Both interaction surfaces within cohesin's hinge domain are essential for its stable chromosomal association.
ABSTRACT: The cohesin complex that mediates sister chromatid cohesion contains three core subunits: Smc1, Smc3, and Scc1. Heterotypic interactions between Smc1 and Smc3 dimerization domains create stable V-shaped Smc1/Smc3 heterodimers with a hinge at the center and nucleotide-binding domains (NBDs) at the ends of each arm. Interconnection of each NBD through their association with the N- and C-terminal domains of Scc1 creates a tripartite ring, within which sister DNAs are thought to be entrapped (the ring model). Crystal structures show that the Smc1/Smc3 hinge has a toroidal shape, with independent "north" and "south" interaction surfaces on an axis of pseudosymmetry. The ring model predicts that sister chromatid cohesion would be lost by transient hinge opening.We find that mutations within either interface weaken heterodimerization of isolated half hinges in vitro but do not greatly compromise formation of cohesin rings in vivo. They do, however, reduce the residence time of cohesin on chromosomes and cause lethal defects in sister chromatid cohesion. This demonstrates that mere formation of rings is insufficient for cohesin function. Stable cohesion requires cohesin rings that cannot easily open.Either the north or south hinge interaction surface is sufficient for the assembly of V-shaped Smc1/Smc3 heterodimers in vivo. Any tendency of Smc proteins with weakened hinges to dissociate will be suppressed by interconnection of their NBDs by Scc1. We suggest that transient hinge dissociation caused by the mutations described here is incompatible with stable sister chromatid cohesion because it permits chromatin fibers to escape from cohesin rings.
Project description:Through their association with a kleisin subunit (Scc1), cohesin's Smc1 and Smc3 subunits are thought to form tripartite rings that mediate sister chromatid cohesion. Unlike the structure of Smc1/Smc3 and Smc1/Scc1 interfaces, that of Smc3/Scc1 is not known. Disconnection of this interface is thought to release cohesin from chromosomes in a process regulated by acetylation. We show here that the N-terminal domain of yeast Scc1 contains two ? helices, forming a four-helix bundle with the coiled coil emerging from Smc3's adenosine triphosphatase head. Mutations affecting this interaction compromise cohesin's association with chromosomes. The interface is far from Smc3 residues, whose acetylation prevents cohesin's dissociation from chromosomes. Cohesin complexes holding chromatids together in vivo do indeed have the configuration of hetero-trimeric rings, and sister DNAs are entrapped within these.
Project description:The cohesin subunits Smc1, Smc3 and Scc1 form large tripartite rings which mediate sister chromatid cohesion and chromatin structure. These are thought to entrap DNA with the help of the associated proteins SA1/2 and Pds5A/B. Structural information is available for parts of cohesin, but analyses of entire cohesin complexes are limited by their flexibility. Here we generated a more rigid 'bonsai' cohesin by truncating the coiled coils of Smc1 and Smc3 and used single-particle electron microscopy, chemical crosslinking-mass spectrometry and in silico modelling to generate three-dimensional models of cohesin bound to Pds5B. The HEAT-repeat protein Pds5B forms a curved structure around the nucleotide-binding domains of Smc1 and Smc3 and bridges the Smc3-Scc1 and SA1-Scc1 interfaces. These results indicate that Pds5B forms an integral part of the cohesin ring by contacting all other cohesin subunits, a property that may reflect the complex role of Pds5 proteins in controlling cohesin-DNA interactions.
Project description:Cohesins establish sister chromatid cohesion during S phase and are removed when cohesin Scc1 is cleaved by separase at anaphase onset. During this process, cohesin Smc3 undergoes a cycle of acetylation: Smc3 acetylation by Eco1 in S phase stabilizes cohesin association with chromosomes, and its deacetylation by Hos1 in anaphase allows re-use of Smc3 in the next cell cycle. Here we find that Smc3 deacetylation by Hos1 has a more immediate effect in the early anaphase of budding yeast. Hos1 depletion significantly delayed sister chromatid separation and segregation. Smc3 deacetylation facilitated removal of cohesins from chromosomes without changing Scc1 cleavage efficiency, promoting dissolution of cohesion. This action is probably due to disengagement of Smc1-Smc3 heads prompted by de-repression of their ATPase activity. We suggest Scc1 cleavage per se is insufficient for efficient dissolution of cohesion in early anaphase; subsequent Smc3 deacetylation, triggered by Scc1 cleavage, is also required.
Project description:Sister chromatid cohesion is thought to involve entrapment of sister DNAs by a tripartite ring composed of the cohesin subunits Smc1, Smc3, and Scc1. Establishment of cohesion during S phase depends on acetylation of Smc3's nucleotide-binding domain (NBD) by the Eco1 acetyl transferase. It is destroyed at the onset of anaphase due to Scc1 cleavage by separase. In yeast, Smc3 acetylation is reversed at anaphase by the Hos1 deacetylase as a consequence of Scc1 cleavage. Smc3 molecules that remain acetylated after mitosis due to Hos1 inactivation cannot generate cohesion during the subsequent S phase, implying that cohesion establishment depends on de novo acetylation during DNA replication. By inducing Smc3 deacetylation in postreplicative cells due to Hos1 overexpression, we provide evidence that Smc3 acetylation contributes to the maintenance of sister chromatid cohesion. A cycle of Smc3 NBD acetylation is therefore an essential aspect of the chromosome cycle in eukaryotic cells.
Project description:BACKGROUND:The Cohesin complex that holds sister chromatins together until anaphase is comprised of three core subunits: Smc1 and Smc3, two long-rod-shaped proteins with an ABC-like ATPase head (nucleotide-binding domain [NBD]) and a dimerization domain linked by a 50 nm long intramolecular antiparallel coiled-coil, and Scc1, an ?-kleisin subunit interconnecting the NBD domains of Smc1 and Smc3. Cohesin's stable association with chromosomes is thought to involve entrapment of chromatin fibers by its tripartite Smc1-Smc3-Scc1 ring via a poorly understood mechanism dependent on a separate Scc2/4 loading complex. A key issue concerns where entrapment initially takes place: at sites where cohesin is found stably associated or at distinct "loading" sites from which it translocates. RESULTS:In this study, we find transition state mutant versions (Smc1E1158Q and SmcE1155Q) defective in disengagement of their nucleotide binding domains (NBDs), unlike functional cohesin, colocalize with Scc2/4 at core centromeres, sites that catalyze wild-type cohesin's recruitment to sequences 20 kb or more away. In addition to Scc2/4, the unstable association of transition state complexes with core centromeres requires Scc1's association with Smc1 and Smc3 NBDs, ATP-driven NBD engagement, cohesin's Scc3 subunit, and its hinge domain. CONCLUSION:We propose that cohesin's association with chromosomes is driven by two key events. NBD engagement driven by ATP binding produces an unstable association with specific loading sites like core centromeres, whereas subsequent ATP hydrolysis triggers DNA entrapment, which permits translocation along chromatin fibers.
Project description:Faithful transmission of chromosomes during eukaryotic cell division requires sister chromatids to be paired from their generation in S phase until their separation in M phase. Cohesion is mediated by the cohesin complex, whose Smc1, Smc3 and Scc1 subunits form a tripartite ring that entraps both DNA double strands. Whereas centromeric cohesin is removed in late metaphase by Scc1 cleavage, metazoan cohesin at chromosome arms is displaced already in prophase by proteolysis-independent signalling. Which of the three gates is triggered by the prophase pathway to open has remained enigmatic. Here, we show that displacement of human cohesin from early mitotic chromosomes requires dissociation of Smc3 from Scc1 but no opening of the other two gates. In contrast, loading of human cohesin onto chromatin in telophase occurs through the Smc1-Smc3 hinge. We propose that the use of differently regulated gates for loading and release facilitates unidirectionality of DNA's entry into and exit from the cohesin ring.
Project description:Sister chromatid cohesion is mediated by cohesin, whose Smc1, Smc3, and kleisin (Scc1) subunits form a ring structure that entraps sister DNAs. The ring is opened either by separase, which cleaves Scc1 during anaphase, or by a releasing activity involving Wapl, Scc3, and Pds5, which bind to Scc1 and open its interface with Smc3. We present crystal structures of Pds5 from the yeast L. thermotolerans in the presence and absence of the conserved Scc1 region that interacts with Pds5. Scc1 binds along the spine of the Pds5 HEAT repeat fold and is wedged between the spine and C-terminal hook of Pds5. We have isolated mutants that confirm the observed binding mode of Scc1 and verified their effect on cohesin by immunoprecipitation and calibrated ChIP-seq. The Pds5 structure also reveals architectural similarities to Scc3, the other large HEAT repeat protein of cohesin and, most likely, Scc2.
Project description:The ring-shaped cohesin complex topologically entraps chromosomes and regulates chromosome segregation, transcription, and DNA repair. The cohesin core consists of the structural maintenance of chromosomes 1 and 3 (Smc1-Smc3) heterodimeric ATPase, the kleisin subunit sister chromatid cohesion 1 (Scc1) that links the two ATPase heads, and the Scc1-bound adaptor protein Scc3. The sister chromatid cohesion 2 and 4 (Scc2-Scc4) complex loads cohesin onto chromosomes. Mutations of cohesin and its regulators, including Scc2, cause human developmental diseases termed cohesinopathy. Here, we report the crystal structure of Chaetomium thermophilum (Ct) Scc2 and examine its interaction with cohesin. Similar to Scc3 and another Scc1-interacting cohesin regulator, precocious dissociation of sisters 5 (Pds5), Scc2 consists mostly of helical repeats that fold into a hook-shaped structure. Scc2 binds to Scc1 through an N-terminal region of Scc1 that overlaps with its Pds5-binding region. Many cohesinopathy mutations target conserved residues in Scc2 and diminish Ct Scc2 binding to Ct Scc1. Pds5 binding to Scc1 weakens the Scc2-Scc1 interaction. Our study defines a functionally important interaction between the kleisin subunit of cohesin and the hook of Scc2. Through competing with Scc2 for Scc1 binding, Pds5 might contribute to the release of Scc2 from loaded cohesin, freeing Scc2 for additional rounds of loading.
Project description:STAG/SA proteins are specific cohesin complex subunits that maintain sister chromatid cohesion in mitosis and meiosis. Two members of this family, STAG1/SA1 and STAG2/SA2,double dagger are classified as mitotic cohesins, as they are found in human somatic cells and in Xenopus laevis as components of the cohesin(SA1) and cohesin(SA2) complexes, in which the shared subunits are Rad21/SCC1, SMC1 and SMC3 proteins. A recently reported third family member, STAG3, is germinal cell-specific and is a subunit of the meiotic cohesin complex. To date, the meiosis-specific cohesin complex has been considered to be responsible for sister chromatid cohesion during meiosis. We studied replacement of the mitotic by the meiotic cohesin complex during mouse germinal cell maturation, and we show that mammalian STAG2 and Rad21 are also involved in several meiosis stages. Immunofluorescence results suggest that a cohesin complex containing Rad21 and STAG2 cooperates with a STAG3-specific complex to maintain sister chromatid cohesion during the diplotene stage of meiosis.
Project description:Cohesin is a protein complex that forms a ring around sister chromatids thus holding them together. The ring is composed of three proteins: Smc1, Smc3 and Scc1. The roles of three additional proteins that associate with the ring, Scc3, Pds5 and Wpl1, are not well understood. It has been proposed that these three factors form a complex that stabilizes the ring and prevents it from opening. This activity promotes sister chromatid cohesion but at the same time poses an obstacle for the initial entrapment of sister DNAs. This hindrance to cohesion establishment is overcome during DNA replication via acetylation of the Smc3 subunit by the Eco1 acetyltransferase. However, the full mechanistic consequences of Smc3 acetylation remain unknown. In the current work, we test the requirement of Scc3 and Pds5 for the stable association of cohesin with DNA. We investigated the consequences of Scc3 and Pds5 depletion in vivo using degron tagging in budding yeast. The previously described DHFR-based N-terminal degron as well as a novel Eco1-derived C-terminal degron were employed in our study. Scc3 and Pds5 associate with cohesin complexes independently of each other and require the Scc1 "core" subunit for their association with chromosomes. Contrary to previous data for Scc1 downregulation, depletion of either Scc3 or Pds5 had a strong effect on sister chromatid cohesion but not on cohesin binding to DNA. Quantity, stability and genome-wide distribution of cohesin complexes remained mostly unchanged after the depletion of Scc3 and Pds5. Our findings are inconsistent with a previously proposed model that Scc3 and Pds5 are cohesin maintenance factors required for cohesin ring stability or for maintaining its association with DNA. We propose that Scc3 and Pds5 specifically function during cohesion establishment in S phase.