The INO80 Chromatin Remodeling Complex Functions in Sister Chromatid Cohesion

Here we identify a defect in sister chromatid cohesion in the Saccharomyces serevisiae arp8 mutant, which impairs the chromatin remodeling activity of the INO80 complex, and we report the direct association of Ino80 with centromeres and cohesin-associated regions. In early S phase, Ino80 is recruited to replication forks along with Ctf18 and PCNA, both of which are involved in the establishment of sister chromatid cohesion. The arp8 mutation perturbs the association of Ctf18 and PCNA but not of cohesin with replication forks. We propose that the INO80 complex is required for the proper establishment of sister chromatid cohesion.     

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.     

Sororin,the Cell Cycle and Sister Chromatid Cohesion

Sister chromatid cohesion is essential for the maintenance of genome integrity. Errors in regulation of cohesion result in increased sensitivity to DNA damage, mis-segregation of chromosomes, and loss of genetic information. We recently showed that sororin is an essential regulator of sister chromatid cohesion in vertebrates. Interestingly, we identified sororin in a screen for proteins whose levels are controlled by the Anaphase Promoting Complex (APC), a cell cycle –regulated ubiquitin ligase. Ubiquitination by the APC and the resulting degradation ensure that sororin levels are low throughout G1 and only rise during S phase. We speculate that this regulation is an essential part of the mechanism that ensures that cohesion is established only after there are in fact two sister chromatids to tie together. Cohesion thus established can then be used both to mediate recombinational DNA repair, as well as to ensure accurate sister chromatid segregation in anaphase. Both of these roles are essential to genome stability.     

The Sister Chromatid Cohesion Pathway Suppresses Multiple Chromosome Gain and Chromosome Amplification

Gain or loss of chromosomes resulting in aneuploidy can be important factors in cancer and adaptive evolution. Although chromosome gain is a frequent event in eukaryotes, there is limited information on its genetic control. Here we measured the rates of chromosome gain in wild-type yeast and sister chromatid cohesion (SCC) compromised strains. SCC tethers the newly replicated chromatids until anaphase via the cohesin complex. Chromosome gain was measured by selecting and characterizing copper-resistant colonies that emerged due to increased copies of the metallothionein gene CUP1. Although all defective SCC diploid strains exhibited increased rates of chromosome gain, there were 15-fold differences between them. Of all mutants examined, a hypomorphic mutation at the cohesin complex caused the highest rate of chromosome gain while disruption of WPL1, an important regulator of SCC and chromosome condensation, resulted in the smallest increase in chromosome gain. In addition to defects in SCC, yeast cell type contributed significantly to chromosome gain, with the greatest rates observed for homozygous mating-type diploids, followed by heterozygous mating type, and smallest in haploids. In fact, wpl1-deficient haploids did not show any difference in chromosome gain rates compared to wild-type haploids. Genomic analysis of copper-resistant colonies revealed that the “driver” chromosome for which selection was applied could be amplified to over five copies per diploid cell. In addition, an increase in the expected driver chromosome was often accompanied by a gain of a small number of other chromosomes. We suggest that while chromosome gain due to SCC malfunction can have negative effects through gene imbalance, it could also facilitate opportunities for adaptive changes. In multicellular organisms, both factors could lead to somatic diseases including cancer.     

The STRUCTURAL MAINTENANCE OF CHROMOSOMES 5/6 Complex Promotes Sister Chromatid Alignment and Homologous Recombination after DNA Damage in Arabidopsis thaliana

Sister chromatids are often arranged as incompletely aligned entities in interphase nuclei of Arabidopsis thaliana. The STRUCTURAL MAINTENANCE OF CHROMOSOMES (SMC) 5/6 complex, together with cohesin, is involved in double-strand break (DSB) repair by sister chromatid recombination in yeasts and mammals. Here, we analyzed the function of genes in Arabidopsis. The wild-type allele of SMC5 is essential for seed development. Each of the two SMC6 homologs of Arabidopsis is required for efficient repair of DNA breakage via intermolecular homologous recombination in somatic cells. Alignment of sister chromatids is enhanced transiently after X-irradiation (and mitomycin C treatment) in wild-type nuclei. In the smc5/6 mutants, the x-ray–mediated increase in sister chromatid alignment is much lower and delayed. The reduced S phase–established cohesion caused by a knockout mutation in one of the α-kleisin genes, SYN1, also perturbed enhancement of sister chromatid alignment after irradiation, suggesting that the S phase–established cohesion is a prerequisite for correct DSB-dependent cohesion. The radiation-sensitive51 mutant, deficient in heteroduplex formation during DSB repair, showed wild-type frequencies of sister chromatid alignment after X-irradiation, implying that the irradiation-mediated increase in sister chromatid alignment is a prerequisite for, rather than a consequence of, DNA strand exchange between sister chromatids. Our results suggest that the SMC5/6 complex promotes sister chromatid cohesion after DNA breakage and facilitates homologous recombination between sister chromatids.     

Arabidopsis Separase Functions beyond the Removal of Sister Chromatid Cohesion during Meiosis

Separase is a capase family protease that is required for the release of sister chromatid cohesion during meiosis and mitosis. Proteolytic cleavage of the α-kleisin subunit of the cohesin complex at the metaphase-to-anaphase transition is essential for the proper segregation of chromosomes. In addition to its highly conserved role in cleaving the α-kleisin subunit, separase appears to have acquired additional diverse activities in some organisms, including involvement in mitotic and meiotic anaphase spindle assembly and elongation, interphase spindle pole body positioning, and epithelial cell reorganization. Results from the characterization of Arabidopsis (Arabidopsis thaliana) separase (ESP) demonstrated that meiotic expression of ESP RNA interference blocked the proper removal of cohesin from chromosomes and resulted in the presence of a mixture of fragmented chromosomes and intact bivalents. The presence of large numbers of intact bivalents raised the possibility that separase may also have multiple roles in Arabidopsis. In this report, we show that meiotic expression of ESP RNA interference blocks the removal of cohesin during both meiosis I and II, results in alterations in nonhomologous centromere association, disrupts the radial microtubule system after telophase II, and affects the proper establishment of nuclear cytoplasmic domains, resulting in the formation of multinucleate microspores.The proper segregation of chromosomes during mitosis and meiosis is dependent on the systematic formation and subsequent removal of sister chromatid cohesion, which is required for homologous chromosome pairing, recombination, and repair (for review, see Onn et al., 2008; Peters et al., 2008). It is also required for the pairwise alignment of chromosomes on the metaphase I spindle and for the generation of tension across centromeres, thereby ensuring their bipolar attachment. In mitosis, cohesion is maintained by the cohesin complex, which consists of four evolutionally conserved proteins: Sister Chromatid Cohesion1 (SCC1), SCC3, Structural Maintenance of Chromosome1 (SMC1), and SMC3 (for review, see Nasmyth and Haering, 2005). During meiosis, SCC1 is largely replaced by its meiotic homolog REC8.The establishment of sister chromatid cohesion in yeast involves a multistep process (Milutinovich et al., 2007) that begins during telophase of the previous cell cycle when cohesin subunits associate with the chromatin, ultimately becoming enriched at discrete loci termed cohesin-associated regions (Blat and Kleckner, 1999; Laloraya et al., 2000). Cohesion is established during S-phase in a process that requires the Chromosome Transmission Fidelity protein (Ctf7), which is also known as Eco1 (Skibbens et al., 1999; Toth et al., 1999) and involves the replication fork (Kenna and Skibbens, 2003; Lengronne et al., 2006). In budding yeast (Saccharomyces cerevisiae) and fission yeast (Schizosaccharomyces pombe), cohesin complexes remain on the chromosomes until mitotic anaphase (Uhlmann et al., 1999, 2000; Tomonaga et al., 2000). In contrast, in vertebrates, most cohesin complexes are released from the chromosomes during prophase in a separase-independent process (Waizenegger et al., 2000; Losada et al., 2002). The small fraction of cohesin that remains primarily in centromeric regions is released to start anaphase (Sumara et al., 2000). The release of chromosome cohesion at the metaphase-to-anaphase transition is triggered by the Cys protease, separase (ESP1), which specifically cleaves the α-kleisin subunit (Ciosk et al., 1998; Uhlmann et al., 1999, 2000; Buonomo et al., 2000; Hauf et al., 2001). Prior to the metaphase-to-anaphase transition, securin inhibits the protease activity of separase. At the onset of anaphase, securin is degraded by the anaphase-promoting complex/cyclosome freeing separase, which cleaves SCC1, facilitating the release of cohesion and chromosome separation (Cohen-Fix et al., 1996; Ciosk et al., 1998).Studies on the distribution of cohesin proteins during meiosis in a number of organisms, including yeast, Caenorhabditis elegans, mammals, and Arabidopsis (Arabidopsis thaliana), have shown that similar to the situation during mitosis in animal cells, a significant amount of cohesin is either removed from or redistributed on prophase chromosomes in a separase-independent process (Pasierbek et al., 2001; Cai et al., 2003; Eijpe et al., 2003; Lee et al., 2003; Yu and Koshland, 2005). The final resolution of chiasmata, formed as the result of homologous chromosome recombination, and the separation of homologous chromosomes depends on separase cleavage of the meiotic α-kleisin subunit, REC8, along chromosome arms at anaphase I (Buonomo et al., 2000; Kitajima et al., 2003). Centromeric cohesion is protected by the conserved SGO family of proteins until anaphase II when separase cleavage of REC8 facilitates the separation of sister chromatids (Rabitsch et al., 2003; Katis et al., 2004; McGuinness et al., 2005).In addition to its highly conserved role in cleaving the α-kleisin subunit, separase appears to have acquired additional diverse activities in different organisms (Queralt and Uhlmann, 2005). For example, separase plays a role in DNA repair by promoting the redistribution of cohesin complexes to sites of DNA damage during mitotic interphase in budding and fission yeast (Nagao et al., 2004; Strom et al., 2004). Separase is also important for mitotic anaphase spindle assembly and elongation (Jensen et al., 2001; Papi et al., 2005; Baskerville et al., 2008), interphase spindle pole body positioning (Nakamura et al., 2002), and spindle formation during meiosis in yeast (Buonomo et al., 2003). It is also important for the proper positioning of the centrosomes during the first asymmetric mitotic division, eggshell development in C. elegans (Siomos et al., 2001; Rappleye et al., 2002), and for epithelial cell reorganization and dynamics in Drosophila melanogaster (Pandey et al., 2005). In zebra fish, a separase mutation causes genome instability and increased susceptibility to epithelial cancer (Shepard et al., 2007).Results from the characterization of Arabidopsis separase suggested that the protein also has multiple roles in plants (Liu and Makaroff, 2006). Seeds homozygous for a T-DNA insert in Arabidopsis ESP exhibited embryo arrest at the globular stage with the endosperm exhibiting a weak titan-like phenotype. Furthermore, expression of ESP RNA interference (RNAi) from the meiosis-specific DMC1 promoter disrupted the proper removal of the SYN1 cohesin protein from chromosomes during meiosis and resulted in the presence of a mixture of fragmented chromosomes and intact bivalents. The presence of large numbers of intact bivalents led the authors to suggest that in addition to its requirement for the removal of cohesin, ESP may also be required for either the proper attachment of the kinetochores to the spindle or spindle function. These findings, along with the observations that separase appears to have multiple roles in other organisms, led us to conduct a detailed characterization of meiosis in ESP RNAi plants.In this report, we show that meiotic expression of ESP RNAi blocks the release of sister chromatid cohesion during both meiosis I and II, results in nonhomologous centromere association, disrupts the radial microtubule system (RMS) after telophase II, and affects the proper establishment of nuclear cytoplasmic domains. Unlike the large majority of plant meiotic mutants that have been characterized to date, reduction of ESP levels during meiosis leads to the formation of multinucleate microspores.     

The Smc5/Smc6/MAGE Complex Confers Resistance to Caffeine and Genotoxic Stress in Drosophila melanogaster

The SMC5/6 protein complex consists of the Smc5, Smc6 and Non-Smc-Element (Nse) proteins and is important for genome stability in many species. To identify novel components in the DNA repair pathway, we carried out a genetic screen to identify mutations that confer reduced resistance to the genotoxic effects of caffeine, which inhibits the ATM and ATR DNA damage response proteins. This approach identified inactivating mutations in CG5524 and MAGE, homologs of genes encoding Smc6 and Nse3 in yeasts. The fact that Smc5 mutants are also caffeine-sensitive and that Mage physically interacts with Drosophila homologs of Nse proteins suggests that the structure of the Smc5/6 complex is conserved in Drosophila. Although Smc5/6 proteins are required for viability in S. cerevisiae, they are not essential under normal circumstances in Drosophila. However, flies carrying mutations in Smc5, Smc6 and MAGE are hypersensitive to genotoxic agents such as ionizing radiation, camptothecin, hydroxyurea and MMS, consistent with the Smc5/6 complex serving a conserved role in genome stability. We also show that mutant flies are not compromised for pre-mitotic cell cycle checkpoint responses. Rather, caffeine-induced apoptosis in these mutants is exacerbated by inhibition of ATM or ATR checkpoint kinases but suppressed by Rad51 depletion, suggesting a functional interaction involving homologous DNA repair pathways that deserves further scrutiny. Our insights into the SMC5/6 complex provide new challenges for understanding the role of this enigmatic chromatin factor in multi-cellular organisms.     

Coordination of DNA damage responses via the Smc5/Smc6 complex

The detection of DNA damage activates DNA repair pathways and checkpoints to allow time for repair. Ultimately, these responses must be coordinated to ensure that cell cycle progression is halted until repair is completed. Several multiprotein complexes containing members of the structural maintenance of chromosomes family of proteins have been described, including the condensin and cohesin complexes, that are critical for chromosomal organization. Here we show that the Smc5/Smc6 (Smc5/6) complex is required for a coordinated response to DNA damage and normal chromosome integrity. Fission yeast cells lacking functional Smc6 initiate a normal checkpoint response to DNA damage, culminating in the phosphorylation and activation of the Chk1 protein kinase. Despite this, cells enter a lethal mitosis, presumably without completion of DNA repair. Another subunit of the complex, Nse1, is a conserved member of this complex and is also required for this response. We propose that the failure to maintain a checkpoint response stems from the lack of ongoing DNA repair or from defective chromosomal organization, which is the signal to maintain a checkpoint arrest. The Smc5/6 complex is fundamental to genome integrity and may function with the condensin and cohesin complexes in a coordinated manner.     

Chromosomal association of the Smc5/6 complex reveals that it functions in differently regulated pathways

The SMC protein complexes safeguard genomic integrity through their functions in chromosome segregation and repair. The chromosomal localization of the budding yeast Smc5/6 complex determined here reveals that the complex works specifically on the duplicated genome in differently regulated pathways. The first controls the association to centromeres and chromosome arms in unchallenged cells, the second regulates the association to DNA breaks, and the third directs the complex to the chromosome arm that harbors the ribosomal DNA arrays. The chromosomal interaction pattern predicts a function that becomes more important with increasing chromosome length and that the complex's role in unchallenged cells is independent of DNA damage. Additionally, localization of Smc6 to collapsed replication forks indicates an involvement in their rescue. Altogether this shows that the complex maintains genomic integrity in multiple ways, and evidence is presented that the Smc5/6 complex is needed during replication to prevent the accumulation of branched chromosome structures.     

A Role for the RSC Chromatin Remodeler in Regulating Cohesion of Sister Chromatid Arms

The precise segregation of chromosomes is critical for the proliferation and development of living organisms. Defects in this process can result in tumorigenesis and hereditary diseases. The four-subunit cohesin complex plays an essential role in chromosome segregation and genome integrity. Recently, we reported that the association of cohesin with centromeres and chromosome arms is differentially regulated by the ATP-dependent RSC chromatin-remodeling complex. Here, we propose two models to explain why the cell should have evolved special mechanisms for centromeric and sister arm cohesion and why RSC differentially regulates these processes.     

Absence of the Spindle Assembly Checkpoint Restores Mitotic Fidelity upon Loss of Sister Chromatid Cohesion

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The Cohesin Subunit Rad21 Is Required for Synaptonemal Complex Maintenance,but Not Sister Chromatid Cohesion,during Drosophila Female Meiosis

Replicated sister chromatids are held in close association from the time of their synthesis until their separation during the next mitosis. This association is mediated by the ring-shaped cohesin complex that appears to embrace the sister chromatids. Upon proteolytic cleavage of the α-kleisin cohesin subunit at the metaphase-to-anaphase transition by separase, sister chromatids are separated and segregated onto the daughter nuclei. The more complex segregation of chromosomes during meiosis is thought to depend on the replacement of the mitotic α-kleisin cohesin subunit Rad21/Scc1/Mcd1 by the meiotic paralog Rec8. In Drosophila, however, no clear Rec8 homolog has been identified so far. Therefore, we have analyzed the role of the mitotic Drosophila α-kleisin Rad21 during female meiosis. Inactivation of an engineered Rad21 variant by premature, ectopic cleavage during oogenesis results not only in loss of cohesin from meiotic chromatin, but also in precocious disassembly of the synaptonemal complex (SC). We demonstrate that the lateral SC component C(2)M can interact directly with Rad21, potentially explaining why Rad21 is required for SC maintenance. Intriguingly, the experimentally induced premature Rad21 elimination, as well as the expression of a Rad21 variant with destroyed separase consensus cleavage sites, do not interfere with chromosome segregation during meiosis, while successful mitotic divisions are completely prevented. Thus, chromatid cohesion during female meiosis does not depend on Rad21-containing cohesin.     

Topoisomerase II Suppresses the Temperature Sensitivity of Saccharomyces cerevisiae pds5 Mutants,but not the Defect in Sister Chromatid Cohesion

Sister chromatid cohesion enables chromosomes to achieve bipolar attachment to the mitotic spindle and its dissolution is required for chromosome segregation. The cohesin complex serves as the primary molecular glue responsible for cohesion. Pds5p binds to the same chromosomal loci as the cohesin complex but plays a distinct role as a regulator of cohesion maintenance. Catenation between sister chromatids must also be removed by Topoisomerase II (Top2p) enzymatic activity to enable chromosome segregation. We identified TOP2 as a high-copy suppressor of the temperature sensitivity of pds5 mutants. TOP2 suppression is specific for pds5 mutants as it does not suppress mutants in the cohesin complex. TOP2 suppresses mini-chromosome loss in pds5 mutants indicating that it rescues a chromosome segregation defect. Surprisingly, TOP2 over-expression fails to suppress the cohesion defect of pds5 mutants, suggesting that it suppresses an additional and as yet uncharacterized defect in pds5 mutants that is essential for viability. A catalytically dead TOP2 allele suppresses pds5 temperature sensitivity, suggesting that suppression is unrelated to Top2p enzymatic function. Consistent with this idea, when the pds5 mutant is combined with the top2-4 mutant, which accumulates DNA catenanes due to defective enzymatic activity, the double mutants exhibit synthetic sickness indicating that increased catenation is toxic to pds5 cells. Our results suggest that Pds5p and Top2p cooperate to promote proper chromosome segregation by a mechanism unrelated to either cohesion or catenation/decatenation.     

Nse5/6 inhibits the Smc5/6 ATPase and modulates DNA substrate binding

Eukaryotic cells employ three SMC (structural maintenance of chromosomes) complexes to control DNA folding and topology. The Smc5/6 complex plays roles in DNA repair and in preventing the accumulation of deleterious DNA junctions. To elucidate how specific features of Smc5/6 govern these functions, we reconstituted the yeast holo‐complex. We found that the Nse5/6 sub‐complex strongly inhibited the Smc5/6 ATPase by preventing productive ATP binding. This inhibition was relieved by plasmid DNA binding but not by short linear DNA, while opposing effects were observed without Nse5/6. We uncovered two binding sites for Nse5/6 on Smc5/6, based on an Nse5/6 crystal structure and cross‐linking mass spectrometry data. One binding site is located at the Smc5/6 arms and one at the heads, the latter likely exerting inhibitory effects on ATP hydrolysis. Cysteine cross‐linking demonstrated that the interaction with Nse5/6 anchored the ATPase domains in a non‐productive state, which was destabilized by ATP and DNA. Under similar conditions, the Nse4/3/1 module detached from the ATPase. Altogether, we show how DNA substrate selection is modulated by direct inhibition of the Smc5/6 ATPase by Nse5/6.     

Intersection Between the Regulators of Sister Chromatid Cohesion Establishment and Maintenance in Budding Yeast Indicates a Multi-Step Mechanism

Sister chromatid cohesion is established during S phase and maintained until anaphase. The cohesin complex (Mcd1p/Scc1p, Smc1p, Smc3p Irr1p/Scc3p in budding yeast) serves a structural role as it is required at all times when cohesion exists. Pds5p co-localizes temporally and spatially with cohesin on chromosomes but is thought to serve as a regulator of cohesion maintenance during mitosis. In contrast, Ctf7p/Eco1p is required during S phase for establishment but is not required during mitosis. Here we provide genetic and biochemical evidence that the pathways of cohesion establishment and maintenance are intimately linked. Our results show that mutants in ctf7 and pds5 are synthetically lethal. Moreover, over-expression of either CTF7 or PDS5 exhibits reciprocal suppression of the other mutant’s temperature sensitivity. The suppression by CTF7 is specific for pds5 mutants as CTF7 over-expression increases the temperature sensitivity of an mcd1 mutant but has no effect on smc1 or smc3 mutants. Three additional findings provide new insights into the process of cohesion establishment. First, over-expression of ctf7 alleles deficient in acetylase activity exhibit significantly reduced suppression of the pds5 mutant but exacerbated toxicity to the mcd1 mutant. Second, using chromosome spreads and chromatin immuno-precipitation, we find neither cohesin complex nor Pds5p chromosomal localization is altered in ctf7 mutants. Finally, biochemical analysis reveals that Ctf7p and Pds5p co-immunoprecipitate, which physically links these regulators of cohesion establishment and maintenance. We propose a model whereby Ctf7p and Pds5p co-operate to facilitate efficient establishment by mediating changes in cohesin complex on chromosomes after its deposition.     

Functional interplay between cohesin and Smc5/6 complexes

Chromosomes are subjected to massive reengineering as they are replicated, transcribed, repaired, condensed, and segregated into daughter cells. Among the engineers are three large protein complexes collectively known as the structural maintenance of chromosome (SMC) complexes: cohesin, condensin, and Smc5/6. As their names suggest, cohesin controls sister chromatid cohesion, condensin controls chromosome condensation, and while precise functions for Smc5/6 have remained somewhat elusive, most reports have focused on the control of recombinational DNA repair. Here, we focus on cohesin and Smc5/6 function. It is becoming increasingly clear that the functional repertoires of these complexes are greater than sister chromatid cohesion and recombination. These SMC complexes are emerging as interrelated and cooperating factors that control chromosome dynamics throughout interphase. However, they also release their embrace of sister chromatids to enable their segregation at anaphase, resetting the dynamic cycle of SMC-chromosome interactions.