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.     

Roles of Human AND-1 in Chromosome Transactions in S Phase

Coordinated execution of DNA replication, checkpoint activation, and postreplicative chromatid cohesion is intimately related to the replication fork machinery. Human AND-1/chromosome transmission fidelity 4 is localized adjacent to replication foci and is required for efficient DNA synthesis. In S phase, AND-1 is phosphorylated in response to replication arrest in a manner dependent on checkpoint kinase, ataxia telangiectasia-mutated, ataxia telangiectasia-mutated and Rad3-related protein, and Cdc7 kinase but not on Chk1. Depletion of AND-1 increases DNA damage, delays progression of S phase, leads to accumulation of late S and/or G₂ phase cells, and induces cell death in cancer cells. It also elevated UV-radioresistant DNA synthesis and caused premature recovery of replication after hydroxyurea arrest, indicating that lack of AND-1 compromises checkpoint activation. This may be partly due to the decreased levels of Chk1 protein in AND-1-depleted cells. Furthermore, AND-1 interacts with cohesin proteins Smc1, Smc3, and Rad21/Scc1, consistent with proposed roles of yeast counterparts of AND-1 in sister chromatid cohesion. Depletion of AND-1 leads to significant inhibition of homologous recombination repair of an I-SceI-driven double strand break. Based on these data, we propose that AND-1 coordinates multiple cellular events in S phase and G₂ phase, such as DNA replication, checkpoint activation, sister chromatid cohesion, and DNA damage repair, thus playing a pivotal role in maintenance of genome integrity.Replication fork is not only the site of DNA synthesis but also the center for coordinated execution of various chromosome transactions. The preparation for replication forks starts in the G₁ phase, when the prereplicative complex composed of origin recognition and minichromosome maintenance assembles on the chromosome. At the G₁-S boundary, Cdc45, GINS complex, and other factors join the prereplicative complex to generate a complex capable of initiating DNA replication. A series of phosphorylation events mediated by cyclin-dependent kinase and Cdc7 kinase play crucial roles in this process and facilitate the generation of active replication forks (1–6). Purification of the putative replisome complex in yeast indicated the presence of the checkpoint mediator Mrc1 and fork protection complex proteins Tof1 and Csm3 in the replication fork machinery (7), consistent with a previous report on the genome-wide analyses with chromatin immunoprecipitation analyses on chip (microarray) (8). Mcm10 is another factor present in the isolated complex, required for loading of replication protein A (RPA)² and primase-DNA polymerase α onto the replisome complex (7, 9, 10).Replication fork machinery can cope with various stresses, including shortage of the cellular nucleotide pool and replication fork blockages that interfere with its progression. Stalled replication forks activate checkpoint pathways, leading to cell cycle arrest, DNA repair, restart of DNA replication, or cell death in some cases (11–14). Single-stranded DNAs coated with RPA at the stalled replication forks are recognized by the ATR-ATR-interacting protein kinase complex and Rad17 for loading of the Rad9-Rad1-Hus1 checkpoint clamp (14–16). Factors present in the replisome complex are also known to be required for checkpoint activation. Claspin, Tim, and Tipin functionally and physically associate with sensor and effector kinases and serve as mediator/adaptors (17–23). Mcm7, a component of the replicative DNA helicase in eukaryotes, was reported to associate with the checkpoint clamp loader Rad17 (24) and to have a distinct function in checkpoint (24, 25). We recently reported that Cdc7 kinase, known to be required for DNA replication initiation, plays a role in activation of DNA replication checkpoint possibly through regulating Claspin phosphorylation (26). Thus, it appears that DNA replication and checkpoint activation functionally and physically interact with each other.Another crucial cellular event for maintenance of genome stability is sister chromatid cohesion. The cohesin complex, a conserved apparatus required for sister chromatid cohesion, contains Smc1, Smc3, and Rad21/Scc1/Mcd1 proteins. The assembled cohesin complexes are loaded onto chromatin prior to DNA replication in G₁ phase and link the sister chromosomes during S and G₂ phase until mitosis when they separate (27, 28). The mitotic cohesion defects are not rescued by supplementing cohesin in G₂ phase, and it has been suggested that establishment of sister chromatid cohesion is coupled with DNA replication (29, 30). Indeed, yeast mutants in some replisome components show defect in sister chromosome cohesion or undergo chromosome loss (31–33). Cdc7 kinase is also required for efficient mitotic chromosome cohesion (34, 35).Human AND-1 is the putative homolog of budding yeast CTF4/Pob1/CHL15 and fission yeast Mcl1/Slr3. The budding yeast counterpart was identified as a replisome component described above (7), which travels along with the replication fork (29). CTF4 is nonessential for viability, but its interactions with primase, Rad2 (FEN1 family of nuclease), and Dna2 have implicated CTF4 in lagging strand synthesis and/or Okazaki fragment processing (36–39). Yeast CTF4 and Mcl1 are involved in chromosome cohesion (33, 40, 41) and genetically interact with a cohesin, Mcd1/Rad21 (40, 42). Recently, it was reported that human AND-1 protein interacts with human primase-DNA polymerase α and Mcm10 and is required for DNA synthesis (43).Here we confirm that human AND-1 protein is required for DNA replication and efficient progression of S phase, and we further show that it facilitates replication checkpoint. Depletion of AND-1 causes accumulation of DNA damage and cell cycle arrest at late S to G₂ phase, ultimately leading to cell death. Furthermore, we also show that human AND-1 physically interacts with cohesin proteins Smc1, Smc3, Rad21/Scc1, suggesting a possibility that AND-1 may physically and functionally link replisome and cohesin complexes in vivo. Recent studies indicate that sister chromatid cohesion is required for recombinational DNA repair (44–47). Thus, we examined the requirement of AND-1 for repair of artificially induced double-stranded DNA breaks and showed that AND-1 depletion leads to significant reduction of the double strand break repair. Possible roles of AND-1 in coordination of various chromosome transactions at a replication fork and in maintenance of genome integrity during S phase will be discussed.     

Smc5-Smc6-Dependent Removal of Cohesin from Mitotic Chromosomes

The function of the essential cohesin-related Smc5-Smc6 complex has remained elusive, though hypomorphic mutants have defects late in recombination, in checkpoint maintenance, and in chromosome segregation. Recombination and checkpoints are not essential for viability, and Smc5-Smc6-null mutants die in lethal mitoses. This suggests that the chromosome segregation defects may be the source of lethality in irradiated Smc5-Smc6 hypomorphs. We show that in smc6 mutants, following DNA damage in interphase, chromosome arm segregation fails due to an aberrant persistence of cohesin, which is normally removed by the Separase-independent pathway. This postanaphase persistence of cohesin is not dependent on DNA damage, since the synthetic lethality of smc6 hypomorphs with a topoisomerase II mutant, defective in mitotic chromosome structure, is also due to the retention of cohesin on undamaged chromosome arms. In both cases, Separase overexpression bypasses the defect and restores cell viability, showing that defective cohesin removal is a major determinant of the mitotic lethality of Smc5-Smc6 mutants.Three essential SMC (structural maintenance of chromosomes) complexes control chromosome dynamics: condensin, cohesin, and the Smc5-Smc6 complex (37). They are composed of SMC heterodimers: Smc2 and -4 in condensin, Smc1 and -3 in cohesin, and Smc5 and -6 in Smc5-Smc6. These are large ATPases with globular N and C termini, which are separated by long coiled-coil domains. The termini interact through an ABC-like coordination of ATP through Walker A and B motifs, with the coiled-coils bending at a flexible “hinge” that acts as the SMC dimerization domain. Each complex contains a number of unique non-Smc subunits, which are likely to contribute to its unique function. Among these is a kleisin subunit, which interacts with both the SMC subunits, closing a potential ring-shaped structure (55, 61).Condensin is localized to chromosomes primarily during mitosis and is essential for mitotic chromosome condensation. Conversely, cohesin is localized primarily to interphase chromosomes and has been postulated to form a ring-shaped structure around sister chromatids to ensure their cohesion, which is important for DNA repair by homologous recombination (HR). As its name suggests, the function of the Smc5-Smc6 complex is relatively poorly understood.Scc2/4 loads cohesin onto chromosomes in G₁, and sister chromatid cohesion is established during replication via the action of the acetyltransferase Eco1. Cohesin must be removed before chromosome segregation, where cleavage of the kleisin subunit Scc1 by the protease Separase is critical (51). In Saccharomyces cerevisiae, Separase-mediated Scc1 cleavage is essential for the removal of cohesin from all loci. In mammals, most cohesin is removed from chromosome arms early in mitosis in a Separase-independent process regulated by cohesin phosphorylation (28, 76). At anaphase, Separase-dependent removal of cohesin at the kinetochores ensures sister chromatid separation. In Schizosaccharomyces pombe, cohesin is thought to be regulated in a manner similar to that in mammals; only a small fraction of the Scc1 homolog Rad21 is cleaved by Separase (70), suggesting that most cohesin is removed by a Separase-independent mechanism.Cohesin-mediated sister chromatid cohesion is required for HR (64). Cohesin is recruited to double-stranded DNA breaks (DSBs) (66) and enforces cohesion genome wide after DNA damage in S. cerevisiae (65, 74). The acetyltransferase activity of Eco1 is essential for genomewide damage-induced cohesion, acting via the acetylation of Smc3 (6, 73, 81). In human cells, small interfering RNA (siRNA) studies have suggested a requirement for Smc5-Smc6 to recruit cohesin to DSBs (57), but this is not the case in S. cerevisiae (65), so the functional relationship between these related complexes also remains to be determined.In S. cerevisiae, Smc5-Smc6 is loaded onto chromatin by the cohesin loader Scc2/4 at loci that overlap with cohesin, including at DSBs (36). Smc5-Smc6-null mutants of S. pombe die in aberrant mitoses (27, 75), though the cause of this is unknown. Genetic analyses of Smc5-Smc6 in these yeasts have focused on its role in DNA repair by utilizing viable hypomorphic mutants that are highly sensitive to DNA damage. Studies with two hypomorphic smc6 mutants, bearing the smc6-X and smc6-74 mutations, have shown that Smc5-Smc6 is required for a late stage of HR subsequent to the recruitment of the Rad51/Rad52 recombination proteins and the formation of recombination intermediates (2). smc6-74 is a mutation (A151T) in the arginine finger motif of the N-terminal globular domain, while smc6-X is a mutation (R706C) in the hinge domain. Overexpression of Brc1, a multi-BRCT domain protein, suppresses the DNA damage sensitivities of several Smc5-Smc6 mutants but does not suppress smc6-X (45, 75). smc6-74 mutants, but not smc6-X mutants, are also defective in an early response to replication fork stalling, involving the recruitment of Rad52 but not Rad51 (30).As with cohesin, the HR defects in Smc5-Smc6 hypomorphic mutants are likely to result from a more general role in chromosome organization than acting as a recombinase. Smc5-Smc6 is required for HR following irradiation or recovery from hydroxyurea (HU)-induced replication arrest (2, 18, 27, 34, 35, 71, 75). However, in contrast to the sustained checkpoint arrest of irradiated HR mutants, S. pombe Smc5-Smc6 hypomorphs, such as that with the smc6-74 mutation, enter highly aberrant mitoses following DNA damage. For DSBs induced by ionizing radiation, smc6 mutants progress into mitosis with wild-type kinetics, but, as shown by pulsed-field gel electrophoresis (PFGE), the chromosomes are highly fragmented (75). In each case, the mitotic defects are blocked by an earlier (upstream) HR defect (2, 27, 43). The chromosome segregation and recombination defects are apparent on each of the three S. pombe chromosomes and are not limited to the ribosomal DNA present on both ends of chromosome III.These aberrant mitoses of Smc5-Smc6 mutants following DNA damage either block segregation completely (the “cut” phenotype, where the division septum bisects the nucleus) or result in partially segregated chromosomes that are incompletely resolved along the division plane, with an elongated mitotic spindle. Since Smc5-Smc6 is required to maintain a damage induced checkpoint arrest, the aberrant mitoses of Smc5-Smc6 mutants could result from attempting to segregate incompletely repaired chromosomes. Alternatively, defects may reflect a role for Smc5-Smc6 in promoting chromosome segregation that is revealed in hypomorphic mutants following exogenous DNA damage but is evident in null mutants without DNA damage or with low-level endogenous lesions. Notably, while viable, the hypomorphic mutants show a high level of spontaneous aneuploidy, which is also consistent with defects in chromosome segregation (35, 75).Another characteristic of smc6 mutants in S. pombe is a strong synthetic lethality with a temperature-sensitive (ts) allele of topoisomerase II (Top2), top2-191, at a permissive temperature for top2-191 of 30°C. This lethality is due to a failure of chromosome segregation that resembles mitoses in irradiated smc6-74 cells (75). top2-191 is a A802V mutation (63), and cells with this mutation show no defects in cell cycle progression at 30°C. At 36°C, top2-191 cells enter mitosis with normal kinetics but fail to segregate chromosomes. The defects of top2-191 cells at the restrictive temperature of 36°C manifest exclusively in mitosis without an interphase delay and include defective chromosome condensation. Therefore, the top2-191 allele may not affect the postreplicative decatenation activity of Top2 in S. pombe. Rather, the smc6-top2-191 interaction may be related to the structural role played by Top2 in mitotic chromosome architecture (12, 14, 79).In vertebrate cells, defective decatenation caused by Top2 inhibition with drugs such as etoposide or doxorubicin block the rejoining of molecules cleaved by Top2. This leaves DSBs that elicit a G₂ DNA damage checkpoint response in many cell types (13, 16, 17, 38). Conversely, human cells in which Top2 has been deleted enter mitosis but show disordered chromosomes that fail to segregate (12). Thus, in S. pombe, top2-191 has a terminal phenotype more closely related to that of human cells with Top2 deleted than to that of cells with chemically inhibited Top2 that are blocked midway in the decatenation reaction.Here we have investigated the mitotic role of Smc5-Smc6 in S. pombe. We find that Smc5-Smc6 is required for the removal of cohesin from damaged chromosome arms prior to anaphase and from undamaged chromosomes when the mitotic function of Top2 is compromised. We show that a defect in cohesin removal is a major determinant of lethality in smc6 mutants and highlight the importance of coordinating Smc5-Smc6 and cohesin function in the maintenance of genome integrity.     

Preimplantation Mouse Embryos Depend on Inhibitory Phosphorylation of Separase To Prevent Chromosome Missegregation

Separase is a critical protease that catalyzes the cleavage of sister chromatid cohesins to allow the separation of sister chromatids in the anaphase. Its activity must be inhibited prior to the onset of the anaphase. Two inhibitory mechanisms exist in vertebrates that block the protease activity. One mechanism is through binding and inhibition by securin, and another is phosphorylation on Ser1126 (in humans [Ser1121 in mice]). These two mechanisms are largely redundant. However, phosphorylation on Ser1121 is critical for the prevention of premature sister separation in embryonic germ cells. As a result, Ser1121-to-Ala mutation leads to depletion of germ cells in development and subsequently to infertility in mice. Here, we report that the same mutation also causes embryogenesis failure between the 8- and 16-cell stages in mice. Our results indicate a critical role of separase phosphorylation in germ cell development as well as in early embryogenesis. Thus, deregulation of separase may be a significant contributor to infertility in humans.Sister chromatids are held together by a multisubunit complex called cohesin composed of Smc1 and -3 and Scc1 and -3 (24). To separate the sister chromosomes, cohesin complexes are removed in a two-step process. First, cohesins on chromosome arms are removed by Plk1- and Aurora B-mediated phosphorylation before the anaphase (4, 8, 19, 20, 31, 35). Second, the centromere-localized cohesins, which are protected by Sgo and PP2A from phosphorylation-mediated removal (13, 21, 28, 29, 32), are cleaved by a protease called separase at the onset of the anaphase (33, 34). Prior to the anaphase, separase is inhibited by securin and by phosphorylation, which is most likely catalyzed by cyclin B1/Cdk1. phosphorylation by cyclin B1/Cdk1 per se is not inhibitory to separase. Rather, the phosphorylation allows the binding of cyclin B1/Cdk1 as an inhibitor to separase (5). Two phosphorylation sites in separase, Ser1126 and Thr1326 (Ser1121 and Thr1321 in mice, respectively), that are important for the inhibition have been identified (30). Activation of separase depends on the function of the E3 ubiquitin ligase anaphase-promoting complex/cyclosome (APC/C), since both securin and cyclin B1 are substrates of APC/C (1-3, 15, 16, 23, 25, 27, 36). Given that APC/C is inhibited by the spindle assembly checkpoint, separation of sister chromatids therefore cannot occur until the checkpoint is satisfied. Thus, the spindle assembly checkpoint prevents premature sister separation and ensures chromosomal stability.Missegregation of chromosomes has dire consequences. It causes genetic imbalances that may transform cells and lead to cancer development in somatic tissues. In germ lines, missegregation in either meiosis I, mainly manifested as nondisjunction of homologue chromosomes, or meiosis II, manifested as premature sister chromatid separation, will generate aneuploid gametes, directly affecting the fecundity of an organism (26, 37). Although the molecular mechanisms underlying chromosome segregation errors in meiosis are still not clear (6), deregulation of separase, either directly or indirectly, is likely a significant contributor.We previously showed that securin and separase phosphorylation are redundant in almost all somatic tissues, as mice lacking either separase inhibitory mechanism are essentially normal (9, 22). However, phosphorylation of separase is uniquely required during germ line development (9). Mice carrying a nonphosphorylatable separase (S1121A) allele are sterile, largely due to depletion of germ cells during embryogenesis. The failure of the germ cells to reach sexually mature stages in the mutant mice prevented us from assessing the function of the inhibitory phosphorylation of separase in meiosis. Here we report our analysis of mice with an oocyte-specific S1121A mutation in separase. We found that these mice were still infertile. However, the infertility was not a result of meiotic errors caused by the mutant separase but was rather a failure of early embryogenesis of zygotes carrying the mutant allele prior to the 16-cell stage.     

Roles of Hop1 and Mek1 in Meiotic Chromosome Pairing and Recombination Partner Choice in Schizosaccharomyces pombe

Synaptonemal complex (SC) proteins Hop1 and Mek1 have been proposed to promote homologous recombination in meiosis of Saccharomyces cerevisiae by establishment of a barrier against sister chromatid recombination. Therefore, it is interesting to know whether the homologous proteins play a similar role in Schizosaccharomyces pombe. Unequal sister chromatid recombination (USCR) was found to be increased in hop1 and mek1 single and double deletion mutants in assays for intrachromosomal recombination (ICR). Meiotic intergenic (crossover) and intragenic (conversion) recombination between homologous chromosomes was reduced. Double-strand break (DSB) levels were also lowered. Notably, deletion of hop1 restored DSB repair in rad50S meiosis. This may indicate altered DSB repair kinetics in hop1 and mek1 deletion strains. A hypothesis is advanced proposing transient inhibition of DSB processing by Hop1 and Mek1 and thus providing more time for repair by interaction with the homologous chromosome. Loss of Hop1 and Mek1 would then result in faster repair and more interaction with the sister chromatid. Thus, in S. pombe meiosis, where an excess of sister Holliday junction over homologous Holliday junction formation has been demonstrated, Hop1 and Mek1 possibly enhance homolog interactions to ensure wild-type level of crossover formation rather than inhibiting sister chromatid interactions.Sexual reproduction in eukaryotes involves formation of haploid gametes from diploid cells by one round of DNA replication, pairing of the homologous chromosomes, and recombination and then by the two meiotic divisions (53). In fungi the gametes differentiate into haploid spores, which germinate to form vegetative cells. Crossover (CO) formation between homologous chromosomes and DNA repair processes between sister chromatids are required for spore viability (10, 55, 58).In vegetative cells homologous recombination (HR) is important for repair of DNA damage and stalled replication forks, with the sister chromatid as the preferred partner (28). Many of the enzymes involved in mitotic HR also contribute to meiotic recombination. In addition, meiosis-specific cytological structures and enzymes enhance recombination frequency (meiotic induction) and shift partner preference from sister chromatids to homologous chromosomes (3, 47, 64, 74). In detail the steps of HR vary between different types of sequence organization (allelic versus sister versus ectopic), between different types of DNA damage, between meiotic and mitotic cells, and between species (10, 55, 58).Meiotic recombination, including CO formation, is initiated by DNA double-strand breaks (DSBs). In Saccharomyces cerevisiae and other eukaryotes, DSBs are formed by Spo11. Many cofactors are required (29). The Schizosaccharomyces pombe homolog is Rec12, also requiring auxiliary factors whose elimination leads to loss of meiotic DSB formation (12). The 5′ single-strand ends at DSBs are processed by nucleases. In S. cerevisiae the MRX complex made up by the proteins Rad50, Mre11, and Xrs2 is required for this resection, as well as for DSB formation. The corresponding MRN complex of S. pombe (Rad50, Rad32, and Nbs1) is not required for DSB formation but is essential for DSB repair (43, 72). Deletion of rad50, rad32, or ctp1 (homologous to SAE2/COM1 in S. cerevisiae and CtIP in humans) leads to very low spore viability. These proteins are also essential for DSB processing (23, 24, 32, 43, 60, 62).Free DNA 3′ ends at DSBs are recruited for invasion of a sister or homologous chromatid by the strand transfer proteins Rad51 and Dmc1, again involving many accessory proteins (16). This results in the central intermediates of HR: heteroduplex DNA consisting of single strands originating from different chromatids and Holliday junctions (HJs). In S. cerevisiae HJs form preferably between homologs with a two- to sixfold excess over intersister HJs (64). Surprisingly, meiotic HJs form with about a fourfold excess between sisters in S. pombe (11). Eventually the intermediates are resolved into crossover (CO) and noncrossover (NCO) events. COs show exchange of the flanking sequences of the two chromatids involved and usually carry a patch of conversion (unilateral transfer of DNA sequences from one chromatid to its interacting partner) near the DSB site. NCOs are conversion events without associated COs (22). In S. pombe loss of core HR functions leads to very low spore viability: deletion of rad51 but not of dmc1 (20), double mutation of rad54 and rdh54 (7), inactivation of the endonuclease activity encoded by mus81 and eme1 (5, 52), and combined deletion of rad22 and rti1 (homologs of RAD52 of S. cerevisiae). But, differently from the other core functions, Rad22 and Rti1 are not required for CO and NCO (50).Early in meiotic prophase of many eukaryotes, axial elements (called lateral elements in later stages) form along sister chromatids, and pairing of homologous chromosomes is initiated, leading to juxtaposition of the homologous chromosomes along their whole length in the synaptonemal complex (SC) (54). In S. pombe no SC is formed, but linear elements (LEs), resembling axial elements of other eukaryotes, are formed. LEs do not form continuously along the chromosomes (1) but load the proteins Rec10, Hop1, and Mek1 (36, 44, 57), which are homologs of, or at least related, to the S. cerevisiae proteins Red1, Hop1, and Mek1, respectively, localizing to axial/lateral elements (2, 67). Hop1 carries a HORMA domain, also present in proteins associating with axial elements and regulating the progress of recombination in higher eukaryotes: Arabidopsis thaliana (61), Caenorhabditis elegans (9, 41), and mammals (18).In S. cerevisiae localization of Hop1 and Mek1 (meiosis-specific protein kinase) to axial elements is dependent on Red1 (2, 67). Mutation of the three S. cerevisiae genes results in reduction of DSB formation, CO and conversion frequencies, and spore viability (26, 31, 59). Direct comparison of unequal sister chromatid recombination (USCR) frequencies in an assay excluding the scoring of intrachromatid recombination (ICR) revealed no increase in the hop1 null mutant but about fourfold increases in the red1 and mek1 null mutants (69). The S. cerevisiae Hop1, Red1, and Mek1 proteins are involved in biasing meiotic DSB repair to occur between homologous chromosomes rather than between sister chromatids (47). Activated Mek1 kinase is required for the inhibition of sister chromatid-mediated DSB repair by Rad51, when the DMC1 gene is deleted and the meiotic recombination checkpoint is activated (4, 27, 38, 47). For Mek1 activation, phosphorylation of Hop1 by the Mec1/Tel1 kinases is also required (6).Less is known about the S. pombe proteins. Hop1 of S. pombe was identified as a nonsignificant hit by sequence comparison with full-length S. cerevisiae Hop1 and contains an N-terminal HORMA domain and a central zinc finger motif like Hop1 in S. cerevisiae. In addition they share a short homology block toward the C terminus (36). The Mek1 protein of S. pombe shares 34% identity and 54% similarity with its S. cerevisiae counterpart along the whole sequence. It contains an FHA domain in the N-terminal part like the other members of its family of checkpoint kinases and is involved in regulation of the meiotic cell cycle (57). Hop1 and Mek1 are strongly expressed in meiosis but not expressed or only slightly expressed in vegetative cells (42, 57). In prophase both proteins localize to LEs as defined by colocalization with the LE component Rec10 (36). Deletion of the distant RED1 homolog rec10 abolishes LE formation (36, 44) and strongly reduces meiotic recombination (17, 70). Rec10, but not Hop1 and Mek1, is required for localization of Rec7 (a distant homolog of S. cerevisiae Rec114) to meiotic chromosomes (34). Rec7 and Rec10 are required for Rec12 activity (12, 29).Obtaining information on the functions of Hop1 and Mek1 in S. pombe was the aim of the work presented here, especially on their possible roles in homolog versus sister discrimination for DSB repair. Deletion mutants have been studied with respect to spore viability and the frequencies of CO and conversion. They have also been assessed for genetic recombination events between sister chromatids in the known PS1 assay (63) and the newly developed VL1 assay (for details, see Fig. ​Fig.3).3). Physical analysis of DSB formation and repair has been performed in meiotic time course experiments. It is proposed that S. pombe Hop1 and Mek1 are promoting interactions between homologous chromosomes rather than inhibiting interactions between sister chromatids.Open in a separate windowFIG. 3.PS1 and VL1 assay systems for intrachromosomal recombination. Strains with constructs carrying repeated DNA sequences have been assayed for prototroph formation either by intrachromatid recombination (ICR, yielding prototrophs only in PS1) or by unequal sister chromatid recombination (USCR, in PS1 and VL1). Crosses of the constructs were performed with strains carrying a deletion of the ade6 gene to exclude other homologous recombination events. (A) The PS1 assay involves copies of the ade6 gene inactivated by either the hot spot mutation M26 or the mutation 469. The repeated sequences are separated by the ura4⁺ marker (63). ICR (left) or USCR (right) between the repeated sequences can lead to formation of adenine prototrophs that have lost the ura4⁺ marker by crossover (CO) or single-strand annealing (SSA) events. Adenine prototrophs maintaining the ura4⁺ marker can derive from noncrossover (NCO) events. Both types of pairing may lead to CO or NCO products. (B) The newly constructed VL1 assay (see the supplemental material) involves different truncations of the ade6 gene separated by the hyg^R marker (also called hphMX6), conferring hygromycin resistance. The left truncation carries a 3′ portion of ade6; the right truncation carries a 5′ portion of ade6. While the gray parts of the truncations are not overlapping, the white sections of 500-bp length are of almost identical sequence, allowing for homologous pairing. CO and SSA products resulting from ICR retain only the central portion of ade6 and remain auxotrophic. Adenine prototrophic CO and NCO products resulting from USCR both retain hygromycin resistance. Note that NCO events may arise through loop formation of one sister chromatid and pairing with a single block (500 bp) of the repeated ade6 sequence (39).     

Mek1 Suppression of Meiotic Double-Strand Break Repair Is Specific to Sister Chromatids,Chromosome Autonomous and Independent of Rec8 Cohesin Complexes

During meiosis, recombination is directed to occur between homologous chromosomes to create connections necessary for proper segregation at meiosis I. Partner choice is determined at the time of strand invasion and is mediated by two recombinases: Rad51 and the meiosis-specific Dmc1. In budding yeast, interhomolog bias is created in part by the activity of a meiosis-specific kinase, Mek1, which is localized to the protein cores of condensed sister chromatids. Analysis of meiotic double-strand break (DSB) repair in haploid and disomic haploid strains reveals that Mek1 suppresses meiotic intersister DSB repair by working directly on sister chromatids. Rec8 cohesin complexes are not required, however, either for suppression of intersister DSB repair or for the repair itself. Regulation of DSB repair in meiosis is chromosome autonomous such that unrepaired breaks on haploid chromosomes do not prevent interhomolog repair between disomic homologs. The pattern of DSB repair in haploids containing Dmc1 and/or Rad51 indicates that Mek1 acts on Rad51-specific recombination processes.IN eukaryotes, meiosis is a specialized type of cell division that produces the gametes required for sexual reproduction. In meiosis, one round of DNA replication is followed by two rounds of chromosome segregation, termed meiosis I and II. As a result of the two divisions, four haploid cells are produced, each containing half the number of chromosomes as the diploid parent. Proper segregation at meiosis I requires connections between homologous chromosomes that are created by a combination of sister chromatid cohesion and recombination (Petronczki et al. 2003). In vegetative cells, cohesion is mediated by multisubunit ring-shaped complexes that are removed by proteolysis of the kleisin subunit, Mcd1/Scc1 (Onn et al. 2008). In meiotic cells, introduction of a meiosis-specific kleisin subunit, Rec8, allows for a two-step removal of cohesion with loss of arm cohesion at anaphase I and centromere cohesion at anaphase II (Klein et al. 1999). Missegregation of chromosomes during meiosis causes abnormal chromosome numbers in gametes that may lead to infertility and genetic disorders such as trisomy 21 or Down''s syndrome.In mitotically dividing budding yeast cells, recombination is mediated by an evolutionarily conserved RecA-like recombinase, Rad51, and occurs preferentially between sister chromatids (Kadyk and Hartwell 1992). In contrast, recombination during meiosis is initiated by the deliberate formation of double-strand breaks (DSBs) by an evolutionarily conserved, topoisomerase-like protein, Spo11, and occurs preferentially between homologous chromosomes (Jackson and Fink 1985; Schwacha and Kleckner 1997; Keeney 2001). After DSB formation, the 5′ ends on either side of the breaks are resected, resulting in 3′ single stranded (ss) tails. Rad51, and the meiosis-specific recombinase Dmc1, bind to the 3′ ssDNA tails to form protein/DNA filaments that promote strand invasion of homologous chromosomes. DNA synthesis and ligation result in the formation of double Holliday junctions, which are then preferentially resolved into crossovers (Allers and Lichten 2001; Hunter 2007).The precise roles that the Rad51 and Dmc1 recombinase activities play in meiotic recombination have been unclear because experiments have indicated both overlapping and distinct functions for the two proteins (Sheridan and Bishop 2006; Hunter 2007). While both rad51Δ and dmc1Δ mutants reduce interhomolog recombination, other studies suggest that Rad51, in complex with the accessory protein Rad54, is involved primarily in intersister DSB repair. In contrast, Dmc1, in conjunction with the accessory protein Rdh54/Tid1 (a paralog of Rad54), effects DSB repair in meiotic cells by invasion of nonsister chromatids (Dresser et al. 1997; Schwacha and Kleckner 1997; Shinohara et al. 1997a,b; Arbel et al. 1999; Bishop et al. 1999; Hayase et al. 2004; Sheridan and Bishop 2006).The preference for recombination to occur between homologous chromosomes during meiosis is created in part by Dmc1. DSBs accumulate in dmc1Δ diploids due to a failure in strand invasion (Bishop et al. 1992; Hunter and Kleckner 2001). In the efficiently sporulating SK1 strain background, these unrepaired breaks trigger the meiotic recombination checkpoint, resulting in prophase arrest (Lydall et al. 1996; Roeder and Bailis 2000). In dmc1Δ mutants, Rad51 is present at DSBs, yet there is no strand invasion of sister chromatids (Bishop 1994; Shinohara et al. 1997a). These results suggest that in addition to Dmc1 promoting interhomolog strand invasion, Rad51 activity must also be suppressed.Recent studies have shown that during meiosis Rad51 recombinase activity is inhibited by two different mechanisms that decrease the formation of Rad51/Rad54 complexes: (1) binding of the meiosis-specific Hed1 protein to Rad51, thereby excluding interaction with Rad54, and (2) reduction in the affinity of Rad54 for Rad51 due to phosphorylation of Rad54 by Mek1 (Tsubouchi and Roeder 2006; Busygina et al. 2008; Niu et al. 2009). Mek1 is a meiosis-specific kinase that is activated in response to DSBs (Niu et al. 2005, 2007; Carballo et al. 2008). In addition to phosphorylating Rad54, Mek1 phosphorylation of an as yet undetermined substrate is required to suppress Rad51/Rad54-mediated strand invasion of sister chromatids (Niu et al. 2009).To dissect the mechanism by which Mek1 suppresses meiotic intersister DSB repair, we took advantage of the ability of yeast cells to undergo haploid meiosis. The lack of homologous chromosomes in haploid cells makes it possible to examine sister-chromatid-specific events in the absence of interhomolog recombination. De Massy et al. (1994) previously observed a delay in DSB repair in haploid cells and proposed that this delay was due to a constraint in using sister chromatids. We have shown that this delay is dependent on MEK1 and utilized the haploid system to determine various biological parameters required to suppress meiotic intersister DSB repair. Our results indicate that Rad51 and Dmc1 recombinase activities have distinct roles during meiosis and that interhomolog bias is established specifically on sister chromatids through regulation of Rad51, not Dmc1. rec8Δ diploids exhibit defects in meiotic DSB repair (Klein et al. 1999; Brar et al. 2009). Given that cohesin complexes are specific for sister chromatids, we investigated the role of REC8 in intersister DSB repair and found it is required neither for suppressing intersister DSB repair during meiosis nor for the repair itself.     

Chromosome Dynamics during Mitosis

The primary goal of mitosis is to partition duplicated chromosomes into daughter cells. Eukaryotic chromosomes are equipped with two distinct classes of intrinsic machineries, cohesin and condensins, that ensure their faithful segregation during mitosis. Cohesin holds sister chromatids together immediately after their synthesis during S phase until the establishment of bipolar attachments to the mitotic spindle in metaphase. Condensins, on the other hand, attempt to “resolve” sister chromatids by counteracting cohesin. The products of the balancing acts of cohesin and condensins are metaphase chromosomes, in which two rod-shaped chromatids are connected primarily at the centromere. In anaphase, this connection is released by the action of separase that proteolytically cleaves the remaining population of cohesin. Recent studies uncover how this series of events might be mechanistically coupled with each other and intricately regulated by a number of regulatory factors.In eukaryotic cells, genomic DNA is packaged into chromatin and stored in the cell nucleus, in which essential chromosomal processes, including DNA replication and gene expression, take place (Fig. 1, interphase). At the onset of mitosis, the nuclear envelope breaks down and chromatin is progressively converted into a discrete set of rod-shaped structures known as metaphase chromosomes (Fig. 1, metaphase). In each chromosome, a pair of sister kinetochores assembles at its centromeric region, and their bioriented attachment to the mitotic spindle acts as a prerequisite for equal segregation of sister chromatids. The linkage between sister chromatids is dissolved at the onset of anaphase, allowing them to be pulled apart to opposite poles of the cell (Fig. 1, anaphase). At the end of mitosis, the nuclear envelope reassembles around two sets of segregated chromatids, leading to the production of genetically identical daughter cells (Fig. 1, telophase).Open in a separate windowFigure 1.Overview of chromosome dynamics during mitosis. In addition to the crucial role of kinetochore–spindle interactions, an intricate balance between cohesive and resolving forces acting on sister chromatid arms (top left, inset) underlies the process of chromosome segregation. See the text for major events in chromosome segregation.Although the centromere–kinetochore region plays a crucial role in the segregation process, sister chromatid arms also undergo dynamic structural changes to facilitate their own separation. Conceptually, such structural changes are an outcome of two balancing forces, namely, cohesive and resolving forces (Fig. 1, top left, inset). The cohesive force holds a pair of duplicated arms until proper timing of separation, otherwise daughter cells would receive too many or too few copies of chromosomes. The resolving force, on the other hand, counteracts the cohesive force, reorganizing each chromosome into a pair of rod-shaped chromatids. From this standpoint, the pathway of chromosome segregation is regarded as a dynamic process, in which the initially robust cohesive force is gradually weakened and eventually dominated by the resolving force. Almost two decades ago, genetic and biochemical studies for the behavior of mitotic chromosomes converged productively, culminating in the discovery of cohesin (Guacci et al. 1997; Michaelis et al. 1997; Losada et al. 1998) and condensin (Hirano et al. 1997; Sutani et al. 1999), which are responsible for the cohesive and resolving forces, respectively. The subsequent characterizations of these two protein complexes have not only transformed our molecular understanding of chromosome dynamics during mitosis and meiosis, but also provided far-reaching implications in genome stability, as well as unexpected links to human diseases. In this article, I summarize recent progress in our understanding of mitotic chromosome dynamics with a major focus on the regulatory networks surrounding cohesin and condensin. I also discuss emerging topics and attempt to clarify outstanding questions in the field.     

Use of Quantitative Mass Spectrometric Analysis to Elucidate the Mechanisms of Phospho-priming and Auto-activation of the Checkpoint Kinase Rad53 in Vivo

The cell cycle checkpoint kinases play central roles in the genome maintenance of eukaryotes. Activation of the yeast checkpoint kinase Rad53 involves Rad9 or Mrc1 adaptor-mediated phospho-priming by Mec1 kinase, followed by auto-activating phosphorylation within its activation loop. However, the mechanisms by which these adaptors regulate priming phosphorylation of specific sites and how this then leads to Rad53 activation remain poorly understood. Here we used quantitative mass spectrometry to delineate the stepwise phosphorylation events in the activation of endogenous Rad53 in response to S phase alkylation DNA damage, and we show that the two Rad9 and Mrc1 adaptors, the four N-terminal Mec1-target TQ sites of Rad53 (Rad53-SCD1), and Rad53-FHA2 coordinate intimately for optimal priming phosphorylation to support substantial Rad53 auto-activation. Rad9 or Mrc1 alone can mediate surprisingly similar Mec1 target site phosphorylation patterns of Rad53, including previously undetected tri- and tetraphosphorylation of Rad53-SCD1. Reducing the number of TQ motifs turns the SCD1 into a proportionally poorer Mec1 target, which then requires the presence of both Mrc1 and Rad9 for sufficient priming and auto-activation. The phosphothreonine-interacting Rad53-FHA domains, particularly FHA2, regulate phospho-priming by interacting with the checkpoint mediators but do not seem to play a major role in the phospho-SCD1-dependent auto-activation step. Finally, mutation of all four SCD1 TQ motifs greatly reduces Rad53 activation but does not eliminate it, and residual Rad53 activity in this mutant is dependent on Rad9 but not Mrc1. Altogether, our results provide a paradigm for how phosphorylation site clusters and checkpoint mediators can be involved in the regulation of signaling relay in protein kinase cascades in vivo and elucidate an SCD1-independent Rad53 auto-activation mechanism through the Rad9 pathway. The work also demonstrates the power of mass spectrometry for in-depth analyses of molecular mechanisms in cellular signaling in vivo.Eukaryotic cells are most vulnerable to exogenous DNA-damaging agents during the S phase of the cell cycle, when unprogrammed DNA lesions interfere with the tightly choreographed DNA replication process. DNA damage during this phase leads to the activation of two overlapping checkpoint pathways in Saccharomyces cerevisiae, the DNA replication checkpoint and the intra-S-phase DNA damage checkpoint (1, 2). Phospho-priming for auto-activation of the central checkpoint kinase Rad53 by the upstream kinase Mec1/Tel1 depends on Mrc1 as an adaptor in the DNA replication checkpoint pathway and Rad9 as an adaptor in the DNA damage checkpoint pathway (3–10). Rad53, a well-accepted model system for studying the function and regulation of Chk2-like kinases, contains two forkhead-associated (FHA)¹ domains (FHA1 and -2) and two SQ/TQ cluster domains (SCD1 and -2) enriched in Mec1/Tel1-target phosphorylation sites (11–13).Mrc1 normally is a replisome component that functionally couples DNA Pol ε with Cdc45 and MCM helicase during replication fork progression (14, 15). As the replication forks are stalled by replication stress, the recruited checkpoint sensor kinase Mec1 phosphorylates the SCD of Mrc1, which abolishes its N-terminal interaction with Pol ε and enables Mrc1 to recruit Rad53 and promote Rad53 phosphorylation by Mec1 as an initial step in the activation of Rad53 in the Mrc1 branch (6, 14, 16). Alanine substitution of all Mec1 target sites of Mrc1 (designated the mrc1-AQ allele) has been shown to selectively disable its checkpoint function for Rad53 activation without affecting its DNA replication functions (4). In response to DNA damage, Rad9 is able to associate with damaged chromatin via its BRCT and Tudor domains, which tether it to Ser129-phosphorylated histone H2A (γH2A) and Lys79-methylated histone H3, respectively (17, 18). Alternatively, the recruitment of Rad9 onto damaged DNA could also be facilitated by its phosphorylation by CDK1, which enables the specific interaction of Rad9 with Dpb11, allowing the formation of the ternary complex of Dpb11, Mec1, and Rad9 (19, 20). Similar to Mrc1, Mec1 activates the adaptor function of Rad9 by phosphorylation of its SCD, which then binds to the Rad53-FHA domains to promote Rad53 phosphorylation by Mec1 (3, 5, 10).Beyond serving as scaffolds to recruit Rad53, Mrc1 and Rad9 have been shown to promote Rad53 phosphorylation by Mec1 in a dose-dependent manner in vitro (3, 16), underlining their adaptor role to enhance the enzyme–substrate (Mec1–Rad53) interaction. However, how they can specifically regulate the priming phosphorylation at specific sites and how this then leads to Rad53 activation remains poorly understood. Finally, hyperphosphorylated Rad9 has also been shown to catalyze the auto-phosphorylation of recombinant Rad53 (21), but it remains to be examined whether and how this occurs in vivo.The activation of SCD-FHA containing kinases such as human Chk2 and fission yeast Cds1 has been suggested to involve a two-step phosphorylation process: first, SCD phosphorylation by an ATM/ATR-like kinase leads to intermolecular binding to the FHA domain of another Chk2/Cds1 monomer, which then results in dimerization/oligomerization-dependent auto-phosphorylation within the kinase activation loop (22–26). In addition to the characteristic N-terminal SCD-FHA module of Chk2-like kinases, Rad53 contains another SCD2-FHA2 module C-terminal to its kinase domain. Similar to its orthologues, Rad53 activation has been proposed to depend on SCD1 phosphorylation (but not SCD2 phosphorylation) and partially redundant functions of the two FHA domains (9, 27–29). However, although Rad53-FHA1 can interact with SCD1 in a phospho-threonine (pT)-dependent manner in vitro (9, 28), it appears to be required for Rad53 activation only in G2/M-arrested cells (27, 29). In contrast, the FHA2 domain, which seems to be more important overall for Rad53 activation, does not appreciably bind phospho-SCD1 peptides in vitro (27, 28). Thus, the mechanisms by which Mrc1, Rad9, SCD1 phosphorylation, and FHA domains interact during checkpoint-dependent Rad53 priming and auto-activation remain to be elucidated.Quantitative mass spectrometric analysis has revolutionized the functional analysis of cellular signaling pathways, including site-specific phosphorylation events of key signaling molecules (30–33), but an important caveat is that MS studies often involve protein tags or nonphysiological expression levels that can interfere with normal protein functions. For example, the integration of a triple HA tag into the endogenous RAD53 gene locus has been shown to reduce Rad53 protein levels, resulting in significantly altered checkpoint activity (34). In this study we used quantitative MS analyses to dissect the stepwise phosphorylation events of endogenous, untagged Rad53 in response to MMS-induced alkylation DNA damage and replication stress during the S phase. Together with functional analyses, our results delineate how the two Mec1 adaptors Rad9 and Mrc1 can coordinate with the four SCD1 priming sites (T5, T8, T12, and T15) to regulate the phospho-priming of Rad53 by Mec1. In addition, an SCD1-priming independent Rad53 auto-activation mechanism and the specific roles of the FHA domains during Rad53 hyperphosphorylation are also elucidated in this work.     

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.     

Cohesins determine the attachment manner of kinetochores to spindle microtubules at meiosis I in fission yeast

During mitosis, sister kinetochores attach to microtubules that extend to opposite spindle poles (bipolar attachment) and pull the chromatids apart at anaphase (equational segregation). A multisubunit complex called cohesin, including Rad21/Scc1, plays a crucial role in sister chromatid cohesion and equational segregation at mitosis. Meiosis I differs from mitosis in having a reductional pattern of chromosome segregation, in which sister kinetochores are attached to the same spindle (monopolar attachment). During meiosis, Rad21/Scc1 is largely replaced by its meiotic counterpart, Rec8. If Rec8 is inactivated in fission yeast, meiosis I is shifted from reductional to equational division. However, the reason rec8Delta cells undergo equational rather than random division has not been clarified; therefore, it has been unclear whether equational segregation is due to a loss of cohesin in general or to a loss of a specific requirement for Rec8. We report here that the equational segregation at meiosis I depends on substitutive Rad21, which relocates to the centromeres if Rec8 is absent. Moreover, we demonstrate that even if sufficient amounts of Rad21 are transferred to the centromeres at meiosis I, thereby establishing cohesion at the centromeres, rec8Delta cells never recover monopolar attachment but instead secure bipolar attachment. Thus, Rec8 and Rad21 define monopolar and bipolar attachment, respectively, at meiosis I. We conclude that cohesin is a crucial determinant of the attachment manner of kinetochores to the spindle microtubules at meiosis I in fission yeast.     

Setting the stage for cohesion establishment by the replication fork

Comment on: Rudra S, et al. Cell Cycle 2012; 2114-21The complex process of semi-conservative DNA replication involves a mechanism whereby the leading and lagging strands with opposite polarity serve as templates for concerted synthesis of complementary base pairs.¹ Lagging-strand synthesis creates discontinuous Okazaki fragments that require timely processing of the 5′ flaps, so that adjacent nascent DNA strands are ligated together to insure genomic stability. While the genetic and molecular requirements of Okazaki fragment maturation have been studied in much detail, the precise temporal and spatial relationship of lagging-strand processing to sister chromatid cohesion remains unclear.² The newly replicated daughter duplex DNA molecules (i.e., the sister chromatids) become tethered during DNA replication and remain paired in order to permit proper segregation of the chromosomes to respective poles during mitosis and nuclear division. Elegant genetic studies in yeast have implicated posttranslational modification of cohesins (specialized protein complexes responsible for tethering sister pairs) by Ctf7/Eco1 acetylase as a key regulatory step in the process, enabling cohesins to perform their function in capturing the newly synthesized sister chromatids. Previous work suggested that genetic and physical interactions among the yeast acetyltransferase Ctf7/Eco1, helicase Chl1, Flap Endonuclease (Fen1) and accessory replication factors [e.g., RFC (clamp loader) and PCNA (clamp)] play an integral role in cohesion establishment. Based on these pieces of evidence, several models to explain the relationship between replication fork dynamics and sister chromatid cohesion have been proposed; however, our understanding of the precise timing of cohesin acetylation and the passage of the replication fork machinery has remained murky at best. Given the importance of proper chromosome segregation for chromosomal stability and the suppression of developmental disorders and tumorigenesis, a comprehensive understanding of the molecular acrobatics involved in sister chromatid cohesion is highly important.In a recent study, the temporal relationship between sister chromatid establishment and lagging-strand synthesis was illuminated.³ The authors have elucidated the link between the catalytic functions of DNA unwinding, flap processing and acetylation, which supports a model of cohesion deposition and establishment that occurs after the passage of the replication fork, similar to how genomic DNA becomes chromatinized. This is a significant advance from an earlier and very popular model of sister chromatid cohesion predicted that Ctf7/Eco1 acetylated cohesin proteins before the encounter by the DNA replication fork, which was thought to permit fork progression and the proper cohesion state for sister chromatid tethering (for review, see ref. ²). Instead, the genetic evidence presented by the Skibbens lab supports a model whereby cohesion establishment is temporally coupled to lagging-strand processing.³ In support of the genetic proof, Rudra and Skibbens went on to show that both Ctf7/Eco1 and Chl1 are associated with the lagging-strand processing nuclease Fen1. Altogether, the experimental results implicate a post-fork establishment model that is analogous to how histone protein complexes are deposited onto newly synthesized sister chromatids and become posttranslationally modified to confer epigenetic status.The discovery from the Skibbens lab that cohesion establishment is closely orchestrated with Okazaki fragment processing prompts a new line of inquiry about the control of flap processing by acetylation and its dual purpose for proper sister chromatid cohesion and replication fidelity in eukaryotes (Fig. 1). The catalytic activity of human FEN-1^4,5 and a functionally related endonuclease known as Dna2⁴ have been shown to be modulated by p300 acetylation, which suggested a model for creating long flap intermediates to promote genomic stability and suppress mutagenesis. Given evidence that ChlR1 is implicated in the genetic disorder Warsaw Breakage syndrome and that the human homolog of yeast Chl1⁶ interacts with the RFC complex and Fen1,⁷ it will be informative to determine if acetyltransferases such as the human orthologs Esco1 and Esco2, the latter mutated in the cohesinopathy Roberts syndrome,⁸ and perhaps other acetyltransferases (e.g., p300) are master regulators of lagging-strand synthesis that not only affect replication fidelity and genomic stability, but also sister chromatid cohesion. Coordination of sister chromatid cohesion establishment with lagging strand synthesis may also involve replication fork stabilization by the Timeless-Tipin protein complex implicated in replication checkpoint.⁹ Defects in the efficient coupling of lagging-strand synthesis to sister chromatid cohesion may contribute to the chromosomal instability characteristic of age-related diseases and cancer.Open in a separate windowFigure 1. Interplay between acetylation, replication fork dynamics and cohesion establishment important for chromosomal integrity.     

Reconstitution of Rad53 Activation by Mec1 through Adaptor Protein Mrc1

Upon DNA replication stress, stalled DNA replication forks serve as a platform to recruit many signaling proteins, leading to the activation of the DNA replication checkpoint. Activation of Rad53, a key effector kinase in the budding yeast Saccharomyces cerevisiae, is essential for stabilizing DNA replication forks during replication stress. Using an activity-based assay for Rad53, we found that Mrc1, a replication fork-associated protein, cooperates with Mec1 to activate Rad53 directly. Reconstitution of Rad53 activation using purified Mec1 and Mrc1 showed that the addition of Mrc1 stimulated a more than 70-fold increase in the ability of Mec1 to activate Rad53. Instead of increasing the catalytic activity of Mec1, Mrc1 was found to facilitate the phosphorylation of Rad53 by Mec1 via promotion of a stronger enzyme-substrate interaction between them. Further, the conserved C-terminal domain of Mrc1 was found to be required for Rad53 activation. These results thus provide insights into the role of the adaptor protein Mrc1 in activating Rad53 in the DNA replication checkpoint.Faithful replication of the genome is important for the survival of all organisms. During DNA replication, replication stress can arise from a variety of situations, including intrinsic errors made by DNA polymerases, difficulties in replicating repeated DNA sequences, and failures to repair damaged DNA caused by either endogenous oxidative agents or exogenous mutagens such as UV light and DNA-damaging chemicals (1–3). In eukaryotes, there is an evolutionarily conserved DNA replication checkpoint that becomes activated in response to DNA replication stress. It helps to stabilize DNA replication forks, block late replication origin firing, and delay mitosis and ultimately helps recovery from stalled replication forks after DNA repair (4–7). Defects in the DNA replication checkpoint could result in elevated genomic instabilities, cancer development, or cell death (8, 9).Aside from replicating the genome, the DNA replication forks also provide a platform to assemble many signaling proteins that function in the DNA replication checkpoint. In the budding yeast Saccharomyces cerevisiae, Mec1, an ortholog of human ATR,² is a phosphoinositide 3-kinase-like kinase (PIKK) involved in sensing stalled DNA replication forks. Mec1 forms a protein complex with Ddc2 (ortholog of human ATRIP). The Mec1-Ddc2 complex is recruited to stalled replication forks through replication protein A (RPA)-coated single-stranded DNA (10, 11). The Mec3-Rad17-Ddc1 complex, a proliferating cell nuclear antigen (PCNA)-like checkpoint clamp and ortholog of the human 9-1-1 complex, was shown to be loaded onto the single- and double-stranded DNA junction of the stalled replication forks by the clamp loader Rad24-RFC complex (12). Once loaded, the Mec3-Rad17-Ddc1 complex stimulates Mec1 kinase activity (13). Dbp11 and its homolog TopBP1 in vertebrates are known components of the replication machinery (14). In addition to regulating the initiation of DNA replication, they were found to play a role in the DNA replication checkpoint (15–17). They interact with the 9-1-1 complex and directly stimulate Mec1/ATR activity in vitro (18–20). Thus, the assembly of multiple protein complexes at stalled DNA replication forks appears to facilitate activation of the DNA replication checkpoint (13, 18).Mrc1 (for mediator of replication checkpoint) was originally identified to be important for cells to respond to hydroxyurea in S. cerevisiae and Schizosaccharomyces pombe (21, 22). Mrc1 is a component of the DNA replisome and travels with the replication forks along chromosome during DNA synthesis (23–25). Deletion of MRC1 causes defects in DNA replication, indicating its role in the normal progression of DNA replication (23). Interestingly, when DNA replication is blocked by hydroxyurea, Mrc1 undergoes Mec1- and Rad3 (S. pombe ortholog of Mec1)-dependent phosphorylation (21, 22). In S. cerevisiae, mutations of Mrc1 at the (S/T)Q sites, which are consensus phosphorylation sites of the Mec1/ATR family kinases, abolishes hydroxyurea-induced Mrc1 phosphorylation in vivo, suggesting a direct phosphorylation of Mrc1 by Mec1 (21, 22).Rad53 and Cds1, homologs of human Chk2, are the major effector kinases in the DNA replication checkpoints in S. cerevisiae and S. pombe, respectively. Activation of Rad53 is a hallmark of DNA replication checkpoint activation and is important for the maintenance of DNA replication forks in response to DNA replication stress (5, 6). Thus, it is important to understand how Rad53 activity is controlled. Interestingly, mutation of all the (S/T)Q sites of Mrc1 not only abolishes the phosphorylation of Mrc1 by Mec1 but also compromises hydroxyurea-induced Rad53 activation in S. cerevisiae (21). Similarly, mutation of the TQ sites of Mrc1 in S. pombe was shown to abolish the binding between Cds1 and Mrc1 as well as Cds1 activation (22). Further, mutation of specific TQ sites of Mrc1 in S. pombe abolishes its binding to Cds1 in vitro and the activation of Cds1 in vivo (26). Thus, Mec1/Rad3-dependent phosphorylation of Mrc1 is responsible for Mrc1 binding to Rad53/Cds1, which is essential for Rad53/Cds1 activation.An intriguing property of the Chk2 family kinases is their ability to undergo autophosphorylation and activation in the absence of other proteins in vitro (27, 28). First, autophosphorylation of a conserved threonine residue in the activation loop of Chk2 family kinase was found to be an essential part of their activation processes (26, 29–31). Second, a direct and trans-phosphorylation of the N-terminal TQ sites of the Chk2 family kinases by the Mec1/ATR family kinases is also important for their activation in vivo. Analogous to the requirement of N-terminal TQ site phosphorylation of Chk2 by ATR in human (32), the activation of Rad53/Cds1 in vivo requires phosphorylation of TQ sites in their N termini by Mec1/Rad3 (33, 34).Considering that Mec1, Mrc1, and many other proteins are recruited at stalled DNA replication forks and have been shown to be involved in DNA replication checkpoint activation, a key question remains unresolved: what is the minimal system that is capable of activating Rad53 directly? Given the direct physical interaction between Mrc1 and Rad53 and the requirement of Mrc1 and Mec1 in vivo, it is likely that they both play a role in Rad53 activation. Furthermore, what is the molecular mechanism of Rad53 activation by its upstream activators? To address these questions, a faithful reconstitution of the activation of Rad53 using purified proteins is necessary. In this study, we developed an activity-based assay consisting of the Dun1 kinase, a downstream substrate of Rad53, and Sml1, as a substrate of Dun1, to quantitatively measure the activity of Rad53. Using this coupled kinase assay from Rad53 to Dun1 and then to Sml1, we screened for Mrc1 and its associated factors to see whether they could directly activate Rad53 in vitro. Our results showed that Mec1 and Mrc1 collaborate to constitute a minimal system in direct activation of Rad53.