Pds5B is required for cohesion establishment and Aurora B accumulation at centromeres

Cohesin mediates sister chromatid cohesion and contributes to the organization of interphase chromatin through DNA looping. In vertebrate somatic cells, cohesin consists of Smc1, Smc3, Rad21, and either SA1 or SA2. Three additional factors Pds5, Wapl, and Sororin bind to cohesin and modulate its dynamic association with chromatin. There are two Pds5 proteins in vertebrates, Pds5A and Pds5B, but their functional specificity remains unclear. Here, we demonstrate that Pds5 proteins are essential for cohesion establishment by allowing Smc3 acetylation by the cohesin acetyl transferases (CoATs) Esco1/2 and binding of Sororin. While both proteins contribute to telomere and arm cohesion, Pds5B is specifically required for centromeric cohesion. Furthermore, reduced accumulation of Aurora B at the inner centromere region in cells lacking Pds5B impairs its error correction function, promoting chromosome mis‐segregation and aneuploidy. Our work supports a model in which the composition and function of cohesin complexes differs between different chromosomal regions.     

HIV uses autophagy for its own means

Replicated sister chromatids are held together until mitosis by cohesin, a conserved multisubunit complex comprised of Smc1, Smc3, Scc1, and Scc3, which in vertebrate cells exists as two closely related homologues (SA1 and SA2). Here, we show that cohesin^SA1 and cohesin^SA2 are differentially required for telomere and centromere cohesion, respectively. Cells deficient in SA1 are unable to establish or maintain cohesion between sister telomeres after DNA replication in S phase. The same phenotype is observed upon depletion of the telomeric protein TIN2. In contrast, in SA2-depleted cells telomere cohesion is normal, but centromere cohesion is prematurely lost. We demonstrate that loss of telomere cohesion has dramatic consequences on chromosome morphology and function. In the absence of sister telomere cohesion, cells are unable to repair chromatid breaks and suffer sister telomere loss. Our studies elucidate the functional distinction between the Scc3 homologues in human cells and further reveal an essential role for sister telomere cohesion in genomic integrity.     

The many functions of cohesin—different rings to rule them all?

EMBO J 31 9, 2076–2089 March132012EMBO J 31 9, 2090–2102 March132012It is well known that somatic and germ cells use different cohesin complexes to mediate sister chromatid cohesion, but why different isoforms of cohesin also co-exist within somatic vertebrate cells has remained a mystery. Two papers in this issue of The EMBO Journal have begun to address this question by analysing mouse cells lacking SA1, an isoform of a specific cohesin subunit.When one cell divides into two, many things have to go right for the two daughter cells to receive identical copies of their mother cell''s genome. It has long been recognized that sister chromatid cohesion, the physical connection established during DNA replication between newly synthesized sister DNA molecules, is one of these essential prerequisites for proper chromosome segregation. It is this cohesion that enables the bi-orientation of chromosomes on the mitotic or meiotic spindle, and thus makes their symmetrical segregation possible. Cohesion is mediated by cohesin, a multi-subunit protein complex, which is thought to connect sister DNA molecules by embracing them as a ring (Figure 1; reviewed in Peters et al, 2008). It is well established that cohesin complexes differ between somatic and germ cells, where they are needed for the proper separation of sister chromatids and of homologous chromosomes, respectively. What has been largely ignored, however, is that even within somatic vertebrate cells there are different forms of cohesin, containing mutually exclusive variable subunits: either SA1 or the closely related SA2 protein (also known as STAG1 and STAG2, respectively), and either Pds5A or the related Pds5B subunit (Peters et al, 2008). Why is that? Two papers from the Losada lab (Remeseiro et al, 2012a, 2012b) have begun to address this question by generating mouse cells lacking the SA1 gene, revealing unexpected insights into the functions of SA1 subunit-containing cohesin complexes (cohesin-SA1).Open in a separate windowFigure 1Schematic drawing illustrating how the SA1 and SA2 proteins interact in a mutually exclusive manner with three core subunits of cohesin (Smc1, Smc3, Rad21) that form a ring-like structure. It has been proposed that these complexes mediate cohesion by trapping the two sister DNA molecules inside the cohesin ring (above), and that cohesin rings might affect chromatin structure by forming or stabilizing intra-chromatid loops (below). Cohesin is thought to influence gene regulation at least in part by mediating chromatin looping.Although cohesin is best known for its role in sister chromatid cohesion, it is clearly also needed for homologous recombination-mediated DNA repair and for gene regulation. Much of what we know about these functions comes from studies in yeast and fruit flies, organisms with only a single SA1/SA2-related mitotic subunit (Scc3 in budding yeast), and only a single Pds5 subunit. It is therefore plausible that, like many other genes during vertebrate evolution, SA1/SA2 and Pds5A/Pds5B have arisen by gene duplication to constitute paralogs, with functional differences between them assumed to be subtle. Consistently, absence of either Pds5A or Pds5B causes only mild, if any, defects in sister chromatid cohesion, and mice lacking either protein can develop to term, although they die shortly after birth owing to multiple organ defects (Zhang et al, 2007, 2009). First indications that the situation may be different for the Scc3-related subunits came from Canudas and Smith (2009), who reported that RNAi depletion of SA1 and SA2 from HeLa cells caused defects in telomere and centromere cohesion, respectively. The generation of mice lacking either one or both alleles of the SA1 gene has now allowed a more systematic and thorough analysis of SA1 function (Remeseiro et al, 2012a, 2012b).One of the most striking results obtained in these studies is that most mice lacking SA1 die around day 12 of embryonic development, clearly showing that the function of SA1 cannot be fulfilled by SA2, despite the fact that SA2 is substantially more abundant in somatic cells than SA1 (Holzmann et al, 2010). What could this SA1-specific function be? Losada and colleagues report observations, which imply that SA1 does not have just one, but possibly several important functions in different processes. First, the authors confirm the previous observation that SA1 is required for cohesion specifically at telomeres, while likely collaborating with SA2 in chromosome arms or centromeric regions. Furthermore, telomeres have an unusual morphology in mitotic chromosomes lacking SA1 (Remeseiro et al, 2012a), reminiscent of a fragile-site phenotype previously reported in telomeres with DNA replication defects (Sfeir et al, 2009), and SA1 is indeed required for efficient telomere duplication. Depletion of sororin, a protein that is required for cohesin''s ability to mediate sister chromatid cohesion, also causes a fragile-site phenotype at telomeres. These findings imply that SA1''s role in telomere cohesion is important for efficient telomere replication, perhaps, as the authors speculate, because telomere cohesion may help to stabilize or re-start stalled replication forks, or because cohesion-dependent homologous recombination might be involved in repair of DNA double strand breaks created by collapsed replication forks. Interestingly, cells lacking SA1 frequently show chromosome bridges in anaphase, often fail to divide, and either die or become bi-nucleated. The exact origin of chromosome bridges is difficult to determine, but previous studies have found such bridges often associated with fragile sites on chromosomes; treatment with low doses of DNA replication inhibitors was shown to increase the frequency of such bridges (Chan et al, 2009), and similar observations were indeed made by Remeseiro et al (2012a) in mouse embryonic fibroblasts. It is therefore plausible that the telomere cohesion defect observed in SA1-lacking cells leads to incomplete telomere replication, which in turn results in the formation of anaphase chromosome bridges and subsequent cytokinesis defects. Losada and colleagues further speculate that these chromosome segregation defects could underlie the increased frequency of spontaneous development of various tumours in mice containing just one instead of two SA1 alleles (Remeseiro et al, 2012a). This is an attractive interpretation since tetraploidy and aneuploidy are thought to contribute to the rate with which tumour cells can evolve; however, Losada and colleagues report SA1 deficiency to cause defects also in other cohesin functions, which may therefore as well contribute to tumour formation.To further understand why SA1 cannot be fulfilled by SA2, Losada and colleagues also analysed the distribution of these proteins in the non-repetitive parts of the mouse genome by chromatin immunoprecipitation coupled to deep sequencing (ChIP-seq). The results of these experiments, published in the second of the two papers (Remeseiro et al, 2012b), raise the interesting possibility that cohesin-SA1 associates more frequently with gene promoters than cohesin-SA2. However, the fact that different antibodies have to be used for any ChIP-based comparison of the distribution of two proteins makes it difficult to know to what degree observed differences might be due to different antibody efficiency. Obviously, such limitations do not exist if the distribution of one and the same protein is analysed under different conditions, and in such an experimental setting, Remeseiro et al indeed make some striking observations. When SA1 is absent, SA2 does not detectably change in abundance, but its distribution in the genome does, in that more than half of all SA2-binding sites in SA1-deficient cells differ from those bound in wild-type cells. Most SA2-binding sites in SA1-deficient cells are in intergenic regions, and CTCF, a zinc finger protein often co-localizing with cohesin and implicated in its gene regulation function (Peters et al, 2008), appears to be absent at many of these sites. It presently remains a mystery why cohesin-SA2 changes its distribution so dramatically in the absence of SA1, but the observation that gene promoters are more frequently occupied by cohesin in the presence of SA1 than in its absence raises the possibility that cohesin-SA1 may have a specific role in gene regulation. This possibility is particularly interesting in light of a recent study that found hardly any change in gene expression upon re-expression of SA2 in SA2-deficient human glioblastoma cells (Solomon et al, 2011), despite the fact that cohesin is thought to regulate numerous genes. With this in mind, Remeseiro et al analysed gene expression in mouse cells and indeed found 549 genes to be mis-regulated in the absence of SA1, in striking contrast to the above-mentioned comparison of human SA2-deficient or proficient cells that found only 19 genes to change in expression levels (Solomon et al, 2011). Obviously direct comparisons will be essential to analyse further the specific roles of SA1 and SA2 in gene regulation, but the current evidence raises the interesting possibility that SA1 may have a particularly important role in gene regulation, whereas cohesin-SA2 is dedicated to creating arm and centromeric cohesive structures for chromosome segregation.That is not to say that cohesin-SA1 cannot mediate sister chromatid cohesion. It almost certainly can, as it is essential for cohesion at telomeres (Canudas and Smith, 2009; Remeseiro et al, 2012a). Likewise, it would be wrong to assume that we now fully understand why SA1 and SA2 co-exist in somatic vertebrate cells, and what their precise functions is. There are many things we do not understand. For example, if SA2 has little or no role in gene regulation, as the Solomon et al (2011) study implies, why does SA2 nevertheless interact directly with CTCF (Xiao et al, 2011), its gene regulation collaborator? How do cohesin-SA1 and cohesin-SA2 complexes further differ in their genomic distributions and their functions depending on whether they contain either Pds5A or Pds5B, constituting really not just two but four distinct cohesin complexes? The work by Losada and colleagues represents an important step towards understanding these questions, but there is still a long and presumably exciting way to go to understand how different cohesin complexes control the mammalian genome.     

Cohesin Acetylation Promotes Sister Chromatid Cohesion Only in Association with the Replication Machinery

Acetylation of the Smc3 subunit of cohesin is essential to establish functional cohesion between sister chromatids. Smc3 acetylation is catalyzed by members of the Eco family of acetyltransferases, although the mechanism by which acetylation is regulated and how it promotes cohesion are largely unknown. In vertebrates, the cohesin complex binds to chromatin during mitotic exit and is converted to a functional form during or shortly after DNA replication. The conserved proliferating cell nuclear antigen-interacting protein box motif in yeast Eco1 is required for function, and cohesin is acetylated during the S phase. This has led to the notion that acetylation of cohesin is stimulated by interaction of Eco1 with the replication machinery. Here we show that in vertebrates Smc3 acetylation occurs independently of DNA replication. Smc3 is readily acetylated before replication is initiated and after DNA replication is complete. However, we also show that functional acetylation occurs only in association with the replication machinery: disruption of the interaction between XEco2 and proliferating cell nuclear antigen prevents cohesion establishment while having little impact on the overall levels of Smc3 acetylation. These results demonstrate that Smc3 acetylation can occur throughout interphase but that only acetylation in association with the replication fork promotes sister chromatid cohesion. These data reveal how the generation of cohesion is limited to the appropriate time and place during the cell cycle and provide insight into the mechanism by which acetylation ensures cohesion.     

Distinct Functions of Human Cohesin-SA1 and Cohesin-SA2 in Double-Strand Break Repair

Cohesin is an essential multiprotein complex that mediates sister chromatid cohesion critical for proper segregation of chromosomes during cell division. Cohesin is also involved in DNA double-strand break (DSB) repair. In mammalian cells, cohesin is involved in both DSB repair and the damage checkpoint response, although the relationship between these two functions is unclear. Two cohesins differing by one subunit (SA1 or SA2) are present in somatic cells, but their functional specificities with regard to DNA repair remain enigmatic. We found that cohesin-SA2 is the main complex corecruited with the cohesin-loading factor NIPBL to DNA damage sites in an S/G₂-phase-specific manner. Replacing the diverged C-terminal region of SA1 with the corresponding region of SA2 confers this activity on SA1. Depletion of SA2 but not SA1 decreased sister chromatid homologous recombination repair and affected repair pathway choice, indicating that DNA repair activity is specifically associated with cohesin recruited to damage sites. In contrast, both cohesin complexes function in the intra-S checkpoint, indicating that cell cycle-specific damage site accumulation is not a prerequisite for cohesin''s intra-S checkpoint function. Our findings reveal the unique ways in which cohesin-SA1 and cohesin-SA2 participate in the DNA damage response, coordinately protecting genome integrity in human cells.     

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.     

Characterization of the Interaction between the Cohesin Subunits Rad21 and SA1/2

The cohesin complex is responsible for the fidelity of chromosomal segregation during mitosis. It consists of four core subunits, namely Rad21/Mcd1/Scc1, Smc1, Smc3, and one of the yeast Scc3 orthologs SA1 or SA2. Sister chromatid cohesion is generated during DNA replication and maintained until the onset of anaphase. Among the many proposed models of the cohesin complex, the ''core'' cohesin subunits Smc1, Smc3, and Rad21 are almost universally displayed as tripartite ring. However, other than its supportive role in the cohesin ring, little is known about the fourth core subunit SA1/SA2. To gain deeper insight into the function of SA1/SA2 in the cohesin complex, we have mapped the interactive regions of SA2 and Rad21 in vitro and ex vivo. Whereas SA2 interacts with Rad21 through a broad region (301–750 aa), Rad21 binds to SA proteins through two SA-binding motifs on Rad21, namely N-terminal (NT) and middle part (MP) SA-binding motif, located at 60–81 aa of the N-terminus and 383–392 aa of the MP of Rad21, respectively. The MP SA-binding motif is a 10 amino acid, α-helical motif. Deletion of these 10 amino acids or mutation of three conserved amino acids (L³⁸⁵, F³⁸⁹, and T³⁹⁰) in this α-helical motif significantly hinders Rad21 from physically interacting with SA1/2. Besides the MP SA-binding motif, the NT SA-binding motif is also important for SA1/2 interaction. Although mutations on both SA-binding motifs disrupt Rad21-SA1/2 interaction, they had no apparent effect on the Smc1-Smc3-Rad21 interaction. However, the Rad21-Rad21 dimerization was reduced by the mutations, indicating potential involvement of the two SA-binding motifs in the formation of the two-ring handcuff for chromosomal cohesion. Furthermore, mutant Rad21 proteins failed to significantly rescue precocious chromosome separation caused by depletion of endogenous Rad21 in mitotic cells, further indicating the physiological significance of the two SA-binding motifs of Rad21.     

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.     

Calpain-1 cleaves Rad21 to promote sister chromatid separation

Defining the mechanisms of chromosomal cohesion and dissolution of the cohesin complex from chromatids is important for understanding the chromosomal missegregation seen in many tumor cells. Here we report the identification of a novel cohesin-resolving protease and describe its role in chromosomal segregation. Sister chromatids are held together by cohesin, a multiprotein ring-like complex comprised of Rad21, Smc1, Smc3, and SA2 (or SA1). Cohesin is known to be removed from vertebrate chromosomes by two distinct mechanisms, namely, the prophase and anaphase pathways. First, PLK1-mediated phosphorylation of SA2 in prophase leads to release of cohesin from chromosome arms, leaving behind centromeric cohesins that continue to hold the sisters together. Then, at the onset of anaphase, activated separase cleaves the centromeric cohesin Rad21, thereby opening the cohesin ring and allowing the sister chromatids to separate. We report here that the calcium-dependent cysteine endopeptidase calpain-1 is a Rad21 peptidase and normally localizes to the interphase nuclei and chromatin. Calpain-1 cleaves Rad21 at L192, in a calcium-dependent manner. We further show that Rad21 cleavage by calpain-1 promotes separation of chromosome arms, which coincides with a calcium-induced partial loss of cohesin at several chromosomal loci. Engineered cleavage of Rad21 at the calpain-cleavable site without activation of calpain-1 can lead to a loss of sister chromatid cohesion. Collectively, our work reveals a novel function of calpain-1 and describes an additional pathway for sister chromatid separation in humans.     

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.     

The DNA-binding protein CST associates with the cohesin complex and promotes chromosome cohesion

Sister chromatid cohesion (SCC), the pairing of sister chromatids after DNA replication until mitosis, is established by loading of the cohesin complex on newly replicated chromatids. Cohesin must then be maintained until mitosis to prevent segregation defects and aneuploidy. However, how SCC is established and maintained until mitosis remains incompletely understood, and emerging evidence suggests that replication stress may lead to premature SCC loss. Here, we report that the ssDNA-binding protein CTC1-STN1-TEN1 (CST) aids in SCC. CST primarily functions in telomere length regulation but also has known roles in replication restart and DNA repair. After depletion of CST subunits, we observed an increase in the complete loss of SCC. In addition, we determined that CST associates with the cohesin complex. Unexpectedly, we did not find evidence of altered cohesin loading or mitotic progression in the absence of CST; however, we did find that treatment with various replication inhibitors increased the association between CST and cohesin. Because replication stress was recently shown to induce SCC loss, we hypothesized that CST may be required to maintain or remodel SCC after DNA replication fork stalling. In agreement with this idea, SCC loss was greatly increased in CST-depleted cells after exogenous replication stress. Based on our findings, we propose that CST aids in the maintenance of SCC at stalled replication forks to prevent premature cohesion loss.     

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.     

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.     

Pds5 cooperates with cohesin in maintaining sister chromatid cohesion

BACKGROUND: Sister chromatid cohesion depends on a complex called cohesin, which contains at least four subunits: Smc1, Smc3, Scc1 and Scc3. Cohesion is established during DNA replication, is partially dismantled in many, but not all, organisms during prophase, and is finally destroyed at the metaphase-to-anaphase transition. A quite separate protein called Spo76 is required for sister chromatid cohesion during meiosis in the ascomycete Sordaria. Spo76-like proteins are highly conserved amongst eukaryotes and a homologue in Aspergillus nidulans, called BimD, is required for the completion of mitosis. The isolation of the cohesin subunit Smc3 as a suppressor of BimD mutations suggests that Spo76/BimD might function in the same process as cohesin. RESULTS: We show here that the yeast homologue of Spo76, called Pds5, is essential for establishing sister chromatid cohesion and maintaining it during metaphase. We also show that Pds5 co-localizes with cohesin on chromosomes, that the chromosomal association of Pds5 and cohesin is interdependent, that Scc1 recruits Pds5 to chromosomes in G1 and that its cleavage causes dissociation of Pds5 from chromosomes at the metaphase-to-anaphase transition. CONCLUSIONS: Our data show that Pds5 functions as part of the same process as cohesin. Sequence similarities and secondary structure predictions indicate that Pds5 consists of tandemly repeated HEAT repeats, and might therefore function as a protein-protein interaction scaffold, possibly in the cohesin-DNA complex assembly.     

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.     

Loss of ChlR1 Helicase in Mouse Causes Lethality Due to the Accumulation of Aneuploid Cells Generated by Cohesion Defects and Placental Malformation

Human DDX11 and DDX12 are closely related genes encoding the helicases ChlR1 and ChlR2, which belong to the CHL1 DNA helicase family. Recently, it was shown that human ChlR1 interacts with components of the cohesin complex and is required for proper centromeric cohesion. To establish the function of ChlR1 in development we made a mutant mouse lacking Ddx11, the single mouse ChlR gene. The absence of Ddx11 resulted in embryonic lethality at E10.5. The mutant embryos were smaller in size, malformed and exhibited sparse cellularity in comparison to normal or heterozygous litter mates. Importantly, loss of Ddx11 resulted in the inability to form a proper placenta, indicating that ChlR1 is essential for placental formation. Detailed analysis of cells isolated from Ddx11-/- embryos revealed a G2/M cell cycle delay, an increased frequency of chromosome missegregation, decreased chromosome cohesion, and increased aneuploidy. To examine whether ChlR proteins are required for arm cohesion and for loading of the cohesin complex, further studies were preformed in ChlR1 siRNA treated cells. These studies revealed that ChlR1 is required for proper sister chromatid arm cohesion and that cohesin complexes bind more loosely to chromatin in the absence of ChlR1. Taken together, these studies provide the first data indicating that the ChlR1 helicase is essential for proper binding of the cohesin complex to both the centromere and the chromosome arms, and indicate that ChlR1 is essential for embryonic development and the prevention of aneuploidy in mammals.     

Structural insights into the regulation of cohesion establishment by Wpl1

Correct segregation of duplicated chromosomes to daughter cells during mitosis requires the action of the cohesin complex. This tripartite ring‐shaped molecule is involved in holding replicated sister chromatids together from S phase until anaphase onset. Establishment of stable cohesion involves acetylation of the Smc3 component of cohesin during replication by the Eco1 acetyltransferase. This has been proposed to antagonise the activity of another member of the cohesin complex, Wpl1. Here, we describe the X‐ray structure of the conserved Wapl domain, and demonstrate that it binds the ATPase head of the Smc3 protein. We present data that suggest that Wpl1 may be involved in regulating the ATPase activity of cohesin, and that this may be subject to the acetylation state of Smc3. In addition, we present a structure of the Wapl domain bound to a functionally relevant segment of the Smc3 ATPase.