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 topological interaction between cohesin rings and a circular minichromosome

Sister chromatid cohesion depends on a multiprotein cohesin complex containing two SMC subunits, Smc1 and Smc3, that dimerize to form V-shaped molecules with ABC-like ATPase heads at the tips of their two arms. Cohesin's Smc1 and Smc3 "heads" are connected by an alpha kleisin subunit called Scc1, forming a tripartite ring with a diameter around 40 nm. We show here that some cohesin remains tightly bound to circular minichromosomes after their purification from yeast cells and that cleavage either of cohesin's ring or of the minichromosome's DNA destroys their association. This suggests that the stable association between cohesin and chromatin detected here is topological rather than physical, which is consistent with the notion that DNA is trapped inside cohesin rings.     

Preferential cleavage of chromatin-bound cohesin after targeted phosphorylation by Polo-like kinase

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.     

Prophase pathway‐dependent removal of cohesin from human chromosomes requires opening of the Smc3–Scc1 gate

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.     

Cohesin: a catenase with separate entry and exit gates?

Cohesin confers both intrachromatid and interchromatid cohesion through formation of a tripartite ring within which DNA is thought to be entrapped. Here, I discuss what is known about the four stages of the cohesin ring cycle using the ring model as an intellectual framework. I postulate that cohesin loading onto chromosomes, catalysed by a separate complex called kollerin, is mediated by the entry of DNA into cohesin rings, whereas dissociation, catalysed by Wapl and several other cohesin subunits (an activity that will be called releasin here), is mediated by the subsequent exit of DNA. I suggest that the ring's entry and exit gates may be separate, with the former and latter taking place at Smc1-Smc3 and Smc3-kleisin interfaces, respectively. Establishment of cohesion during S phase involves neutralization of releasin through acetylation of Smc3 at a site close to the putative exit gate of DNA, which locks rings shut until opened irreversibly by kleisin cleavage through the action of separase, an event that triggers the metaphase to anaphase transition.     

The dissociation of cohesin from chromosomes in prophase is regulated by Polo-like kinase

The separation of sister chromatids in anaphase depends on the dissociation of cohesin from chromosomes. In vertebrates, some cohesin is removed from chromosomes at the onset of anaphase by proteolytic cleavage. In contrast, the bulk of cohesin is removed from chromosomes already in prophase and prometaphase by an unknown mechanism that does not involve cohesin cleavage. We show that Polo-like kinase is required for the cleavage-independent dissociation of cohesin from chromosomes in Xenopus. Cohesin phosphorylation depends on Polo-like kinase and reduces the ability of cohesin to bind to chromatin. These results suggest that Polo-like kinase regulates the dissociation of cohesin from chromosomes early in mitosis.     

Separase biosensor reveals that cohesin cleavage timing depends on phosphatase PP2A(Cdc55) regulation

In anaphase, sister chromatids separate abruptly and are then segregated by the mitotic spindle. The protease separase triggers sister separation by cleaving the Scc1/Mcd1 subunit of the cohesin ring that holds sisters together. Polo-kinase phosphorylation of Scc1 promotes its cleavage, but the underlying regulatory circuits are unclear. We developed a separase biosensor in Saccharomyces cerevisiae that provides a quantitative indicator of cohesin cleavage in single cells. Separase is abruptly activated and cleaves most cohesin within 1?min, after which anaphase begins. Cohesin near centromeres and telomeres is cleaved at the same rate and time. Protein phosphatase PP2A(Cdc55) inhibits cohesin cleavage by counteracting polo-kinase phosphorylation of Scc1. In early anaphase, the previously described separase inhibition of PP2A(Cdc55) promotes cohesin cleavage. Thus, separase acts directly on Scc1 and also indirectly, through inhibition of PP2A(Cdc55), to stimulate cohesin cleavage, providing a feedforward loop that may contribute to a robust and timely anaphase.     

SMC complexes and topoisomerase II work together so that sister chromatids can work apart

The pairing of sister chromatids in interphase facilitates error-free homologous recombination (HR). Sister chromatids are held together by cohesin, one of three Structural Maintenance of Chromosomes (SMC) complexes. In mitosis, chromosome condensation is controlled by another SMC complex, condensin, and the type II topoisomerase (Top2). In prophase, cohesin is stripped from chromosome arms, but remains at centromeres until anaphase, whereupon it is removed via proteolytic cleavage. The third SMC complex, Smc5/6, is generally described as a regulator of HR-mediated DNA repair. However, cohesin and condensin are also required for DNA repair, and HR genes are not essential for cell viability, but the SMC complexes are. Smc5/6 null mutants die in mitosis, and in fission yeast, Smc5/6 hypomorphs show lethal mitoses following genotoxic stress, or when combined with a Top2 mutant, top2-191. We found these mitotic defects are due to retention of cohesin on chromosome arms. We also show that Top2 functions in the cohesin cycle, and accumulating data suggests this is not related to its decatenation activity. Thus the SMC complexes and Top2 functionally interact, and any DNA repair function ascribed to Smc5/6 is likely a reflection of a more fundamental role in the regulation of chromosome structure.     

Cut1/separase-dependent roles of multiple phosphorylation of fission yeast cohesin subunit Rad21 in post-replicative damage repair and mitosis

Cohesin is a multiprotein complex essential for sister-chromatid cohesion. It plays a pivotal role in proper chromosome segregation and DNA damage repair. The mitotic behavior of cohesin is controlled through its phosphorylation, which possibly induces the dissociation of cohesin from chromosomes and enhances its susceptibility to separase. Here, we report using mass spectrometry and anti-phospho antibodies that the central domain of Rad21, the separase-target subunit of Schizosaccharomyces pombe cohesin, is regulated by various kinase-induced phosphorylation at nine residues, indicating the multiple roles for S. pombe cohesin. In vegetative and non-dividing G0 cells, Rad21 is phosphorylated by unknown S/TP-consensus kinases, in mitotic and non-mitotic cells by polo/Plo1 and CDK, and in DNA-damaged cells by Rad3/ATR. While mitotic phosphorylation is implicated in the dissociation of Rad21 and its cleavage by separase in anaphase, the Rad3/ATR-dependent damage-induced phosphorylation occurs intensively at the time of repair completion, and only in post-replicative cells. This damage-induced Rad21 phosphorylation is involved in the recovery process of cells from checkpoint arrest, and needed for the removal of cohesin by separase after the completion of damage repair. These complex phospho-regulations of Rad21 indicate the functional significance of cohesin in cell adaptation to a variety of cellular conditions.     

Cohesin gene mutations in tumorigenesis: from discovery to clinical significance

Cohesin is a multi-protein complex composed of four core subunits (SMC1A, SMC3, RAD21, and either STAG1 or STAG2) that is responsible for the cohesion of sister chromatids following DNA replication until its cleavage during mitosis thereby enabling faithful segregation of sister chromatids into two daughter cells. Recent cancer genomics analyses have discovered a high frequency of somatic mutations in the genes encoding the core cohesin subunits as well as cohesin regulatory factors (e.g. NIPBL, PDS5B, ESPL1) in a select subset of human tumors including glioblastoma, Ewing sarcoma, urothelial carcinoma, acute myeloid leukemia, and acute megakaryoblastic leukemia. Herein we review these studies including discussion of the functional significance of cohesin inactivation in tumorigenesis and potential therapeutic mechanisms to selectively target cancers harboring cohesin mutations. [BMB Reports 2014; 47(6): 299-310]     

Casein Kinase 1 and Phosphorylation of Cohesin Subunit Rec11 (SA3) Promote Meiotic Recombination through Linear Element Formation

Proper meiotic chromosome segregation, essential for sexual reproduction, requires timely formation and removal of sister chromatid cohesion and crossing-over between homologs. Early in meiosis cohesins hold sisters together and also promote formation of DNA double-strand breaks, obligate precursors to crossovers. Later, cohesin cleavage allows chromosome segregation. We show that in fission yeast redundant casein kinase 1 homologs, Hhp1 and Hhp2, previously shown to regulate segregation via phosphorylation of the Rec8 cohesin subunit, are also required for high-level meiotic DNA breakage and recombination. Unexpectedly, these kinases also mediate phosphorylation of a different meiosis-specific cohesin subunit Rec11. This phosphorylation in turn leads to loading of linear element proteins Rec10 and Rec27, related to synaptonemal complex proteins of other species, and thereby promotes DNA breakage and recombination. Our results provide novel insights into the regulation of chromosomal features required for crossing-over and successful reproduction. The mammalian functional homolog of Rec11 (STAG3) is also phosphorylated during meiosis and appears to be required for fertility, indicating wide conservation of the meiotic events reported here.     

Cohesin acetylation and Wapl‐Pds5 oppositely regulate translocation of cohesin along DNA

Cohesin is a ring‐shaped protein complex that plays a crucial role in sister chromatid cohesion and gene expression. The dynamic association of cohesin with chromatin is essential for these functions. However, the exact nature of cohesin dynamics, particularly cohesin translocation, remains unclear. We evaluated the dynamics of individual cohesin molecules on DNA and found that the cohesin core complex possesses an intrinsic ability to traverse DNA in an adenosine triphosphatase (ATPase)‐dependent manner. Translocation ability is suppressed in the presence of Wapl‐Pds5 and Sororin; this suppression is alleviated by the acetylation of cohesin and the action of mitotic kinases. In Xenopus laevis egg extracts, cohesin is translocated on unreplicated DNA in an ATPase‐ and Smc3 acetylation‐dependent manner. Cohesin movement changes from bidirectional to unidirectional when cohesin faces DNA replication; otherwise, it is incorporated into replicating DNA without being translocated or is dissociated from replicating DNA. This study provides insight into the nature of individual cohesin dynamics and the mechanisms by which cohesin achieves cohesion in different chromatin contexts.     

Functional characterization of cohesin subunit SCC1 in Trypanosoma brucei and dissection of mutant phenotypes in two life cycle stages

In yeast and metazoa, structural maintenance of chromosome (SMC) complexes play key roles in chromosome segregation, architecture and DNA repair. The main function of the cohesin complex is to hold replicated sister chromatids together until segregation at anaphase, which is dependent on proteolytic cleavage of the cohesin subunit SCC1. Analysis of trypanosomatid genomes showed that the core cohesin and condensin complexes are conserved, but SMC5/6 is absent. To investigate the functional conservation of cohesin in eukaryotes distantly related to yeast and metazoa, we characterized the Trypanosoma brucei SCC1 orthologue. TbSCC1 is expressed prior to DNA synthesis at late G1, remains in the nucleus throughout S- and G2-phases of the cell cycle and disappears at anaphase. Depletion of SCC1 by RNAi or expression of a non-cleavable SCC1 resulted in karyokinesis failure. Using the dominant negative phenotype of non-cleavable SCC1 we investigated checkpoint regulation of cytokinesis in response to mitosis failure at anaphase. In the absence of chromosome segregation, procyclic trypanosomes progressed through cytokinesis to produce one nucleated and one anucleate cell (zoid). In contrast, cytokinesis was incomplete in bloodstream forms, where cleavage was initiated but cells failed to progress to abscission. Kinetoplast duplication was uninterrupted resulting in cells with multiple kinetoplasts and flagella.     

Orchestrating anaphase and mitotic exit: separase cleavage and localization of Slk19

Anaphase in budding yeast is triggered by cleavage of the central subunit, Scc1, of the chromosomal cohesin complex by the protease separase. Here we show that separase also cleaves the kinetochore-associated protein Slk19 at anaphase onset. Separase activity is also required for the proper localization of a stable Slk19 cleavage product to the spindle midzone in anaphase. The cleavage and localization of Slk19 are necessary to stabilize the anaphase spindle, and we show that a stable spindle is a prerequisite for timely exit from mitosis. This demonstrates the cleavage of targets other than cohesin by separase in the orchestration of high-fidelity anaphase.     

Dissociation of cohesin from chromosome arms and loss of arm cohesion during early mitosis depends on phosphorylation of SA2

Cohesin is a protein complex that is required to hold sister chromatids together. Cleavage of the Scc1 subunit of cohesin by the protease separase releases the complex from chromosomes and thereby enables the separation of sister chromatids in anaphase. In vertebrate cells, the bulk of cohesin dissociates from chromosome arms already during prophase and prometaphase without cleavage of Scc1. Polo-like kinase 1 (Plk1) and Aurora-B are required for this dissociation process, and Plk1 can phosphorylate the cohesin subunits Scc1 and SA2 in vitro, consistent with the possibility that cohesin phosphorylation by Plk1 triggers the dissociation of cohesin from chromosome arms. However, this hypothesis has not been tested yet, and in budding yeast it has been found that phosphorylation of Scc1 by the Polo-like kinase Cdc5 enhances the cleavability of cohesin, but does not lead to separase-independent dissociation of cohesin from chromosomes. To address the functional significance of cohesin phosphorylation in human cells, we have searched for phosphorylation sites on all four subunits of cohesin by mass spectrometry. We have identified numerous mitosis-specific sites on Scc1 and SA2, mutated them, and expressed nonphosphorylatable forms of both proteins stably at physiological levels in human cells. The analysis of these cells lines, in conjunction with biochemical experiments in vitro, indicate that Scc1 phosphorylation is dispensable for cohesin dissociation from chromosomes in early mitosis but enhances the cleavability of Scc1 by separase. In contrast, our data reveal that phosphorylation of SA2 is essential for cohesin dissociation during prophase and prometaphase, but is not required for cohesin cleavage by separase. The similarity of the phenotype obtained after expression of nonphosphorylatable SA2 in human cells to that seen after the depletion of Plk1 suggests that SA2 is the critical target of Plk1 in the cohesin dissociation pathway.     

Nuclear Exclusion of Separase Prevents Cohesin Cleavage in Interphase Cells

During mitosis, equal transmission of the duplicated chromosomes demands a strict regulation of separase, which cleaves cohesin and triggers sister chromatid separation in anaphase. Vertebrate separase is inhibited by securin and the inhibitory phosphorylation of separase. However, knockout experiments indicate that securin is dispensable and the inhibitory phosphorylation was observed only in M phase cells. This begs the question how cohesin cleavage by separase is prevented in the absence these two mechanisms. Here we show that separase is excluded from cohesin by the nuclear envelope, which forms in telophase and disassembles in mitosis. The exclusion is achieved passively by its large physical mass and may be backed up by the CRM1-dependent nuclear export. A functional NES motif is identified in separase. We demonstrated that the nuclear envelope is sufficient to prevent active separase from cleaving nuclear cohesin. We propose that the nuclear exclusion is important to prevent cohesin cleavage during interphase in the absence of securin and the phosphorylation inhibition.     

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.     

Live-cell imaging reveals a stable cohesin-chromatin interaction after but not before DNA replication

Cohesin is a multisubunit protein complex that links sister chromatids from replication until segregation. The lack of obvious cohesin-targeting-specific sequences on DNA, as well as cohesin's molecular arrangement as a large ring, has led to the working hypothesis that cohesin acts as a direct topological linker. To preserve the identity of sister chromatids, such a linkage would need to stably persist throughout the entire S and G2 phases of the cell cycle. Unexpectedly, cohesin binds chromatin already in telophase, and a large fraction dissociates from chromosomes during prophase in a phosphorylation-dependent manner, whereas initiation of anaphase requires proteolytic cleavage of only a small fraction of cohesin. These observations raised the question of how and when cohesin interacts with chromatin during the cell cycle. Here, we report a cell-cycle dependence in the stability of cohesin binding to chromatin. Using photobleaching and quantitative live-cell imaging, we identified several cohesin pools with different chromatin binding stabilities. Although all chromatin bound cohesin dissociated after a mean residence time of less than 25 min before replication, about one-third of cohesin was bound much more stably after S phase and persisted until metaphase, consistent with long-lived links mediating cohesion between sister chromatids.     

Specific recruitment of human cohesin to laser-induced DNA damage

Cohesin is a conserved multiprotein complex that plays an essential role in sister chromatid cohesion. During interphase, cohesin is required for the establishment of cohesion following DNA replication. Because cohesin mutants resulted in increased sensitivity to DNA damage, a role for cohesin in DNA repair was also suggested. However, it was unclear whether this was due to general perturbation of cohesion or whether cohesin has a specialized role at the damage site. We therefore used a laser microbeam to create DNA damage at discrete sites in the cell nucleus and observed specific in vivo assembly of proteins at these sites by immunofluorescent detection. We observed that human cohesin is recruited to the damage site immediately after damage induction. Analysis of mutant cells revealed that cohesin recruitment to the damage site is dependent on the DNA double-strand break repair factor Mre11/Rad50 but not ATM or Nbs1. Consistently, Mre11/Rad50 and cohesin interact with each other in an interphase-specific manner. This interaction peaks in S/G(2) phase, during which cohesin is recruited to the DNA damage. Our results demonstrate the S/G(2)-specific and Mre11/Rad50-dependent recruitment of human cohesin to DNA damage, suggesting a specialized subfunction for cohesin in cell cycle-specific DNA double strand break repair.