Scc2 couples replication licensing to sister chromatid cohesion in Xenopus egg extracts

The cohesin complex is a central player in sister chromatid cohesion, a process that ensures the faithful segregation of chromosomes in mitosis and meiosis. Previous genetic studies in yeast show that Scc2/Mis4, a HEAT-repeat-containing protein, is required for the loading of cohesin onto chromatin. In this study, we have identified two isoforms of Scc2 in humans and Xenopus (termed Scc2A and Scc2B), which are encoded by a single gene but have different carboxyl termini created by alternative splicing. Both Scc2A and Scc2B bind to chromatin concomitant with cohesin during DNA replication in Xenopus egg extracts. Simultaneous immunodepletion of Scc2A and Scc2B from the extracts impairs the association of cohesin with chromatin, leading to severe defects in sister chromatid pairing in the subsequent mitosis. The loading of Scc2 onto chromatin is inhibited in extracts treated with geminin but not with p21(CIP1), suggesting that this step depends on replication licensing but not on the initiation of DNA replication. Upon mitotic entry, Scc2 is removed from chromatin through a mechanism that requires cdc2 but not aurora B or polo-like kinase. Our results suggest that vertebrate Scc2 couples replication licensing to sister chromatid cohesion by facilitating the loading of cohesin onto chromatin.     

A screen for cohesion mutants uncovers Ssl3, the fission yeast counterpart of the cohesin loading factor Scc4

Sister-chromatid cohesion is mediated by cohesin, a ring-shape complex made of four core subunits called Scc1, Scc3, Smc1, and Smc3 in Saccharomyces cerevisiae (Rad21, Psc3, Psm1, and Psm3 in Schizosaccharomyces pombe). How cohesin ensures cohesion is unknown, although its ring shape suggests that it may tether sister DNA strands by encircling them . Cohesion establishment is a two-step process. Cohesin is loaded on chromosomes before replication and cohesion is subsequently established during S phase. In S. cerevisiae, cohesin loading requires a separate complex containing the Scc2 and Scc4 proteins. Cohesin rings fail to associate with chromatin and cohesion can not establish when Scc2 is impaired . The mechanism of loading is unknown, although some data suggest that hydrolysis of ATP bound to Smc1/3 is required . Scc2 homologs exist in fission yeast (Mis4), Drosophila, Xenopus, and human . By contrast, no homolog of Scc4 has been identified so far. We report here on the identification of fission yeast Ssl3 as a Scc4-like factor. Ssl3 is in complex with Mis4 and, as a bona fide loading factor, Ssl3 is required in G1 for cohesin binding to chromosomes but dispensable in G2 when cohesion is established. The discovery of a functional homolog of Scc4 indicates that the machinery of cohesin loading is conserved among eukaryotes.     

Postreplicative recruitment of cohesin to double-strand breaks is required for DNA repair

Chromosome stability depends on accurate chromosome segregation and efficient DNA double-strand break (DSB) repair. Sister chromatid cohesion, established during S phase by the protein complex cohesin, is central to both processes. In the absence of cohesion, chromosomes missegregate and G2-phase DSB repair fails. Here, we demonstrate that G2-phase repair also requires the presence of cohesin at the damage site. Cohesin components are shown to be recruited to extended chromosome regions surrounding DNA breaks induced during G2. We find that in the absence of functional cohesin-loading proteins (Scc2/Scc4), the accumulation of cohesin at DSBs is abolished and repair is defective, even though sister chromatids are connected by S phase generated cohesion. Evidence is also provided that DSB induction elicits establishment of sister chromatid cohesion in G2, implicating that damage-recruited cohesin facilitates DNA repair by tethering chromatids.     

Metazoan Scc4 homologs link sister chromatid cohesion to cell and axon migration guidance

Saccharomyces cerevisiae Scc2 binds Scc4 to form an essential complex that loads cohesin onto chromosomes. The prevalence of Scc2 orthologs in eukaryotes emphasizes a conserved role in regulating sister chromatid cohesion, but homologs of Scc4 have not hitherto been identified outside certain fungi. Some metazoan orthologs of Scc2 were initially identified as developmental gene regulators, such as Drosophila Nipped-B, a regulator of cut and Ultrabithorax, and delangin, a protein mutant in Cornelia de Lange syndrome. We show that delangin and Nipped-B bind previously unstudied human and fly orthologs of Caenorhabditis elegans MAU-2, a non-axis-specific guidance factor for migrating cells and axons. PSI-BLAST shows that Scc4 is evolutionarily related to metazoan MAU-2 sequences, with the greatest homology evident in a short N-terminal domain, and protein–protein interaction studies map the site of interaction between delangin and human MAU-2 to the N-terminal regions of both proteins. Short interfering RNA knockdown of human MAU-2 in HeLa cells resulted in precocious sister chromatid separation and in impaired loading of cohesin onto chromatin, indicating that it is functionally related to Scc4, and RNAi analyses show that MAU-2 regulates chromosome segregation in C. elegans embryos. Using antisense morpholino oligonucleotides to knock down Xenopus tropicalis delangin or MAU-2 in early embryos produced similar patterns of retarded growth and developmental defects. Our data show that sister chromatid cohesion in metazoans involves the formation of a complex similar to the Scc2-Scc4 interaction in the budding yeast. The very high degree of sequence conservation between Scc4 homologs in complex metazoans is consistent with increased selection pressure to conserve additional essential functions, such as regulation of cell and axon migration during development.     

Requirement of chromatid cohesion proteins rad21/scc1 and mis4/scc2 for normal spindle-kinetochore interaction in fission yeast

BACKGROUND: Proteins conserved from yeast to human hold two sister chromatids together. The failure to form cohesion in the S phase results in premature separation of chromatids in G2/M. Mitotic kinetochores free from microtubules or the lack of tension are known to activate spindle checkpoint. RESULTS: The loss of chromatid cohesion in fission yeast mutants (mis4-242 and rad21-K1) leads to the activation of Mad2- and Bub1-dependent checkpoint, possibly due to a diminished microtubule-kinetochore interaction. Bub1, a checkpoint kinase, localizes briefly at early mitotic kinetochores in wild-type, whereas the cohesion mutation greatly increases the duration of kinetochore localization. Bub1 is bound to the central centromere region of mitotic cells. These cohesion mutants are hypersensitive to a tubulin poison and are synthetic lethal with dis1 and bir1/cut17, which are defective in microtubule-kinetochore interaction. The formation of specialized centromere chromatin containing CENP-A does not require cohesion. Dominant-negative noncleavable Rad21 fails to activate checkpoint but blocks sister chromatid separation and full spindle elongation in anaphase. CONCLUSIONS: Mis4 and Rad21 (budding yeast Scc2 and Scc1 homologs, respectively) act in establishing the normal spindle-kinetochore interaction in early mitosis and inhibit sister chromatid separation until the cleavage of Rad21 in anaphase. Checkpoint directly or indirectly monitors the states of cohesion in early mitosis. Full spindle extension occurs with unequal nuclear division in cohesion mutants in the absence of Mad2.     

The kleisin subunit of cohesin dictates damage-induced cohesion

Cohesin, the protein complex that mediates sister chromatid cohesion, is required for faithful chromosome segregation and efficient repair of double-strand breaks (DSBs). Cohesion generation is normally restricted to S phase. However, in G2/M, a DSB activates cohesion generation near the DSB and genome-wide. Here, using budding yeast, we show that DSB-induced cohesion occurs when cohesin contains the kleisin subunit, Mcd1 (Scc1), but not when Mcd1 is replaced by its meiotic isoform, Rec8. We exploit this divergence to demonstrate that serine 83 of Mcd1 and the Chk1 kinase are critical determinants for DSB-induced cohesion. We propose that a DSB in G2/M activates Mec1 (ATR), which in turn stimulates Chk1-dependent phosphorylation of Mcd1 at serine 83. Serine 83 phosphorylation promotes chromatin-bound cohesin to become cohesive.     

A SUMO-Dependent Step during Establishment of Sister Chromatid Cohesion

Cohesin is a protein complex that ties sister DNA molecules from the time of DNA replication until the metaphase to anaphase transition. Current models propose that the association of the Smc1, Smc3, and Scc1/Mcd1 subunits creates a ring-shaped structure that entraps the two sister DNAs [1]. Cohesin is essential for correct chromosome segregation and recombinational repair. Its activity is therefore controlled by several posttranslational modifications, including acetylation, phosphorylation, sumoylation, and site-specific proteolysis. Here we show that cohesin sumoylation occurs at the time of cohesion establishment, after cohesin loading and ATP binding, and independently from Eco1-mediated cohesin acetylation. In order to test the functional relevance of cohesin sumoylation, we have developed a novel approach in budding yeast to deplete SUMO from all subunits in the cohesin complex, based on fusion of the Scc1 subunit to a SUMO peptidase Ulp domain (UD). Downregulation of cohesin sumoylation is lethal, and the Scc1-UD chimeras have a failure in sister chromatid cohesion. Strikingly, the unsumoylated cohesin rings are acetylated. Our findings indicate that SUMO is a novel molecular determinant for the establishment of sister chromatid cohesion, and we propose that SUMO is required for the entrapment of sister chromatids during the acetylation-mediated closure of the cohesin ring.     

Cornelia de Lange Syndrome and the link between chromosomal function, DNA repair and developmental gene regulation

Cornelia de Lange Syndrome (CdLS) is a rare multiple malformation disorder with characteristic facial features, growth and cognitive retardation, and many other abnormalities. CdLS individuals were recently shown to have heterozygous mutations in a previously uncharacterised gene, NIPBL, which encodes delangin, a homologue of fungal Scc2-type sister chromatid cohesion proteins and the Drosophila Nipped-B developmental regulator. Nipped-B and vertebrate delangins are also now known to regulate sister chromatid cohesion, probably as part of oligomeric complexes required to load cohesin subunits onto chromatin. CdLS is likely to be one of several developmental disorders resulting from defective expression of a multi-functional protein with roles in chromosome function, gene regulation and double-strand DNA repair - a combination of properties shared by certain bacterial proteins responsible for structural maintenance of chromatin.     

Functional links between <Emphasis Type="Italic">Drosophila</Emphasis> Nipped-B and cohesin in somatic and meiotic cells

Drosophila Nipped-B is an essential protein that has multiple functions. It facilitates expression of homeobox genes and is also required for sister chromatid cohesion. Nipped-B is conserved from yeast to man, and its orthologs also play roles in deoxyribonucleic acid repair and meiosis. Mutation of the human ortholog, Nipped-B-Like (NIPBL), causes Cornelia de Lange syndrome (CdLS), associated with multiple developmental defects. The Nipped-B protein family is required for the cohesin complex that mediates sister chromatid cohesion to bind to chromosomes. A key question, therefore, is whether the Nipped-B family regulates gene expression, meiosis, and development by controlling cohesin. To gain insights into Nipped-B’s functions, we compared the effects of several Nipped-B mutations on gene expression, sister chromatid cohesion, and meiosis. We also examined association of Nipped-B and cohesin with somatic and meiotic chromosomes by immunostaining. Missense Nipped-B alleles affecting the same HEAT repeat motifs as CdLS-causing NIPBL mutations have intermediate effects on both gene expression and mitotic chromatid cohesion, linking these two functions and the role of NIPBL in human development. Nipped-B colocalizes extensively with cohesin on chromosomes in both somatic and meiotic cells and is present in soluble complexes with cohesin subunits in nuclear extracts. In meiosis, Nipped-B also colocalizes with the synaptonemal complex and contributes to maintenance of meiotic chromosome cores. These results support the idea that direct regulation of cohesin function underlies the diverse functions of Nipped-B and its orthologs. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Gause, Webber, and Misulovin provided equal contributions.     

S-phase checkpoint genes safeguard high-fidelity sister chromatid cohesion

Cohesion establishment and maintenance are carried out by proteins that modify the activity of Cohesin, an essential complex that holds sister chromatids together. Constituents of the replication fork, such as the DNA polymerase alpha-binding protein Ctf4, contribute to cohesion in ways that are poorly understood. To identify additional cohesion components, we analyzed a ctf4Delta synthetic lethal screen performed on microarrays. We focused on a subset of ctf4Delta-interacting genes with genetic instability of their own. Our analyses revealed that 17 previously studied genes are also necessary for the maintenance of robust association of sisters in metaphase. Among these were subunits of the MRX complex, which forms a molecular structure similar to Cohesin. Further investigation indicated that the MRX complex did not contribute to metaphase cohesion independent of Cohesin, although an additional role may be contributed by XRS2. In general, results from the screen indicated a sister chromatid cohesion role for a specific subset of genes that function in DNA replication and repair. This subset is particularly enriched for genes that support the S-phase checkpoint. We suggest that these genes promote and protect a chromatin environment conducive to robust cohesion.     

Cohesin rings devoid of scc3 and pds5 maintain their stable association with the DNA

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.     

Cohesin ensures bipolar attachment of microtubules to sister centromeres and resists their precocious separation

The multisubunit protein complex cohesin is required to establish cohesion between sister chromatids during S phase and to maintain it during G2 and M phases. Cohesin is essential for mitosis, and even partial defects cause very high rates of chromosome loss. In budding yeast, cohesin associates with specific sites which are distributed along the entire length of a chromosome but are more dense in the vicinity of the centromere. Real-time imaging of individual centromeres tagged with green fluorescent protein suggests that cohesin bound to centromeres is important for bipolar attachment to microtubules. This cohesin is, however, incapable of resisting the consequent force, which leads to sister centromere splitting and chromosome stretching. Meanwhile, cohesin bound to sequences flanking the centromeres prevents sister chromatids from completely unzipping and is required to pull back together sister centromeres that have already split. Cohesin therefore has a central role in generating a dynamic tension between microtubules and sister chromatid cohesion at centromeres, which lasts until chromosome segregation is finally promoted by separin-dependent cleavage of the cohesin subunit Scc1p.     

The hsSsu72 phosphatase is a cohesin‐binding protein that regulates the resolution of sister chromatid arm cohesion

Cohesin is a multiprotein complex that establishes sister chromatid cohesion from S phase until mitosis or meiosis. In vertebrates, sister chromatid cohesion is dissolved in a stepwise manner: most cohesins are removed from the chromosome arms via a process that requires polo‐like kinase 1 (Plk1), aurora B and Wapl, whereas a minor amount of cohesin, found preferentially at the centromere, is cleaved by separase following its activation by the anaphase‐promoting complex/cyclosome. Here, we report that our budding yeast two‐hybrid assay identified hsSsu72 phosphatase as a Rad21‐binding protein. Additional experiments revealed that Ssu72 directly interacts with Rad21 and SA2 in vitro and in vivo, and associates with sister chromatids in human cells. Interestingly, depletion or mutational inactivation of Ssu72 phosphatase activity caused the premature resolution of sister chromatid arm cohesion, whereas the overexpression of Ssu72 yielded high resistance to this resolution. Interestingly, it appears that Ssu72 regulates the cohesion of chromosome arms but not centromeres, and acts by counteracting the phosphorylation of SA2. Thus, our study provides important new evidence, suggesting that Ssu72 is a novel cohesin‐binding protein capable of regulating cohesion between sister chromatid arms.     

A Conserved Domain in the Scc3 Subunit of Cohesin Mediates the Interaction with Both Mcd1 and the Cohesin Loader Complex

The Structural Maintenance of Chromosome (SMC) complex, termed cohesin, is essential for sister chromatid cohesion. Cohesin is also important for chromosome condensation, DNA repair, and gene expression. Cohesin is comprised of Scc3, Mcd1, Smc1, and Smc3. Scc3 also binds Pds5 and Wpl1, cohesin-associated proteins that regulate cohesin function, and to the Scc2/4 cohesin loader. We mutagenized SCC3 to elucidate its role in cohesin function. A 5 amino acid insertion after Scc3 residue I358, or a missense mutation of residue D373 in the adjacent stromalin conservative domain (SCD) induce inviability and defects in both cohesion and cohesin binding to chromosomes. The I358 and D373 mutants abrogate Scc3 binding to Mcd1. These results define an Scc3 region extending from I358 through the SCD required for binding Mcd1, cohesin localization to chromosomes and cohesion. Scc3 binding to the cohesin loader, Pds5 and Wpl1 are unaffected in I358 mutant and the loader still binds the cohesin core trimer (Mcd1, Smc1 and Smc3). Thus, Scc3 plays a critical role in cohesin binding to chromosomes and cohesion at a step distinct from loader binding to the cohesin trimer. We show that residues Y371 and K372 within the SCD are critical for viability and chromosome condensation but dispensable for cohesion. However, scc3 Y371A and scc3 K372A bind normally to Mcd1. These alleles also provide evidence that Scc3 has distinct mechanisms of cohesin loading to different loci. The cohesion-competence, condensation-incompetence of Y371 and K372 mutants suggests that cohesin has at least one activity required specifically for condensation.     

Cohesin's binding to chromosomes depends on a separate complex consisting of Scc2 and Scc4 proteins

Cohesion between sister chromatids depends on a multisubunit cohesin complex that binds to chromosomes around DNA replication and dissociates from them at the onset of anaphase. Scc2p, though not a cohesin subunit, is also required for sister chromatid cohesion. We show here that Scc2p forms a complex with a novel protein, Scc4p, which is also necessary for sister cohesion. In scc2 or scc4 mutants, cohesin complexes form normally but fail to bind both to centromeres and to chromosome arms. Our data suggest that a major role for the Scc2p/Scc4p complex is to facilitate the loading of cohesin complexes onto chromosomes.     

Sororin is required for stable binding of cohesin to chromatin and for sister chromatid cohesion in interphase

Sister chromatid cohesion depends on cohesin [1-3]. Cohesin associates with chromatin dynamically throughout interphase [4]. During DNA replication, cohesin establishes cohesion [5], and this process coincides with the generation of a cohesin subpopulation that is more stably bound to chromatin [4]. In mitosis, cohesin is removed from chromosomes, enabling sister chromatid separation [6]. How cohesin associates with chromatin and establishes cohesion is poorly understood. By searching for proteins that are associated with chromatin-bound cohesin, we have identified sororin, a protein that was known to be required for cohesion [7]. To obtain further insight into sororin's function, we have addressed when during the cell cycle sororin is required for cohesion. We show that sororin is dispensable for the association of cohesin with chromatin but that sororin is essential for proper cohesion during G2 phase. Like cohesin, sororin is also needed for efficient repair of DNA double-strand breaks in G2. Finally, sororin is required for the presence of normal amounts of the stably chromatin-bound cohesin population in G2. Our data indicate that sororin interacts with chromatin-bound cohesin and functions during the establishment or maintenance of cohesion in S or G2 phase, respectively.     

The mechanism of sister chromatid cohesion

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