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.     

DNA damage response pathway uses histone modification to assemble a double-strand break-specific cohesin domain

The postreplicative repair of double-strand breaks (DSBs) is thought to require sister chromatid cohesion, provided by the cohesin complex along the chromosome arms. A further specialized role for cohesin in DSB repair is suggested by its de novo recruitment to regions of DNA damage in mammals. Here, we show in budding yeast that a single DSB induces the formation of a approximately 100 kb cohesin domain around the lesion. Our analyses suggest that the primary DNA damage checkpoint kinases Mec1p and Tel1p phosphorylate histone H2AX to generate a large domain, which is permissive for cohesin binding. Cohesin binding to the phospho-H2AX domain is enabled by Mre11p, a component of a critical repair complex, and Scc2p, a component of the cohesin loading machinery that is necessary for sister chromatid cohesion. We also provide evidence that the DSB-induced cohesin domain functions in postreplicative repair.     

Cohesin-SA1 deficiency drives aneuploidy and tumourigenesis in mice due to impaired replication of telomeres

Cohesin is a protein complex originally identified for its role in sister chromatid cohesion, although increasing evidence portrays it also as a major organizer of interphase chromatin. Vertebrate cohesin consists of Smc1, Smc3, Rad21/Scc1 and either stromal antigen 1 (SA1) or SA2. To explore the functional specificity of these two versions of cohesin and their relevance for embryonic development and cancer, we generated a mouse model deficient for SA1. Complete ablation of SA1 results in embryonic lethality, while heterozygous animals have shorter lifespan and earlier onset of tumourigenesis. SA1-null mouse embryonic fibroblasts show decreased proliferation and increased aneuploidy as a result of chromosome segregation defects. These defects are not caused by impaired centromeric cohesion, which depends on cohesin-SA2. Instead, they arise from defective telomere replication, which requires cohesion mediated specifically by cohesin-SA1. We propose a novel mechanism for aneuploidy generation that involves impaired telomere replication upon loss of cohesin-SA1, with clear implications in tumourigenesis.     

Postreplicative recruitment of cohesin to double-strand breaks is required for DNA repair

Chromosome stability depends on accurate chromosome segregation and efficient DNA double-strand break (DSB) repair. Sister chromatid cohesion, established during S phase by the protein complex cohesin, is central to both processes. In the absence of cohesion, chromosomes missegregate and G2-phase DSB repair fails. Here, we demonstrate that G2-phase repair also requires the presence of cohesin at the damage site. Cohesin components are shown to be recruited to extended chromosome regions surrounding DNA breaks induced during G2. We find that in the absence of functional cohesin-loading proteins (Scc2/Scc4), the accumulation of cohesin at DSBs is abolished and repair is defective, even though sister chromatids are connected by S phase generated cohesion. Evidence is also provided that DSB induction elicits establishment of sister chromatid cohesion in G2, implicating that damage-recruited cohesin facilitates DNA repair by tethering chromatids.     

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

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

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.     

Ctf18 is required for homologous recombination-mediated double-strand break repair

The efficient repair of double-strand breaks (DSBs) is crucial in maintaining genomic integrity. Sister chromatid cohesion is important for not only faithful chromosome segregation but also for proper DSB repair. During DSB repair, the Smc1–Smc3 cohesin complex is loaded onto chromatin around the DSB to support recombination-mediated DSB repair. In this study, we investigated whether Ctf18, a factor implicated in the establishment of sister chromatid cohesion, is involved in DSB repair in budding yeast. Ctf18 was recruited to HO-endonuclease induced DSB sites in an Mre11-dependent manner and to damaged chromatin in G₂/M phase-arrested cells. The ctf18 mutant cells showed high sensitivity to DSB-inducible genotoxic agents and defects in DSB repair, as well as defects in damage-induced recombination between sister chromatids and between homologous chromosomes. These results suggest that Ctf18 is involved in damage-induced homologous recombination.     

Increased sister chromatid cohesion and DNA damage response factor localization at an enzyme-induced DNA double-strand break in vertebrate cells

The response to DNA damage in vertebrate cells involves successive recruitment of DNA signalling and repair factors. We used light microscopy to monitor the genetic dependencies of such localization to a single, induced DNA double strand break (DSB) in vertebrate cells. We used an inducible version of the rare-cutting I-SceI endonuclease to cut a chromosomally integrated I-SceI site beside a Tet operator array that was visualized by binding a Tet repressor-GFP fusion. Formation of γ-H2AX foci at a single DSB was independent of ATM or Ku70. ATM-deficient cells showed normal kinetics of 53Bp1 recruitment to DSBs, but Rad51 localization was retarded. 53Bp1 and Rad51 foci formation at a single DSB was greatly reduced in H2AX-null DT40 cells. We also observed decreased inter-sister chromatid distances after DSB induction, suggesting that cohesin loading at DSBs causes elevated sister chromatid cohesion. Loss of ATM reduced DSB-induced cohesion, consistent with cohesin being an ATM target in the DSB response. These data show that the same genetic pathways control how cells respond to single DSBs and to multiple lesions induced by whole-cell DNA damage.     

The role of SMC proteins in the responses to DNA damage

The SMC proteins form the cores of three protein complexes in eukaryotes, cohesin, condensin and the Smc5-6 complex. Cohesin holds sister chromatids together after DNA replication and is involved in both the repair of double-strand breaks by homologous recombination and the intra-S-phase checkpoint. Condensin assists in the condensation of chromosomes at mitosis and also has a role in checkpoint control pathways. The Smc5-6 complex is involved in a variety of DNA repair and damage response pathways by as yet unknown mechanisms, but is also associated with repair by homologous recombination.     

Cohesin phosphorylation and mobility of SMC1 at ionizing radiation-induced DNA double-strand breaks in human cells

Cohesin, a hetero-tetrameric complex of SMC1, SMC3, Rad21 and Scc3, associates with chromatin after mitosis and holds sister chromatids together following DNA replication. Following DNA damage, cohesin accumulates at and promotes the repair of DNA double-strand breaks. In addition, phosphorylation of the SMC1/3 subunits contributes to DNA damage-induced cell cycle checkpoint regulation. The aim of this study was to determine the regulation and consequences of SMC1/3 phosphorylation as part of the cohesin complex. We show here that the ATM-dependent phosphorylation of SMC1 and SMC3 is mediated by H2AX, 53BP1 and MDC1. Depletion of RAD21 abolishes these phosphorylations, indicating that only the fully assembled complex is phosphorylated. Comparison of wild type SMC1 and SMC1S966A in fluorescence recovery after photo-bleaching experiments shows that phosphorylation of SMC1 is required for an increased mobility after DNA damage in G2-phase cells, suggesting that ATM-dependent phosphorylation facilitates mobilization of the cohesin complex after DNA damage.     

Mrc1 is required for sister chromatid cohesion to aid in recombination repair of spontaneous damage

The SRS2 gene of Saccharomyces cerevisiae encoding a 3'-->5' DNA helicase is part of the postreplication repair pathway and functions to ensure proper repair of DNA damage arising during DNA replication through pathways that do not involve homologous recombination. Through a synthetic gene array analysis, genes that are essential when Srs2 is absent have been identified. Among these are MRC1, TOF1, and CSM3, which mediate the intra-S checkpoint response. srs2 Delta mrc1 Delta synthetic lethality is due to inappropriate recombination, as the lethality can be suppressed by genetic elimination of homologous recombination. srs2 Delta mrc1 Delta synthetic lethality is dependent on the role of Mrc1 in DNA replication but independent of the role of Mrc1 in a DNA damage checkpoint response. mrc1 Delta, tof1 Delta and csm3 Delta mutants have sister chromatid cohesion defects, implicating sister chromatid cohesion established at the replication fork as an important factor in promoting repair of stalled replication forks through gap repair.     

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.     

A second Wpl1 anti‐cohesion pathway requires dephosphorylation of fission yeast kleisin Rad21 by PP4

Cohesin mediates sister chromatid cohesion which is essential for chromosome segregation and repair. Sister chromatid cohesion requires an acetyl‐transferase (Eso1 in fission yeast) counteracting Wpl1, promoting cohesin release from DNA. We report here that Wpl1 anti‐cohesion function includes an additional mechanism. A genetic screen uncovered that Protein Phosphatase 4 (PP4) mutants allowed cell survival in the complete absence of Eso1. PP4 co‐immunoprecipitated Wpl1 and cohesin and Wpl1 triggered Rad21 de‐phosphorylation in a PP4‐dependent manner. Relevant residues were identified and mapped within the central domain of Rad21. Phospho‐mimicking alleles dampened Wpl1 anti‐cohesion activity, while alanine mutants were neutral indicating that Rad21 phosphorylation would shelter cohesin from Wpl1 unless erased by PP4. Experiments in post‐replicative cells lacking Eso1 revealed two cohesin populations. Type 1 was released from DNA by Wpl1 in a PP4‐independent manner. Type 2 cohesin, however, remained DNA‐bound and lost its cohesiveness in a manner depending on Wpl1‐ and PP4‐mediated Rad21 de‐phosphorylation. These results reveal that Wpl1 antagonizes sister chromatid cohesion by a novel pathway regulated by the phosphorylation status of the cohesin kleisin subunit.     

S-phase and DNA damage activated establishment of Sister chromatid cohesion—importance for DNA repair

By holding sister chromatids together from the moment of their formation until their separation at anaphase, the multi subunit protein complex Cohesin guarantees correct chromosome segregation. This S-phase established chromatid cohesion is also essential for repair of DNA double strand breaks (DSB) in postreplicative cells. In addition, Cohesin has to be recruited to a DSB, and new cohesion has to form in response to the damage for repair. When it became clear that cohesion is created de novo in response to DNA breaks, the term “damage induced cohesion” (DI-cohesion) was coined. It is now established that certain factors are needed for establishment of both S-phase and DI-cohesion, while others have been found to be unique for respective process. In addition, post-translational modifications of Cohesin components that are functionally important for cohesion formation, either during S-phase or in response to damage, have recently been identified. Here, we present and discuss the current models for establishment of S-phase and DI-cohesion in the context of their involvement in DSB repair.     

Damage-induced reactivation of cohesin in postreplicative DNA repair

Cohesin establishes sister-chromatid cohesion during S phase to ensure proper chromosome segregation in mitosis. It also facilitates postreplicative homologous recombination repair of DNA double-strand breaks by promoting local pairing of damaged and intact sister chromatids. In G2 phase, cohesin that is not bound to chromatin is inactivated, but its reactivation can occur in response to DNA damage. Recent papers by Koshland's and Sj?gren's groups describe the critical role of the known cohesin cofactor Eco1 (Ctf7) and ATR checkpoint kinase in damage-induced reactivation of cohesin, revealing an intricate mechanism that regulates sister-chromatid pairing to maintain genome integrity.     

Cohesin Is Limiting for the Suppression of DNA Damage–Induced Recombination between Homologous Chromosomes

Double-strand break (DSB) repair through homologous recombination (HR) is an evolutionarily conserved process that is generally error-free. The risk to genome stability posed by nonallelic recombination or loss-of-heterozygosity could be reduced by confining HR to sister chromatids, thereby preventing recombination between homologous chromosomes. Here we show that the sister chromatid cohesion complex (cohesin) is a limiting factor in the control of DSB repair and genome stability and that it suppresses DNA damage–induced interactions between homologues. We developed a gene dosage system in tetraploid yeast to address limitations on various essential components in DSB repair and HR. Unlike RAD50 and RAD51, which play a direct role in HR, a 4-fold reduction in the number of essential MCD1 sister chromatid cohesion subunit genes affected survival of gamma-irradiated G₂/M cells. The decreased survival reflected a reduction in DSB repair. Importantly, HR between homologous chromosomes was strongly increased by ionizing radiation in G₂/M cells with a single copy of MCD1 or SMC3 even at radiation doses where survival was high and DSB repair was efficient. The increased recombination also extended to nonlethal doses of UV, which did not induce DSBs. The DNA damage–induced recombinants in G₂/M cells included crossovers. Thus, the cohesin complex has a dual role in protecting chromosome integrity: it promotes DSB repair and recombination between sister chromatids, and it suppresses damage-induced recombination between homologues. The effects of limited amounts of Mcd1and Smc3 indicate that small changes in cohesin levels may increase the risk of genome instability, which may lead to genetic diseases and cancer.