controls each of the sequential steps of cytokinesis, including furrow ingression, membrane resolution and cell separation in budding yeast. We use phosphoproteome analysis of mitotic exit to identify Cdk targets that are dephosphorylated at the time of cytokinesis. We then apply a new and widely applicable tool to generate conditionally phosphorylated proteins to identify those whose dephosphorylation is required for cytokinesis. This approach identifies Aip1, Ede1 and Inn1 as cytokinetic regulators. Our results suggest that cytokinesis is coordinately controlled by the master cell cycle regulator Cdk together with its counteracting phosphatase and that it is executed by concerted dephosphorylation of Cdk targets involved in several cell biological processes.

Microtubules of the mitotic spindle form the structural basis for chromosome segregation. In metaphase, microtubules show high dynamic instability, which is thought to aid the 'search and capture' of chromosomes for bipolar alignment on the spindle. Microtubules suddenly become more stable at the onset of anaphase, but how this change in microtubule behaviour is regulated and how important it is for the ensuing chromosome segregation are unknown. Here we show that in the budding yeast Saccharomyces cerevisiae, activation of the phosphatase Cdc14 at anaphase onset is both necessary and sufficient for silencing microtubule dynamics. Cdc14 is activated by separase, the protease that triggers sister chromatid separation, linking the onset of anaphase to microtubule stabilization. If sister chromatids separate in the absence of Cdc14 activity, microtubules maintain high dynamic instability; this correlates with defects in both the movement of chromosomes to the spindle poles (anaphase A) and the elongation of the anaphase spindle (anaphase B). Cdc14 promotes localization of microtubule-stabilizing proteins to the anaphase spindle, and dephosphorylation of the kinetochore component Ask1 contributes to both the silencing of microtubule turnover and successful anaphase A.

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.

Sister chromatid cohesion is resolved at anaphase onset when separase, a site-specific protease, cleaves the Scc1 subunit of the chromosomal cohesin complex that is responsible for holding sister chromatids together. This mechanism to initiate anaphase is conserved in eukaryotes from budding yeast to man. Budding yeast separase recognizes and cleaves two conserved peptide motifs within Scc1. In addition, separase cleaves a similar motif in the kinetochore and spindle protein Slk19. Separase may cleave further substrate proteins to orchestrate multiple cellular events that take place during anaphase. To investigate substrate recognition by budding yeast separase we analyzed the sequence requirements at one of the Scc1 cleavage site motifs by systematic mutagenesis. We derived a cleavage site consensus motif (not(FKRWY))(ACFHILMPVWY)(DE)X(AGSV)R/X. This motif is found in 1,139 of 5,889 predicted yeast proteins. We analyzed 28 candidate proteins containing this motif as well as 35 proteins that contain a core (DE)XXR motif. We could so far not confirm new separase substrates, but we have uncovered other forms of mitotic regulation of some of the proteins. We studied whether determinants other than the cleavage site motif mediate separase-substrate interaction. When the separase active site was occupied with a peptide inhibitor covering the cleavage site motif, separase still efficiently interacted with its substrate Scc1. This suggests that separase recognizes both a cleavage site consensus sequence as well as features outside the cleavage site.

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.

Background: Sister chromatid separation and segregation at anaphase onset are triggered by cleavage of the chromosomal cohesin complex by the protease separase. Separase is regulated by its binding partner securin in two ways: securin is required to support separase activity in anaphase; and, at the same time, securin must be destroyed via ubiquitylation before separase becomes active. The molecular mechanisms underlying this dual regulation of separase by securin are unknown. Results: We show that, in budding yeast, securin supports separase localization. Separase enters the nucleus independently of securin, but securin is required and sufficient to cause accumulation of separase in the nucleus, where its known cleavage targets reside. Securin also ensures that separase gains full proteolytic activity in anaphase. We also show that securin, while present, directly inhibits the proteolytic activity of separase. Securin prevents the binding of separase to its substrates. It also hinders the separase N terminus from interacting with and possibly inducing an activating conformational change at the protease active site 150 kDa downstream at the protein's C terminus. Conclusions: Securin inhibits the proteolytic activity of separase in a 2-fold manner. While inhibiting separase, securin is able to promote nuclear accumulation of separase and help separase to become fully activated after securin's own destruction at anaphase onset.

In the quantitative model of cell-cycle control, progression from G1 through S phase and into mitosis is ordered by thresholds of increasing cyclin-dependent kinase (Cdk) activity. How such thresholds are read out by substrates that respond with the correct phosphorylation timing is not known. Here, using the budding yeast model, we show that the abundant PP2ACdc55 phosphatase counteracts Cdk phosphorylation during interphase and delays phosphorylation of late Cdk substrates. PP2ACdc55 specifically counteracts phosphorylation on threonine residues, and consequently, we find that threonine-directed phosphorylation occurs late in the cell cycle. Furthermore, the late phosphorylation of a model substrate, Ndd1, depends on threonine identity of its Cdk target sites. Our results support a model in which Cdk-counteracting phosphatases contribute to cell-cycle ordering by imposing Cdk thresholds. They also unveil a regulatory principle based on the phosphoacceptor amino acid, which is likely to apply to signaling pathways beyond cell-cycle control.

Substrate dephosphorylation by the cyclin-dependent kinase (Cdk)-opposing phosphatase, Cdc14, is vital for many events during budding yeast mitotic exit. Cdc14 is sequestered in the nucleolus through inhibitory binding to Net1, from which it is released in anaphase following Net1 phosphorylation. Initial Net1 phosphorylation depends on Cdk itself, in conjunction with proteins of the Cdc14 Early Anaphase Release (FEAR) network. Later on, the Mitotic Exit Network (MEN) signaling cascade maintains Cdc14 release. An important unresolved question is how Cdc14 activity can increase in early anaphase, while Cdk activity, that is required for Net1 phosphorylation, decreases and the MEN is not yet active. Here we show that the nuclear rim protein Nur1 interacts with Net1 and, in its Cdk phosphorylated form, inhibits Cdc14 release. Nur1 is dephosphorylated by Cdc14 in early anaphase, relieving the inhibition and promoting further Cdc14 release. Nur1 dephosphorylation thus describes a positive feedback loop in Cdc14 phosphatase activation during mitotic exit, required for faithful chromosome segregation and completion of the cell division cycle., Author Summary During the cell cycle, a specific sequence of events leads to the formation of two daughter cells from one mother cell. Progression through the cell cycle is tightly controlled, with events occurring in the right place at the right time. Exactly how this is achieved is still being elucidated. In budding yeast, the events occurring during the final cell cycle phase – “mitotic exit” – are controlled by the phosphatase Cdc14. It is kept sequestered and inactive until it is needed for mitotic exit, at which time it is rapidly released. In this study, we have identified a new regulator of Cdc14 activity, the protein Nur1. In a series of experiments, we saw that Nur1 acts both upstream and downstream of Cdc14 activation, thereby creating a positive feedback loop. On the one hand, Nur1 contributes to inhibiting Cdc14 until the start of mitotic exit. On the other hand, through the actions of Cdc14 itself, Nur1 is disabled as an opponent of the phosphatase. This creates a robust system, rapidly switching between two opposing states and thus driving forward the mitotic exit transition.

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.

The packaging of centimetre long DNA molecules into compact metaphase chromosomes is essential for genome segregation in anaphase. The chromosomal condensin complex plays a crucial part in this packaging, and important new insight into condensin action in vitro and in vivo has recently been gained.

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.

Replication Factor C (RFC) is a five-subunit protein complex required for eukaryotic DNA replication and repair. The large subunit within this complex contains a C-terminal DNA binding domain which provides specificity for PCNA loading at a primer-template and a second, N-terminal DNA binding domain of unknown function. We isolated the N-terminal DNA binding domain from Drosophila melanogaster and defined the region within this polypeptide required for DNA binding. The DNA determinants most efficiently recognized by both the Drosophila minimal DNA binding domain and the N-terminal half of the human large subunit consist of a double-stranded DNA containing a recessed 5' phosphate. DNA containing a recessed 5' phosphate was preferred 5-fold over hairpined DNA containing a recessed 3' hydroxyl. Combined with existing data, these DNA binding properties suggest a role for the N-terminal DNA binding domain in the recognition of phosphorylated DNA ends.

Using rapid amplification of cDNA ends (RACE), we have cloned the 5'-untranslated region (5'-UTR) of the N-methyl-D-aspartate receptor subunit epsilon(2) from murine forebrain-derived mRNA. We identified two distinct types of cDNA species differing in the presence or absence of one exon sequence. Sequencing of the 5'-non-coding region of the epsilon(2) gene revealed that the epsilon(2) 5'-UTR consists of three untranslated exons located at least 20 kb upstream of exon 4 that contains the ATG codon for initiation of translation. This genomic organization shows a close similarity to the epsilon(3) gene. The transcriptional start site was determined by primer extension assays. Expression of the alternative exon sequence was shown by in situ hybridization in the murine brain. Basal transcriptional activity of the epsilon(2) promoter was detected in different neuronal and non-neuronal cell lines with transient reporter gene expression assays. Potential SP1 and CREB binding sites were found in the promoter region. Specific binding of these transcription factors was demonstrated in electrophoretic mobility shift assays. (C) 1998 Elsevier Science B.V.

Caspase-3 is an ICE-like protease activated during apoptosis induced by different stimuli. Poly(ADP-ribose) polymerase (PARP), the first characterized substrate of caspase-3, shares a region of homology with the large subunit of Replication Factor C (RF-C), a five-subunit complex that is part of the processive eukaryotic DNA polymerase holoenzymes. Caspase-3 cleaves PARP at a DEVD-G motif present in the 140 kDa subunit of RF-C (RFC140) and evolutionarily conserved. We show that cleavage of RFC140 during Fas-mediated apoptosis in Jurkat cells and lymphocytes results in generation of multiple fragments. Cleavage is inhibited by the caspase-3-like protease inhibitor Ac-DEVD-CHO but not the caspase-1/ICE-type protease inhibitor Ac-YVAD-CHO. In addition, recombinant caspase-3 cleaves RFC140 in vitro at least at three different sites in the C-terminal half of the protein. Using amino-terminal microsequencing of radioactive fragments, we identified three sites: DEVD723G, DLVD922S and IETD1117A. We did not detect cleavage of small subunits of RF-C of 36, 37, 38 and 40 kDa by recombinant caspase-3 or by apoptotic Jurkat cell lysates. Cleavage of RFC140 during apoptosis inactivates its function in DNA replication and generates truncated forms that further inhibit DNA replication. These results identify RFC140 as a critical target for caspase-3-like proteases and suggest that caspases could mediate cell cycle arrest.

Anaphase onset is triggered when the protease separase cleaves the cohesive bond that holds replicated sister chromatids together until metaphase. In budding yeast, separase then also regulates mitotic exit. New findings suggest that in vertebrates, separase also participates in cell-cycle regulation, albeit in an unexpected manner.