The cohesin ring concatenates sister DNA molecules

Sister chromatid cohesion, which is essential for mitosis, is mediated by a multi-subunit protein complex called cohesin whose Scc1, Smc1, and Smc3 subunits form a tripartite ring structure. It has been proposed that cohesin holds sister DNAs together by trapping them inside its ring. To test this, we used site-specific cross-linking to create chemical connections at the three interfaces between the ring’s three constituent polypeptides, thereby creating covalently closed cohesin rings. As predicted by the ring entrapment model, this procedure produces dimeric DNA/cohesin structures that are resistant to protein denaturation. We conclude that cohesin rings concatenate individual sister minichromosome DNAs

A quantitative model for cyclin-dependent kinase control of the cell cycle: revisited

The eukaryotic cell division cycle encompasses an ordered series of events. Chromosomal DNA is replicated during S phase of the cell cycle before being distributed to daughter cells in mitosis. Both S phase and mitosis in turn consist of an intricately ordered sequence of molecular events. How cell cycle ordering is achieved, to promote healthy cell proliferation and avert insults on genomic integrity, has been a theme of Paul Nurse's research. To explain a key aspect of cell cycle ordering, sequential S phase and mitosis, Stern & Nurse proposed ‘A quantitative model for cdc2 control of S phase and mitosis in fission yeast’. In this model, S phase and mitosis are ordered by their dependence on increasing levels of cyclin-dependent kinase (Cdk) activity. Alternative mechanisms for ordering have been proposed that rely on checkpoint controls or on sequential waves of cyclins with distinct substrate specificities. Here, we review these ideas in the light of experimental evidence that has meanwhile accumulated. Quantitative Cdk control emerges as the basis for cell cycle ordering, fine-tuned by cyclin specificity and checkpoints. We propose a molecular explanation for quantitative Cdk control, based on thresholds imposed by Cdk-counteracting phosphatases, and discuss its implications

Ipl1/aurora kinase suppresses S-CDK-driven spindle formation during prophase I to ensure chromosome integrity during meiosis

Cells coordinate spindle formation with DNA repair and morphological modifications to chromosomes prior to their segregation to prevent cell division with damaged chromosomes. Here we uncover a novel and unexpected role for Aurora kinase in preventing the formation of spindles by Clb5-CDK (S-CDK) during meiotic prophase I and when the DDR is active in budding yeast. This is critical since S-CDK is essential for replication during premeiotic S-phase as well as double-strand break induction that facilitates meiotic recombination and, ultimately, chromosome segregation. Furthermore, we find that depletion of Cdc5 polo kinase activity delays spindle formation in DDR-arrested cells and that ectopic expression of Cdc5 in prophase I enhances spindle formation, when Ipl1 is depleted. Our findings establish a new paradigm for Aurora kinase function in both negative and positive regulation of spindle dynamics

DNA replication stress restricts ribosomal DNA copy number

Ribosomal RNAs (rRNAs) in budding yeast are encoded by ~100–200 repeats of a 9.1kb sequence arranged in tandem on chromosome XII, the ribosomal DNA (rDNA) locus. Copy number of rDNA repeat units in eukaryotic cells is maintained far in excess of the requirement for ribosome biogenesis. Despite the importance of the repeats for both ribosomal and non-ribosomal functions, it is currently not known how “normal” copy number is determined or maintained. To identify essential genes involved in the maintenance of rDNA copy number, we developed a droplet digital PCR based assay to measure rDNA copy number in yeast and used it to screen a yeast conditional temperature-sensitive mutant collection of essential genes. Our screen revealed that low rDNA copy number is associated with compromised DNA replication. Further, subculturing yeast under two separate conditions of DNA replication stress selected for a contraction of the rDNA array independent of the replication fork blocking protein, Fob1. Interestingly, cells with a contracted array grew better than their counterparts with normal copy number under conditions of DNA replication stress. Our data indicate that DNA replication stresses select for a smaller rDNA array. We speculate that this liberates scarce replication factors for use by the rest of the genome, which in turn helps cells complete DNA replication and continue to propagate. Interestingly, tumors from mini chromosome maintenance 2 (MCM2)-deficient mice also show a loss of rDNA repeats. Our data suggest that a reduction in rDNA copy number may indicate a history of DNA replication stress, and that rDNA array size could serve as a diagnostic marker for replication stress. Taken together, these data begin to suggest the selective pressures that combine to yield a “normal” rDNA copy number

Principles of meiotic chromosome assembly revealed in S. cerevisiae

During meiotic prophase, chromosomes organise into a series of chromatin loops emanating from a proteinaceous axis, but the mechanisms of assembly remain unclear. Here we use Saccharomyces cerevisiae to explore how this elaborate three-dimensional chromosome organisation is linked to genomic sequence. As cells enter meiosis, we observe that strong cohesin-dependent grid-like Hi-C interaction patterns emerge, reminiscent of mammalian interphase organisation, but with distinct regulation. Meiotic patterns agree with simulations of loop extrusion with growth limited by barriers, in which a heterogeneous population of expanding loops develop along the chromosome. Importantly, CTCF, the factor that imposes similar features in mammalian interphase, is absent in S. cerevisiae, suggesting alternative mechanisms of barrier formation. While grid-like interactions emerge independently of meiotic chromosome synapsis, synapsis itself generates additional compaction that matures differentially according to telomere proximity and chromosome size. Collectively, our results elucidate fundamental principles of chromosome assembly and demonstrate the essential role of cohesin within this evolutionarily conserved process

Cohesin Releases DNA through Asymmetric ATPase-Driven Ring Opening

Cohesin stably holds together the sister chromatids from S phase until mitosis. To do so, cohesin must be protected against its cellular antagonist Wapl. Eco1 acetylates cohesin's Smc3 subunit, which locks together the sister DNAs. We used yeast genetics to dissect how Wapl drives cohesin from chromatin and identified mutants of cohesin that are impaired in ATPase activity but remarkably confer robust cohesion that bypasses the need for the cohesin protectors Eco1 in yeast and Sororin in human cells. We uncover a functional asymmetry within the heart of cohesin's highly conserved ABC-like ATPase machinery and find that both ATPase sites contribute to DNA loading, whereas DNA release is controlled specifically by one site. We propose that Smc3 acetylation locks cohesin rings around the sister chromatids by counteracting an activity associated with one of cohesin's two ATPase sites. Tight regulation of DNA entrapment and release by the cohesin complex is crucial for its multiple cellular functions. Elbatsh et al. find that cohesin's release from DNA requires an activity associated with one of its ATPase sites, whereas both sites control cohesin's loading onto DNA

Rapid induction of p21WAF1 but delayed down-regulation of Cdc25A in the TGF-β-induced cell cycle arrest of gastric carcinoma cells

Transforming growth factor-beta (TGF-beta) is a multifunctional polypeptide that inhibits cellular proliferation in most epithelial cells. cdk4 and several cyclin-dependent kinase (cdk) inhibitors (p15(INK4B), p21(WAFI/Cip1) and p27(Kip1)) have been implicated in the TGF-beta-induced cell cycle arrest. More recently, down-regulation of Cdc25A, a cdk activator, was additionally suggested as a mechanism underlying growth inhibition by TGF-beta. The existence of diverse cellular mediators, of TGF-beta, however, raises the question of whether their involvement might occur in a redundant manner or coordinately in a certain cell type. Using two TGF-beta-sensitive gastric carcinoma cell lines (SNU-16 and -620), we addressed the contributory roles of several cdk inhibitors, and of cdk4 and Cdc25A, in TGF-beta-induced cell cycle arrest by comparing their temporal expression pattern in response to TGF-beta. Among the cdk inhibitors examined, p21 mRNA was most rapidly (in less than 1 h) and prominently induced by TGF-beta. In contrast, p15 mRNA was more slowly induced than p21 in SNU-620: cells, and not expressed in SNU-16 cells harbouring homozygous deletion of p15. Western blotting results confirmed the rapid increase of p21 while opposite patterns of p27 expression were observed in the two cell lines. The down-regulation of Cdc25A mRNA occurred, but was more delayed than that of p15 or p21. Until G1 arrest was established, changes in the protein levels of both Cdc25A and cdk4 were marginal. Co-immunoprecipitation with anti-cdk4 antibody showed that induced p21 associates with cdk4, and that its kinase activity is reduced by TGF-beta, which kinetically correlates closely with G1 arrest following TGF-beta treatment of both cell lines. These results suggest that in certain human epithelial cells, p21 may play an early role in TGF-beta-induced cell cycle arrest, and its cooperation with other cdk inhibitors is different depending on cell type. Delayed down-regulation of Cdc25A and cdk4 may contribute to cell adaptation to the quiescent state in the two gastric carcinoma cell lines studied

Lesson from the Stoichiometry Determination of the Cohesin Complex: A Short Protease Mediated Elution Increases the Recovery from Cross-Linked Antibody-Conjugated Beads

Affinity purification of proteins using antibodies coupled to beads and subsequent mass spectrometric analysis has become a standard technique for the identification of protein complexes. With the recent transfer of the isotope dilution mass spectrometry principle (IDMS) to the field of proteomics, quantitative analysesssuch as the stoichiometry determination of protein complexesshave become achievable. Traditionally proteins were eluted from antibody-conjugated beads using glycine at low pH or using diluted acids such as HCl, TFA, or FA, but elution was often found to be incomplete. Using the cohesin complex and the anaphase promoting complex/cyclosome (APC/C) as examples, we show that a short 15-60 min predigestion with a protease such as LysC (modified on-bead digest termed protease elution) increases the elution efficiency 2- to 3-fold compared to standard acid elution protocols. While longer incubation periodssas performed in standard on-bead digestionsled to partial proteolysis of the cross-linked antibodies, no or only insignificant cleavage was observed after 15-60 min protease mediated elution. Using the protease elution method, we successfully determined the stoichiometry of the cohesin complex by absolute quantification of the four core subunits using LC-SRM analysis and 19 reference peptides generated with the EtEP strategy. Protease elution was 3-fold more efficient compared to HCl elution, but measurements using both elution techniques are in agreement with

Bub1 Is a Fission Yeast Kinetochore Scaffold Protein, and Is Sufficient to Recruit other Spindle Checkpoint Proteins to Ectopic Sites on Chromosomes

The spindle checkpoint delays anaphase onset until all chromosomes have attached in a bi-polar manner to the mitotic spindle. Mad and Bub proteins are recruited to unattached kinetochores, and generate diffusible anaphase inhibitors. Checkpoint models propose that Mad1 and Bub1 act as stable kinetochore-bound scaffolds, to enhance recruitment of Mad2 and Mad3/BubR1, but this remains untested for Bub1. Here, fission yeast FRAP experiments confirm that Bub1 stably binds kinetochores, and by tethering Bub1 to telomeres we demonstrate that it is sufficient to recruit anaphase inhibitors in a kinase-independent manner. We propose that the major checkpoint role for Bub1 is as a signalling scaffold