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Underlying higher order chromatin organization are Structural Maintenance of Chromosomes (SMC) complexes, large protein rings that entrap DNA. The molecular mechanism by which SMC complexes organize chromatin is as yet incompletely understood. Two prominent models posit that SMC complexes actively extrude DNA loops (loop extrusion), or that they sequentially entrap two DNAs that come into proximity by Brownian motion (diffusion capture). To explore the implications of these two mechanisms, we perform biophysical simulations of a 3.76 Mb-long chromatin chain, the size of the long Schizosaccharomyces pombe chromosome I left arm. On it, the SMC complex condensin is modeled to perform loop extrusion or diffusion capture. We then compare computational to experimental observations of mitotic chromosome formation. Both loop extrusion and diffusion capture can result in native-like contact probability distributions. In addition, the diffusion capture model more readily recapitulates mitotic chromosome axis shortening and chromatin compaction. Diffusion capture can also explain why mitotic chromatin shows reduced, as well as more anisotropic, movements, features that lack support from loop extrusion. The condensin distribution within mitotic chromosomes, visualized by stochastic optical reconstruction microscopy (STORM), shows clustering predicted from diffusion capture. Our results inform the evaluation of current models of mitotic chromosome formation.

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.

Two models have been put forward for cyclin-dependent kinase (Cdk) control of the cell cycle. In the qualitative model, cell cycle events are ordered by distinct substrate specificities of successive cyclin waves. Alternatively, in the quantitative model, the gradual rise of Cdk activity from G1 phase to mitosis leads to ordered substrate phosphorylation at sequential thresholds. Here, we study the relative contributions of qualitative and quantitative Cdk control in Saccharomyces cerevisiae All S phase and mitotic cyclins can be replaced by a single mitotic cyclin, albeit at the cost of reduced fitness. A single cyclin can also replace all G1 cyclins to support ordered cell cycle progression, fulfilling key predictions of the quantitative model. However, single-cyclin cells fail to polarize or grow buds and thus cannot survive. Our results suggest that budding yeast has become dependent on G1 cyclin specificity to couple cell cycle progression to essential morphogenetic events.

Chromatin is a very long DNA–protein complex that controls the expression and inheritance of the genetic information. Chromatin is stored within the nucleus in interphase and further compacted into chromosomes during mitosis. This process, known as chromosome condensation, is essential for faithful segregation of genomic DNA into daughter cells. Condensin and cohesin, members of the structural maintenance of chromosomes (SMC) family, are fundamental for chromosome architecture, both for establishment of chromatin structure in the interphase nucleus and for the formation of condensed chromosomes in mitosis. These ring-shaped SMC complexes are thought to regulate the interactions between DNA strands by topologically entrapping DNA. How this activity shapes chromosomes is not yet understood. Recent high throughput chromosome conformation capture studies revealed how chromatin is reorganized during the cell cycle and have started to explore the role of SMC complexes in mitotic chromatin architecture. Here, we summarize these findings and discuss the conserved nature of chromosome condensation in eukaryotes. We highlight the unexpected finding that condensin-dependent intra-chromosomal interactions in mitosis increase within a distinctive distance range that is characteristic for an organism, while longer and shorter-range interactions are suppressed. This reveals important molecular insight into chromosome architecture.

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.

The cell cycle is an ordered series of events by which cells grow and divide to give rise to two daughter cells. In eukaryotes, cyclin–cyclin-dependent kinase (cyclin–Cdk) complexes act as master regulators of the cell division cycle by phosphorylating numerous substrates. Their activity and expression profiles are regulated in time. The budding yeast S. cerevisiae was one of the pioneering model organisms to study the cell cycle. Its genetic amenability continues to make it a favorite model to decipher the principles of how changes in cyclin-Cdk activity translate into the intricate sequence of substrate phosphorylation events that govern the cell cycle. In this chapter, we introduce robust and straightforward methods to analyze cell cycle progression in S. cerevisiae. These techniques can be utilized to describe cell cycle events and to address the effects of perturbations on accurate and timely cell cycle progression.

Underlying higher order chromatin organization are Structural Maintenance of Chromosomes (SMC) complexes, large protein rings that entrap DNA. The molecular mechanism by which SMC complexes organize chromatin is as yet incompletely understood. Two prominent models posit that SMC complexes actively extrude DNA loops (loop extrusion), or that they sequentially entrap two DNAs that come into proximity by Brownian motion (diffusion capture). To explore the implications of these two mechanisms, we perform biophysical simulations of a 3.76 Mb-long chromatin chain, the size of the longS. pombechromosome I left arm. On it, the SMC complex condensin is modeled to perform loop extrusion or diffusion capture. We then compare computational to experimental observations of mitotic chromosome formation. Both loop extrusion and diffusion capture can result in native-like contact probability distributions. In addition, the diffusion capture model more readily recapitulates mitotic chromosome axis shortening and chromatin density enrichment. Diffusion capture can also explain why mitotic chromatin shows reduced, as well as more anisotropic, movements, features that lack support from loop extrusion. The condensin distribution within mitotic chromosomes, visualized by stochastic optical reconstruction microscopy (STORM), shows clustering predicted from diffusion capture. Our results inform the evaluation of current models of mitotic chromosome formation.

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.

Summary Amine-reactive Tandem Mass Tag 10plex (TMT10plex) labeling permits multiplexed protein identification and quantitative analysis by tandem mass spectrometry (MS/MS). We have used this technology to label 20 Saccharomyces cerevisiae samples collected in a time-resolved manner from a wild-type and phosphatase mutant background to characterize phosphoproteome dynamics. Here, we provide a detailed protocol for biological and mass spectrometry sample preparation and analysis. For complete details on the use and execution of this protocol, please refer to Touati et al. (2019)., Graphical Abstract, Highlights • Characterization of the phosphoproteome in a time-resolved manner • TMT10plex Mass Tag labelling of budding yeast extracts • Mass spectrometry sample preparation and analysis, Amine-reactive Tandem Mass Tag 10plex (TMT10plex) labeling permits multiplexed protein identification and quantitative analysis by tandem mass spectrometry (MS/MS).We have used this technology to label 20 Saccharomyces cerevisiae samples collected in a time-resolved manner from a wild-type and phosphatase mutant background to characterize phosphoproteome dynamics. Here, we provide a detailed protocol for biological andmass spectrometry sample preparation and analysis.

Background Structural maintenance of chromosomes (SMC) complexes are central organizers of chromatin architecture throughout the cell cycle. The SMC family member condensin is best known for establishing long-range chromatin interactions in mitosis. These compact chromatin and create mechanically stable chromosomes. How condensin contributes to chromatin organization in interphase is less well understood. Results Here, we use efficient conditional depletion of fission yeast condensin to determine its contribution to interphase chromatin organization. We deplete condensin in G2-arrested cells to preempt confounding effects from cell cycle progression without condensin. Genome-wide chromatin interaction mapping, using Hi-C, reveals condensin-mediated chromatin interactions in interphase that are qualitatively similar to those observed in mitosis, but quantitatively far less prevalent. Despite their low abundance, chromatin mobility tracking shows that condensin markedly confines interphase chromatin movements. Without condensin, chromatin behaves as an unconstrained Rouse polymer with excluded volume, while condensin constrains its mobility. Unexpectedly, we find that condensin is required during interphase to prevent ongoing transcription from eliciting a DNA damage response. Conclusions In addition to establishing mitotic chromosome architecture, condensin-mediated long-range chromatin interactions contribute to shaping chromatin organization in interphase. The resulting structure confines chromatin mobility and protects the genome from transcription-induced DNA damage. This adds to the important roles of condensin in maintaining chromosome stability.

The cell cycle is the process by which a cell duplicates its DNA during S-phase and divides its chromosomes during M-phase, creating two genetically identical daughter cells. Cell cycle events are ordered by synthesis and degradation of key cell regulators and by phosphorylation and dephosphorylation of numerous substrates. Phosphorylation can alter the activity, interactions or subcellular localization of a protein. A substrate's phosphorylation status is the readout of competing activities of kinases and phosphatases that target each of its phosphorylation sites. In our recent study (EMBO J. 37, e98745), we performed time-resolved global phosphoproteome analysis of a period during the cell cycle known as mitotic exit. During this time, numerous cell biological events happen in fast succession but in strict order. First, at the metaphase to anaphase transition, the mitotic spindle elongates to pull maximally condensed chromosomes to opposite cell halves. Shortly after that, spindles disassemble and chromosomes decondense, before finally cell division is completed by cytokinesis. Our time-resolved phosphoproteome analysis of this period in budding yeast provided a survey of the principles of phosphoregulation used to order these events.

Summary Eukaryotic cells inherit their genomes in the form of chromosomes, which are formed from the compaction of interphase chromatin by the condensin complex. Condensin is a member of the structural maintenance of chromosomes (SMC) family of ATPases, large ring-shaped protein assemblies that entrap DNA to establish chromosomal interactions. Here, we use the budding yeast Saccharomyces cerevisiae to dissect the role of the condensin ATPase and its relationship with cell-cycle-regulated chromosome binding dynamics. ATP hydrolysis-deficient condensin binds to chromosomes but is defective in chromosome condensation and segregation. By modulating the ATPase, we demonstrate that it controls condensin’s dynamic turnover on chromosomes. Mitosis-specific phosphorylation of condensin’s Smc4 subunit reduces the turnover rate. However, reducing turnover by itself is insufficient to compact chromosomes. We propose that condensation requires fine-tuned dynamic condensin interactions with more than one DNA. These results enhance our molecular understanding of condensin function during chromosome condensation., Graphical Abstract, Highlights • The budding yeast condensin ATPase promotes chromosome condensation • The ATPase controls the dynamic chromosome binding cycle of the complex • Smc4 phosphorylation by cyclin-dependent kinase adjusts the turnover • Chromosome condensation requires fine-tuning of condensin behavior, The condensin complex is a key determinant of mitotic chromosome formation. Thadani et al. study the dynamic binding of condensin to chromosomes. They reveal how condensin turnover is regulated by its ATPase and by cell-cycle phosphorylation. Chromosome condensation in mitosis requires fine-tuning of this dynamic behavior.

Chl1 is a member of the XPD family of 5’-3’ DNA helicases, which perform a variety of roles in genome maintenance and transmission. They possess a variety of unique structural features, including the presence of a highly variable, partially-ordered insertion in the helicase domain 1. Chl1 has been shown to be required for chromosome segregation in yeast due to its role in the formation of persistent chromosome cohesion during S-phase. Here we present structural and biochemical data to show that Chl1 has the same overall domain organisation as other members of the XPD family, but with some conformational alterations. We also present data suggesting the insert domain in Chl1 regulates its DNA binding.

Replication factor C complexes load and unload processivity clamps from DNA and are involved in multiple DNA replication and repair pathways. The RFCC tf18 variant complex is required for activation of the intra‐S‐phase checkpoint at stalled replication forks and aids the establishment of sister chromatid cohesion. Unlike other RFC complexes, RFCC tf18 contains two non‐Rfc subunits, Dcc1 and Ctf8. Here, we present the crystal structure of the Dcc1‐Ctf8 heterodimer bound to the C‐terminus of Ctf18. We find that the C‐terminus of Dcc1 contains three‐winged helix domains, which bind to both ssDNA and dsDNA. We further show that these domains are required for full recruitment of the complex to chromatin, and correct activation of the replication checkpoint. These findings provide the first structural data on a eukaryotic seven‐subunit clamp loader and define a new biochemical activity for Dcc1.

Structural maintenance of chromosomes (SMC) complexes play pivotal roles in controlling chromatin organization. Condensin is an essential SMC complex that compacts chromatin to form condensed chromosomes in mitosis. Complete condensin inactivation is necessary to reveal how condensin converts interphase chromatin into mitotic chromosomes. Here, we have developed a condensin depletion system in fission yeast that combines transcriptional repression with auxin-inducible protein degradation. This achieves efficient condensin depletion without need for a temperature shift. Our system is useful when studying how condensin contributes to chromosome architecture and is applicable to the study of other SMC complexes.

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 Temporal control over protein phosphorylation and dephosphorylation is crucial for accurate chromosome segregation and for completion of the cell division cycle during exit from mitosis. In budding yeast, the Cdc14 phosphatase is thought to be a major regulator at this time, while in higher eukaryotes PP2A phosphatases take a dominant role. Here, we use time-resolved phosphoproteome analysis in budding yeast to evaluate the respective contributions of Cdc14, PP2ACdc55, and PP2ARts1. This reveals an overlapping requirement for all three phosphatases during mitotic progression. Our time-resolved phosphoproteome resource reveals how Cdc14 instructs the sequential pattern of phosphorylation changes, in part through preferential recognition of serine-based cyclin-dependent kinase (Cdk) substrates. PP2ACdc55 and PP2ARts1 in turn exhibit a broad substrate spectrum with some selectivity for phosphothreonines and a role for PP2ARts1 in sustaining Aurora kinase activity. These results illustrate synergy and coordination between phosphatases as they orchestrate phosphoproteome dynamics during mitotic progression., Graphical Abstract, Highlights • Cdc14, PP2ACdc55, and PP2ARts1 phosphatases cooperate during budding yeast mitosis • Cdc14 targets serine Cdk motifs for rapid dephosphorylation • PP2ACdc55 dephosphorylates Cdk and Plk substrates on threonine residues • PP2ARts1 displays regulatory crosstalk with Aurora kinase, By using time-resolved phosphoproteome analysis, Touati et al. illuminate the regulatory network that controls temporal order during chromosome segregation and cell division. The work highlights how three different phosphatases cooperate to shape phosphoproteome dynamics at this time, but each also makes unique contributions.

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.

SummaryThe compaction of diffuse interphase chromatin into stable mitotic chromosomes enables the segregation of replicated DNA to daughter cells. Two new studies characterise, both in vivo and in vitro, the essential contribution of the vertebrate condensin complex to chromosome organisation.

DNA replication of circular genomes generates physically interlinked or catenated sister DNAs. These are resolved through transient DNA fracture by type II topoisomerases to permit chromosome segregation during cell division. Topoisomerase II is similarly required for linear chromosome segregation, suggesting that linear chromosomes also remain intertwined following DNA replication. Indeed, chromosome resolution defects are a frequent cause of chromosome segregation failure and consequent aneuploidies. When and where intertwines arise and persist along linear chromosomes are not known, owing to the difficulty of demonstrating intertwining of linear DNAs. Here, we used excision of chromosomal regions as circular “loop outs” to convert sister chromatid intertwines into catenated circles. This revealed intertwining at replication termination and cohesin-binding sites, where intertwines are thought to arise and persist but not to a greater extent than elsewhere in the genome. Intertwining appears to spread evenly along chromosomes but is excluded from heterochromatin. We found that intertwines arise before replication termination, suggesting that replication forks rotate during replication elongation to dissipate torsion ahead of the forks. Our approach provides previously inaccessible insight into the topology of eukaryotic chromosomes and illuminates a process critical for successful chromosome segregation.

Imagine stowing all 401 km of the London Underground rail network into a suitcase. That's equivalent to cramming four meters of DNA that harbor the genetic blueprint of our lives into a micrometer-sized human cell nucleus. During cell division, this enormous amount of DNA is packed into neatly shaped and structurally stable chromosomes, which allows the faithful segregation of genetic material to daughter cells. This packaging task relies on the work of sophisticated molecular machines ( 1 ). On page 1284 of this issue, Shintomi et al. ( 2 ) report that chromosomes can be assembled based on condensin-containing axes, challenging the long-held view that nucleosomes are essential to chromosome architecture.

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.

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.