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.     

DNA binding to SMC ATPases—trapped for release

The SMC/Rad50/RecN proteins are universal DNA‐associated ABC‐type ATPases with crucial functions in genome maintenance. New insights into Rad50–DNA complex structure and cohesin regulation inspire a speculative look at the entire superfamily. Identification of a continuous DNA binding site across the Rad50 dimer interface (Liu et al, 2016 ; Seifert et al, 2016 ) suggests a similar site in cohesin. The localization of this site hints a DNA‐activated mechanism for cohesin removal from chromosomes.     

Displacement and re-accumulation of centromeric cohesin during transient pre-anaphase centromere splitting

The ring-shaped cohesin complex links sister chromatids until their timely segregation during mitosis. Cohesin is enriched at centromeres where it provides the cohesive counterforce to bipolar tension produced by the mitotic spindle. As a consequence of spindle tension, centromeric sequences transiently split in pre-anaphase cells, in some organisms up to several micrometers. This ‘centromere breathing’ presents a paradox, how sister sequences separate where cohesin is most enriched. We now show that in the budding yeast Saccharomyces cerevisiae, cohesin binding diminishes over centromeric sequences that split during breathing. We see no evidence for cohesin translocation to surrounding sequences, suggesting that cohesin is removed from centromeres during breathing. Two pools of cohesin can be distinguished. Cohesin loaded before DNA replication, which has established sister chromatid cohesion, disappears during breathing. In contrast, cohesin loaded after DNA replication is partly retained. As sister centromeres re-associate after transient separation, cohesin is reloaded in a manner independent of the canonical cohesin loader Scc2/Scc4. Efficient centromere re-association requires the cohesion establishment factor Eco1, suggesting that re-establishment of sister chromatid cohesion contributes to the dynamic behaviour of centromeres in mitosis. These findings provide new insights into cohesin behaviour at centromeres. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

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.     

The cohesin complex is required for the DNA damage‐induced G2/M checkpoint in mammalian cells

Cohesin complexes mediate sister chromatid cohesion. Cohesin also becomes enriched at DNA double‐strand break sites and facilitates recombinational DNA repair. Here, we report that cohesin is essential for the DNA damage‐induced G2/M checkpoint. In contrast to cohesin's role in DNA repair, the checkpoint function of cohesin is independent of its ability to mediate cohesion. After RNAi‐mediated depletion of cohesin, cells fail to properly activate the checkpoint kinase Chk2 and have defects in recruiting the mediator protein 53BP1 to DNA damage sites. Earlier work has shown that phosphorylation of the cohesin subunits Smc1 and Smc3 is required for the intra‐S checkpoint, but Smc1/Smc3 are also subunits of a distinct recombination complex, RC‐1. It was, therefore, unknown whether Smc1/Smc3 function in the intra‐S checkpoint as part of cohesin. We show that Smc1/Smc3 are phosphorylated as part of cohesin and that cohesin is required for the intra‐S checkpoint. We propose that accumulation of cohesin at DNA break sites is not only needed to mediate DNA repair, but also facilitates the recruitment of checkpoint proteins, which activate the intra‐S and G2/M checkpoints.     

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.     

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.     

Structural Proteins of the SMC (Structural Maintenance of Chromosomes) Family and Their Role in Chromatin Reorganization

Structural chromatin proteins of the SMC (Structural Maintenance of Chromosomes) family play an important role in structural DNA reorganization in pro- and eukaryotes. Eukaryotic SMC proteins are the core components of the cohesin and condensin complexes. The cohesin complex is responsible for sister chromatid and homolog cohesion in mitosis and meiosis. The condensin complex uses ATP energy to induce positive coiled-coils in DNA, which results in compaction of the latter and formation of mitotic chromosome scaffold. In addition, the SMC proteins constitute recombination and recombination repair complexes. In hermaphrodites of nematode Caenorhabditis elegans, the SMC protein-containing complex controls dosage compensation and inactivation of the X chromosome genes.     

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.     

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.     

Meiotic cohesin STAG3 is required for chromosome axis formation and sister chromatid cohesion

The cohesin complex is essential for mitosis and meiosis. The specific meiotic roles of individual cohesin proteins are incompletely understood. We report in vivo functions of the only meiosis‐specific STAG component of cohesin, STAG3. Newly generated STAG3‐deficient mice of both sexes are sterile with meiotic arrest. In these mice, meiotic chromosome architecture is severely disrupted as no bona fide axial elements (AE) form and homologous chromosomes do not synapse. Axial element protein SYCP3 forms dot‐like structures, many partially overlapping with centromeres. Asynapsis marker HORMAD1 is diffusely distributed throughout the chromatin, and SYCP1, which normally marks synapsed axes, is largely absent. Centromeric and telomeric sister chromatid cohesion are impaired. Centromere and telomere clustering occurs in the absence of STAG3, and telomere structure is not severely affected. Other cohesin proteins are present, localize throughout the STAG3‐devoid chromatin, and form complexes with cohesin SMC1β. No other deficiency in a single meiosis‐specific cohesin causes a phenotype as drastic as STAG3 deficiency. STAG3 emerges as the key STAG cohesin involved in major functions of meiotic cohesin.     

Histone tail-independent chromatin binding activity of recombinant cohesin holocomplex

Cohesin, an SMC (structural maintenance of chromosomes) protein-containing complex, governs several important aspects of chromatin dynamics, including the essential chromosomal process of sister chromatid cohesion. The exact mechanism by which cohesin achieves the bridging of sister chromatids is not known. To elucidate this mechanism, we reconstituted a recombinant cohesin complex and investigated its binding to DNA fragments corresponding to natural chromosomal sites with high and low cohesin occupancy in vivo. Cohesin displayed uniform but nonspecific binding activity with all DNA fragments tested. Interestingly, DNA fragments with high occupancy by cohesin in vivo showed strong nucleosome positioning in vitro. We therefore utilized a defined model chromatin fragment (purified reconstituted dinucleosome) as a substrate to analyze cohesin interaction with chromatin. The four-subunit cohesin holocomplex showed a distinct chromatin binding activity in vitro, whereas the Smc1p-Smc3p dimer was unable to bind chromatin. Histone tails and ATP are dispensable for cohesin binding to chromatin in this reaction. A model for cohesin association with chromatin is proposed.     

Cohesin's role in pluripotency and reprogramming

Cohesin is required for ES cell self-renewal and iPS-mediated reprogramming of somatic cells. This may indicate a special role for cohesin in the regulation of pluripotency genes, perhaps by mediating long-range chromosomal interactions between gene regulatory elements. However, cohesin is also essential for genome integrity, and its depletion from cycling cells induces DNA damage responses. Hence, the failure of cohesin-depleted cells to establish or maintain pluripotency gene expression could be explained by a loss of long-range interactions or by DNA damage responses that undermine pluripotency gene expression. In recent work we began to disentangle these possibilities by analyzing reprogramming in the absence of cell division. These experiments showed that cohesin was not specifically required for reprogramming, and that the expression of most pluripotency genes was maintained when ES cells were acutely depleted of cohesin. Here we take this analysis to its logical conclusion by demonstrating that deliberately inflicted DNA damage - and the DNA damage that results from proliferation in the absence of cohesin - can directly interfere with pluripotency and reprogramming. The role of cohesin in pluripotency and reprogramming may therefore be best explained by essential cohesin functions in the cell cycle.     

Dalmatian: spotting the difference in cohesin protectors

The cohesin complex prevents separation of chromosomes following their duplication until the appropriate time during cell division. In vertebrates, establishment and maintenance of cohesin‐dependent linkages depend on two distinct proteins, sororin and shugoshin. New findings published in The EMBO Journal show that in Drosophila, the function of both of these cohesin regulators is carried out by a single hybrid protein, Dalmatian.     

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.     

Intermolecular DNA interactions stimulated by the cohesin complex in vitro: implications for sister chromatid cohesion

The establishment of sister chromatid cohesion during S phase and its dissolution at the metaphase-anaphase transition are essential for the faithful segregation of chromosomes in mitosis [1-4]. Recent studies in yeast genetics and Xenopus biochemistry have identified a large protein complex, cohesin, that plays a key role in sister chromatid cohesion [5-10]. The cohesin complex consists of a heterodimeric pair of SMC (structural maintenance of chromosomes) subunits and at least two non-SMC subunits. This structural organization is reminiscent of that of condensin, another major SMC protein complex that drives chromosome condensation in eukaryotic cells [11]. Condensin has been shown to reconfigure and compact DNA in vitro by utilizing the energy of ATP hydrolysis [12]. Very little is known, however, about how cohesin works at a mechanistic level. Here we report the first set of biochemical activities associated with an intact cohesin complex purified from HeLa cell extracts. The cohesin complex binds directly to double-stranded DNA and induces the formation of large protein-DNA aggregates. In the presence of topoisomerase II, cohesin stimulates intermolecular catenation of circular DNA molecules. This activity is in striking contrast to intramolecular knotting directed by condensin [13]. Cohesin also increases the probability of intermolecular ligation of linear DNA molecules in the presence of DNA ligase. Our results are consistent with a model in which cohesin functions as an intermolecular DNA crosslinker and is part of the molecular "glue" that holds sister chromatids together [14].