Cohesin's concatenation of sister DNAs maintains their intertwining

The contribution of DNA catenation to sister chromatid cohesion is unclear partly because it has never been observed directly within mitotic chromosomes. Differential sedimentation-velocity and gel electrophoresis reveal that sisters of 26 kb circular minichromosomes are held together by catenation as well as by cohesin. The finding that chemical crosslinking of cohesin's three subunit interfaces entraps sister DNAs of circular but not linear minichromosomes implies that cohesin functions using a topological principle. Importantly, cohesin holds both catenated and uncatenated DNAs together in this manner. In the vicinity of centromeres, catenanes are resolved by spindle forces, but linkages mediated directly by cohesin resist these forces even after complete decatenation. Crucially, persistence of catenation after S phase depends on cohesin. We conclude that by retarding Topo II-driven decatenation, cohesin mediates sister chromatid cohesion by an indirect mechanism as well as one involving entrapment of sister DNAs inside its tripartite ring.     

From a single double helix to paired double helices and back

The propagation of our genomes during cell proliferation depends on the movement of sister DNA molecules produced by DNA replication to opposite sides of the cell before it divides. This feat is achieved by microtubules in eukaryotic cells but it has long remained a mystery how cells ensure that sister DNAs attach to microtubules with opposite orientations, known as amphitelic attachment. It is currently thought that sister chromatid cohesion has a crucial role. By resisting the forces exerted by microtubules, sister chromatid cohesion gives rise to tension that is thought essential for stabilizing kinetochore-microtubule attachments. Efficient amphitelic attachment is therefore achieved by an error correction mechanism that selectively eliminates connections that do not give rise to tension. Cohesion between sister chromatids is mediated by a multisubunit complex called cohesin which forms a gigantic ring structure. It has been proposed that sister DNAs are held together owing to their becoming entrapped within a single cohesin ring. Cohesion between sister chromatids is destroyed at the metaphase to anaphase transition by proteolytic cleavage of cohesin's Scc1 subunit by a thiol protease called separase, which severs the ring and thereby releases sister DNAs.     

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.     

Structure and stability of cohesin's Smc1-kleisin interaction

A multisubunit complex called cohesin forms a huge ring structure that mediates sister chromatid cohesion, possibly by entrapping sister DNAs following replication. Cohesin's kleisin subunit Scc1 completes the ring, connecting the ABC-like ATPase heads of a V-shaped Smc1/3 heterodimer. Proteolytic cleavage of Scc1 by separase triggers sister chromatid disjunction, presumably by breaking the Scc1 bridge. One half of the SMC-kleisin bridge is revealed here by a crystal structure of Smc1's ATPase complexed with Scc1's C-terminal domain. The latter forms a winged helix that binds a pair of beta strands in Smc1's ATPase head. Mutation of conserved residues within the contact interface destroys Scc1's interaction with Smc1/3 heterodimers and eliminates cohesin function. Interaction of Scc1's N terminus with Smc3 depends on prior C terminus connection with Smc1. There is little or no turnover of Smc1-Scc1 interactions within cohesin complexes in vivo because expression of noncleavable Scc1 after DNA replication does not hinder anaphase.     

Is chromatin remodeling required to build sister-chromatid cohesion?

Chromosome segregation during mitosis and meiosis depends on the linkage of sister DNA molecules after replication. These links, known as sister-chromatid cohesion, are provided by a multi-subunit complex called cohesin. Recent papers suggest that chromatin-remodeling complexes also have a role in the generation of sister-chromatid cohesion. It remains unclear whether they do so by facilitating the recruitment of cohesin to specific chromosomal sequences or by modifying an event at replication forks giving rise to cohesion between sister DNAs.     

How might cohesin hold sister chromatids together?

The sister chromatid cohesion essential for the bi-orientation of chromosomes on mitotic spindles depends on a multi-subunit complex called cohesin. This paper reviews the evidence that cohesin is directly responsible for holding sister DNAs together and considers how it might perform this function in the light of recent data on its structure.     

A matter of choice: the establishment of sister chromatid cohesion

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.     

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.     

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.     

A model for ATP hydrolysis-dependent binding of cohesin to DNA

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.     

A new thermosensitive smc-3 allele reveals involvement of cohesin in homologous recombination in C. elegans

The cohesin complex is required for the cohesion of sister chromatids and for correct segregation during mitosis and meiosis. Crossover recombination, together with cohesion, is essential for the disjunction of homologous chromosomes during the first meiotic division. Cohesin has been implicated in facilitating recombinational repair of DNA lesions via the sister chromatid. Here, we made use of a new temperature-sensitive mutation in the Caenorhabditis elegans SMC-3 protein to study the role of cohesin in the repair of DNA double-strand breaks (DSBs) and hence in meiotic crossing over. We report that attenuation of cohesin was associated with extensive SPO-11-dependent chromosome fragmentation, which is representative of unrepaired DSBs. We also found that attenuated cohesin likely increased the number of DSBs and eliminated the need of MRE-11 and RAD-50 for DSB formation in C. elegans, which suggests a role for the MRN complex in making cohesin-loaded chromatin susceptible to meiotic DSBs. Notably, in spite of largely intact sister chromatid cohesion, backup DSB repair via the sister chromatid was mostly impaired. We also found that weakened cohesins affected mitotic repair of DSBs by homologous recombination, whereas NHEJ repair was not affected. Our data suggest that recombinational DNA repair makes higher demands on cohesins than does chromosome segregation.     

Getting through anaphase: splitting the sisters and beyond

Sister-chromatid cohesion, thought to be primarily mediated by the cohesin complex, is essential for chromosome segregation. The forces holding the two sisters resist the tendency of microtubules to prematurely pull sister DNAs apart and thereby prevent random segregation of the genome during mitosis, and consequent aneuploidy. By counteracting the spindle pulling forces, cohesion between the two sisters generates the tension necessary to stabilize microtubule-kinetochore attachments. Upon entry into anaphase, however, the linkages that hold the two sister DNAs must be rapidly destroyed to allow physical separation of chromatids. Anaphase cells must therefore possess mechanisms that ensure faithful segregation of single chromatids that are now attached stably to the spindle in a manner no longer dependent on tension. In the present review, we discuss the nature of the cohesive forces that hold sister chromatids together, the mechanisms that trigger their physical separation, and the anaphase-specific changes that ensure proper segregation of single chromatids during the later stages of mitosis.     

Sister chromatid cohesion is required for postreplicative double-strand break repair in Saccharomyces cerevisiae.

The repair of DNA double-strand breaks by recombination requires the presence of an undamaged copy that is used as a template during the repair process. Because cells acquire resistance to gamma irradiation during DNA replication and because sister chromatids are the preferred partner for double-strand break repair in mitotic diploid yeast cells, it has long been suspected that cohesion between sister chromatids might be crucial for efficient repair. This hypothesis is consistent with the sensitivity to gamma irradiation of mutants defective in the cohesin complex that holds sister chromatids together from DNA replication until the onset of anaphase (reviewed in) . It is also in accordance with the finding that surveillance mechanisms (checkpoints) that sense DNA damage arrest cell cycle progression in yeast by causing stabilization of the securin Pds1, thereby blocking sister chromatid separation. The hypersensitivity to irradiation of cohesin mutants could, however, be due to a more direct involvement of the cohesin complex in the process of DNA repair. We show here that passage through S phase in the presence of cohesin, and not cohesin per se, is essential for efficient double-strand break repair during G2 in yeast. Proteins needed to load cohesin onto chromosomes (Scc2) and to generate cohesion during S phase (Eco1) are also shown to be required for repair. Our results confirm what has long been suspected but never proven, that cohesion between sister chromatids is essential for efficient double-strand break repair in mitotic cells.     

Yeast cohesin complex embraces 2 micron plasmid sisters in a tri-linked catenane complex

Sister chromatid cohesion, crucial for faithful segregation of replicated chromosomes in eukaryotes, is mediated by the multi-subunit protein complex cohesin. The Saccharomyces cerevisiae plasmid 2 micron circle mimics chromosomes in assembling cohesin at its partitioning locus. The plasmid is a multi-copy selfish DNA element that resides in the nucleus and propagates itself stably, presumably with assistance from cohesin. In metaphase cell lysates, or fractions enriched for their cohesed state by sedimentation, plasmid molecules are trapped topologically by the protein ring formed by cohesin. They can be released from cohesin’s embrace either by linearizing the DNA or by cleaving a cohesin subunit. Assays using two distinctly tagged cohesin molecules argue against the hand-cuff (an associated pair of monomeric cohesin rings) or the bracelet (a dimeric cohesin ring) model as responsible for establishing plasmid cohesion. Our cumulative results most easily fit a model in which a single monomeric cohesin ring, rather than a series of such rings, conjoins a pair of sister plasmids. These features of plasmid cohesion account for its sister-to-sister mode of segregation by cohesin disassembly during anaphase. The mechanistic similarities of cohesion between mini-chromosome sisters and 2 micron plasmid sisters suggest a potential kinship between the plasmid partitioning locus and centromeres.     

Cohesin's DNA Exit Gate Is Distinct from Its Entrance Gate and Is Regulated by Acetylation

Sister chromatid cohesion is mediated by entrapment of sister DNAs by a tripartite ring composed of cohesin's Smc1, Smc3, and α-kleisin subunits. Cohesion requires acetylation of Smc3 by Eco1, whose role is to counteract an inhibitory (antiestablishment) activity associated with cohesin's Wapl subunit. We show that mutations abrogating antiestablishment activity also reduce turnover of cohesin on pericentric chromatin. Our results reveal?a "releasing" activity inherent to cohesin complexes transiently associated with Wapl that catalyzes their dissociation from chromosomes. Fusion of Smc3's nucleotide binding domain to α-kleisin's N-terminal domain also reduces cohesin turnover within pericentric chromatin and permits establishment of Wapl-resistant cohesion in the absence of Eco1. We suggest that releasing activity opens the Smc3/α-kleisin interface, creating a DNA exit gate distinct from its proposed entry gate at the Smc1/3 interface. According to this notion, the function of Smc3 acetylation is to block its dissociation from α-kleisin. The functional implications of regulated ring opening are discussed.     

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.     

Chromosomal cohesin forms a ring

The cohesin complex is essential for sister chromatid cohesion during mitosis. Its Smc1 and Smc3 subunits are rod-shaped molecules with globular ABC-like ATPases at one end and dimerization domains at the other connected by long coiled coils. Smc1 and Smc3 associate to form V-shaped heterodimers. Their ATPase heads are thought to be bridged by a third subunit, Scc1, creating a huge triangular ring that could trap sister DNA molecules. We address here whether cohesin forms such rings in vivo. Proteolytic cleavage of Scc1 by separase at the onset of anaphase triggers its dissociation from chromosomes. We show that N- and C-terminal Scc1 cleavage fragments remain connected due to their association with different heads of a single Smc1/Smc3 heterodimer. Cleavage of the Smc3 coiled coil is sufficient to trigger cohesin release from chromosomes and loss of sister cohesion, consistent with a topological association with chromatin.     

A conserved motif at the C terminus of sororin is required for sister chromatid cohesion

Sororin is a positive regulator of sister chromatid cohesion that interacts with the cohesin complex. Sororin is required for the increased stability of the cohesin complex on chromatin following DNA replication and sister chromatid cohesion during G(2). The mechanism by which sororin ensures cohesion is currently unknown. Because the primary sequence of sororin does not contain any previously characterized structural or functional motifs, we have undertaken a structure-function analysis of the sororin protein. Using a series of mutant derivatives of sororin, we show that the ability of sororin to bind to chromatin is separable from both its role in sister chromatid cohesion and its interaction with the cohesin complex. We also show that derivatives of sororin with deletions or mutations in the conserved C terminus fail to rescue the loss-of-cohesion phenotype caused by sororin RNAi and that these mutations also abrogate the association of sororin with the cohesin complex. Our data suggest that the interaction of the highly conserved motif at the C terminus of sororin with the cohesin complex is critical to its ability to mediate sister chromatid cohesion.     

Establishment of sister chromatid cohesion at the S. cerevisiae replication fork

Two identical sister copies of eukaryotic chromosomes are synthesized during S phase. To facilitate their recognition as pairs for segregation in mitosis, sister chromatids are held together from their synthesis onward by the chromosomal cohesin complex. Replication fork progression is thought to be coupled to establishment of sister chromatid cohesion, facilitating identification of replication products, but evidence for this has remained circumstantial. Here we show that three proteins required for sister chromatid cohesion, Eco1, Ctf4, and Ctf18, are found at, and Ctf4 travels along chromosomes with, replication forks. The ring-shaped cohesin complex is loaded onto chromosomes before S phase in an ATP hydrolysis-dependent reaction. Cohesion establishment during DNA replication follows without further cohesin recruitment and without need for cohesin to re-engage an ATP hydrolysis motif that is critical for its initial DNA binding. This provides evidence for cohesion establishment in the context of replication forks and imposes constraints on the mechanism involved.