DNA damage signaling pathways are initiated in response to chemical reagents and radiation damage, as well as in response to hypoxia. It is implicated that structural maintenance of chromosomes 1 (SMC1) is not only a component of the cohesion complex but also facilitates the activation of DNA damage checkpoint proteins. Here, we studied the mechanism of DNA damage checkpoint activated by ATR–SMC1 pathway when cells are treated with desferrioxamine (DFO), a hypoxia-mimetic reagent. We show that DFO treatment induces phosphorylation of SMC1 at Ser966, NBS1 at Ser343, Chk1 at Ser317, Chk2 at Thr68, and p53 at Ser15. Among these sites, phosphorylation of SMC1, NBS1, and Chk1 by DFO are mediated by ATR as it is greatly reduced in both ATR-deficient human fibroblasts and HCT116 human colon cancer cells in which ATR is heterozygously mutated, whereas these proteins are phosphorylated in cells deficient for ATM and DNA-PKcs. DFO-induced apoptosis is decreased in ATR-mutant HCT116 cells, although p53 is normally activated in those cells. Expression of SMC1 S966A in which Ser966 is substituted to Ala attenuates apoptosis and phosphorylation of Chk1 at Ser317 after DFO treatment, although levels of HIF1α are not significantly changed. These results suggest that DFO induces apoptosis through the ATR–SMC1 arm of the pathway.
ATR; SMC1; Hypoxia
The structural maintenance of chromosomes (Smc) family members Smc5 and Smc6 are both essential in budding and fission yeasts. Yeast smc5/6 mutants are hypersensitive to DNA damage, and Smc5/6 is recruited to HO-induced double-strand breaks (DSBs), facilitating intersister chromatid recombinational repair. To determine the role of the vertebrate Smc5/6 complex during the normal cell cycle, we generated an Smc5-deficient chicken DT40 cell line using gene targeting. Surprisingly, Smc5− cells were viable, although they proliferated more slowly than controls and showed mitotic abnormalities. Smc5-deficient cells were sensitive to methyl methanesulfonate and ionizing radiation (IR) and showed increased chromosome aberration levels upon irradiation. Formation and resolution of Rad51 and gamma-H2AX foci after irradiation were altered in Smc5 mutants, suggesting defects in homologous recombinational (HR) repair of DNA damage. Ku70−/− Smc5− cells were more sensitive to IR than either single mutant, with Rad54−/− Smc5− cells being no more sensitive than Rad54−/− cells, consistent with an HR function for the vertebrate Smc5/6 complex. Although gene targeting occurred at wild-type levels, recombinational repair of induced double-strand breaks was reduced in Smc5− cells. Smc5 loss increased sister chromatid exchanges and sister chromatid separation distances in mitotic chromosomes. We conclude that Smc5/6 regulates recombinational repair by ensuring appropriate sister chromatid cohesion.
SMC1A (structural maintenance of chromosomes 1A), which encodes a structural subunit of the cohesin protein complex, is necessary for the process of sister chromatid cohesion during the cell cycle. Mutation and deregulation of SMC1A are highly relevant to diverse human diseases, including Cornelia de Lange syndrome and malignant carcinomas. In order to further investigate the role of SMC1A in the oncogenesis of lung cancer, SMC1A-specific short hairpin RNA (shRNA)-expressing lentivirus (Lv-shSMC1A) was constructed and used to infect A549 and H1299 cells. SMC1A mRNA and protein expression levels were downregulated in A549 and H1299 cells as demonstrated by real-time PCR and western blot assays. We found that SMC1A inhibition resulted in significantly impaired proliferation and colony formation as well as reduced invasiveness of tumor cells. Notably, Lv-shSMC1A-infected cancer cells exhibited a greater proportion of cells in the G0/G1 phase, but a lower proportion of S phase cells, compared to the parent or Lv-shCon infected cancer cells. Moreover, a greater proportion of sub-G1 apoptotic cells was observed in Lv-shSMC1A-infected cells. These results suggest that SMC1A is a novel proliferation regulator that promotes the growth of lung cancer cells, and that down-regulation of SMC1A expression induces growth suppression of A549 and H1299 cells via G1/S cell cycle phase arrest and apoptosis pathways. Therefore, SMC1A may serve as a new molecular target for lung cancer therapy.
SMC1A; proliferation; shRNA; lung cancer
Cohesin subunit SMC1β is specific and essential for meiosis. Previous studies showed functions of SMC1β in determining the axis-loop structure of synaptonemal complexes (SCs), in providing sister chromatid cohesion (SCC) in metaphase I and thereafter, in protecting telomere structure, and in synapsis. However, several central questions remained unanswered and concern roles of SMC1β in SCC and synapsis and processes related to these two processes. Here we show that SMC1β substantially supports prophase I SCC at centromeres but not along chromosome arms. Arm cohesion and some of centromeric cohesion in prophase I are provided by non-phosphorylated SMC1α. Besides supporting synapsis of autosomes, SMC1β is also required for synapsis and silencing of sex chromosomes. In absence of SMC1β, the silencing factor γH2AX remains associated with asynapsed autosomes and fails to localize to sex chromosomes. Microarray expression studies revealed up-regulated sex chromosome genes and many down-regulated autosomal genes. SMC1β is further required for non-homologous chromosome associations observed in absence of SPO11 and thus of programmed double-strand breaks. These breaks are properly generated in Smc1β−/− spermatocytes, but their repair is delayed on asynapsed chromosomes. SMC1α alone cannot support non-homologous associations. Together with previous knowledge, three main functions of SMC1β have emerged, which have multiple consequences for spermatocyte biology: generation of the loop-axis architecture of SCs, homologous and non-homologous synapsis, and SCC starting in early prophase I.
The generation of mammalian gametes through meiosis comprises two subsequent cell divisions. The first division, meiosis I, features highly specific chromosome structures, and behavior, and requires distinct sets of chromosome-associated proteins. Cohesin proteins, of which some are meiosis-specific, are essential for meiosis, but their particular roles in meiosis are incompletely understood. We show here that SMC1β, a meiosis-specific cohesin, serves key functions already in prophase of meiosis I: SMC1β contributes to keeping sister chromatids in cohesion at their centromeres and supports synapsis of the four sister chromatids present in these cells. SMC1β is required for the synapsis of the X and Y sex chromosomes. The failure of autosomes to properly synapse in absence of SMC1β causes extensive alterations in gene expression. This leads to expression of sex chromosome-linked genes, which are lethal at this stage, explaining the death of spermatocytes in mid-prophase I. Together with the analyses of other cohesin proteins and of phosphorylated forms of SMC3 and SMC1α, this paper describes hitherto undescribed properties and functions of meiotic cohesin in sister chromatid cohesion and synapsis.
The structural maintenance of chromosomes (Smc) proteins regulate nearly all aspects of chromosome biology and are critical for genomic stability. In eukaryotes, six Smc proteins form three heterodimers--Smc1/3, Smc2/4, and Smc5/6--which together with non-Smc proteins form cohesin, condensin, and the Smc5/6 complex, respectively. Cohesin is required for proper chromosome segregation. It establishes and maintains sister-chromatid cohesion until all sister chromatids achieve bipolar attachment to the mitotic spindle. Condensin mediates chromosome condensation during mitosis. The Smc5/6 complex has multiple roles in DNA repair. In addition to their major functions in chromosome cohesion and condensation, cohesin and condensin also participate in the cellular DNA damage response. Here we review recent progress on the functions of all three Smc complexes in DNA repair and their cell cycle regulation by posttranslational modifications, such as acetylation, phosphorylation, and sumoylation. An in-depth understanding of the mechanisms by which these complexes promote DNA repair and genomic stability may help us to uncover the molecular basis of genomic instability in human cancers and devise ways that exploit this instability to treat cancers.
Cohesin; Condensin; Smc5; Smc6; homologous recombination; DNA repair; DNA damage checkpoint; rDNA; SUMO
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.
ATM; SMC1; SMC3; Cohesin; Ionizing radiation; DNA repair
No rapid reliable method exists for identifying ataxia-telangiectasia (A-T) homozygotes or heterozygotes. Heterozygotes are at an increased risk of cancer and are more sensitive to the effects of ionizing radiation (IR) than the general population. We report a rapid flow cytometry (FC)-based ataxia-telangiectasia mutated (ATM) kinase assay that measures ATM-dependent phosphorylation of structural maintenance of chromosomes 1 (SMC1) following DNA damage (FC-pSMC1 assay).
After optimizing conditions with lymphoblastoid cell lines (LCLs), we studied peripheral blood mononuclear cells (PBMCs) isolated from 16 healthy donors (unknowns), 10 obligate A-T heterozygotes, and 6 unrelated A-T patients. One hour after DNA damage (by either IR or bleomycin), the cells were fixed and incubated with a primary antibody to SMC1pSer966. We analyzed the stained cells by FC to determine the difference in geometric mean fluorescence intensity (ΔGMFI) of untreated and treated cells; this difference was expressed as a percentage of daily experimental controls.
The FC-pSMC1 assay reliably distinguished ATM heterozygotes and homozygotes from controls. Average ΔGMFI percentages (SD) of daily controls were, for unknowns, 106.1 (37.6); for A-T heterozygotes, 37.0 (18.7); and for A-T homozygotes; −8.73 (16.2). Values for heterozygotes and homozygotes were significantly different from those of controls (P < 0.0001).
The FC-pSMC1 assay shortens the turnaround time for diagnosing A-T homozygotes from approximately 3 months to approximately 3 h. It also identifies A-T heterozygotes and can be used for pre-natal counseling or for screening individuals in large study cohorts for potential ATM heterozygosity, which can then be confirmed by sequencing.
The cohesion of sister chromatids is mediated by cohesin, a protein complex containing members of the structural maintenance of chromosome (Smc) family. How cohesins tether sister chromatids is not yet understood. Here, we mutate SMC1, the gene encoding a cohesin subunit of budding yeast, by random insertion dominant negative mutagenesis to generate alleles that are highly informative for cohesin assembly and function. Cohesins mutated in the Hinge or Loop1 regions of Smc1 bind chromatin by a mechanism similar to wild-type cohesin, but fail to enrich at cohesin-associated regions (CARs) and pericentric regions. Hence, the Hinge and Loop1 regions of Smc1 are essential for the specific chromatin binding of cohesin. This specific binding and a subsequent Ctf7/Eco1-dependent step are both required for the establishment of cohesion. We propose that a cohesin or cohesin oligomer tethers the sister chromatids through two chromatin-binding events that are regulated spatially by CAR binding and temporally by Ctf7 activation, to ensure cohesins crosslink only sister chromatids.
Complexes containing members of the structural maintenance of chromosomes (Smc) family regulate higher order chromosome architecture in diverse aspects of DNA metabolism including chromosome condensation, sister chromatid cohesion, DNA repair, and global control of transcription. Smc complexes are thought to regulate higher order chromosome folding by tethering together two strands of chromatin. However, the mechanism of tethering is poorly understood in part because of a poor understanding of the function of the core Smc subunits. To gain insight into the structure and function of Smc subunits, we developed a novel strategy of mutagenesis called random insertion dominant negative (RID), which generates informative alleles with high efficiency and should provide an effective tool to study any multi-subunit complex. Using RID we generated novel alleles of a Smc subunit from the cohesin complex. The cohesin complex tethers together newly replicated chromosomes (sister chromatids). The analyses of these RID mutants suggest that the tethering activity of cohesin (and possibly other Smc complexes) is generated by two sequential chromatin-binding events (first the capture of one piece of chromatin followed by the capture of the second piece of chromatin), which are regulated both spatially and temporally. We speculate that the spatial and temporal regulation of cohesin ensures that it tethers together only sister chromatids rather than randomly crosslinking the entire genome.
Cohesin mediates sister chromatid cohesion by topologically entrapping sister DNA molecules inside its ring structure. Cohesin is loaded onto DNA by the Scc2/NIPBL-Scc4/MAU2-loading complex in a manner that depends on the adenosine triphosphatase (ATPase) activity of cohesin’s Smc1 and Smc3 subunits. Subsequent cohesion establishment during DNA replication depends on Smc3 acetylation by Esco1 and Esco2 and on recruitment of sororin, which “locks” cohesin on DNA by inactivating the cohesin release factor Wapl.
Human cohesin ATPase mutants associate transiently with DNA in a manner that depends on the loading complex but cannot be stabilized on chromatin by depletion of Wapl. These mutants cannot be acetylated, fail to interact with sororin, and do not mediate cohesion. The absence of Smc3 acetylation in the ATPase mutants is not a consequence of their transient association with DNA but is directly caused by their inability to hydrolyze ATP because acetylation of wild-type cohesin also depends on ATP hydrolysis.
Our data indicate that cohesion establishment involves the following steps. First, cohesin transiently associates with DNA in a manner that depends on the loading complex. Subsequently, ATP hydrolysis by cohesin leads to entrapment of DNA and converts Smc3 into a state that can be acetylated. Finally, Smc3 acetylation leads to recruitment of sororin, inhibition of Wapl, and stabilization of cohesin on DNA. Our finding that cohesin’s ATPase activity is required for both cohesin loading and Smc3 acetylation raises the possibility that cohesion establishment is directly coupled to the reaction in which cohesin entraps DNA.
•Cohesin initially interacts with chromatin transiently via the loading complex•Cohesin’s ATPase activity is required for dynamic chromatin-cohesin interactions•Smc3 acetylation does not significantly alter cohesin’s ATPase activity•Cohesin’s ATPase activity is required for Smc3 acetylation
Ladurner et al. present evidence that the adenosine triphosphatase (ATPase) activity of the cohesin complex is required for cohesin acetylation, suggesting that sister-chromatid entrapment by cohesin is coupled to the establishment of cohesion.
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.
The cellular concentrations of individual proteins are expected to be kept within an optimal range, but protein expression is often stochastic. Some proteins are known to be in limiting amounts, so that even modest reduction can lead to malfunction. Within the network of genes that determine genome stability, proteins that are limiting impose a risk for the cell, because fluctuation in their amounts may start a cascade of genomic alternations that will influence many biochemical pathways either under normal growth conditions or in response to chromosome damage. We sought to identify genes that are limiting for DSB repair by lowering the dosage of key genes from 4 to 1 in tetraploid Saccharomyces cerevisiae strains. We found that the complex that holds sister chromatid cohesion together (cohesin) is limiting in DSB repair. In addition, when it is reduced modestly, recombination between homologous chromosomes is highly increased, suggesting that the risk for loss of hetrozygosity (LOH) is increased too. These results should also be considered in light of increasing evidence that copy number variation can impact cellular function.
The cohesin complexes play a key role in chromosome segregation during both mitosis and meiosis. They establish sister chromatid cohesion between duplicating DNA molecules during S-phase, but they also have an important role during postreplicative double-strand break repair in mitosis, as well as during recombination between homologous chromosomes in meiosis. An additional function in meiosis is related to the sister kinetochore cohesion, so they can be pulled by microtubules to the same pole at anaphase I. Data about the dynamics of cohesin subunits during meiosis are scarce; therefore, it is of great interest to characterize how the formation of the cohesin complexes is achieved in order to understand the roles of the different subunits within them. We have investigated the spatio-temporal distribution of three different cohesin subunits in prophase I grasshopper spermatocytes. We found that structural maintenance of chromosome protein 3 (SMC3) appears as early as preleptotene, and its localization resembles the location of the unsynapsed axial elements, whereas radiation-sensitive mutant 21 (RAD21) (sister chromatid cohesion protein 1, SCC1) and stromal antigen protein 1 (SA1) (sister chromatid cohesion protein 3, SCC3) are not visualized until zygotene, since they are located in the synapsed regions of the bivalents. During pachytene, the distribution of the three cohesin subunits is very similar and all appear along the trajectories of the lateral elements of the autosomal synaptonemal complexes. However, whereas SMC3 also appears over the single and unsynapsed X chromosome, RAD21 and SA1 do not. We conclude that the loading of SMC3 and the non-SMC subunits, RAD21 and SA1, occurs in different steps throughout prophase I grasshopper meiosis. These results strongly suggest the participation of SMC3 in the initial cohesin axis formation as early as preleptotene, thus contributing to sister chromatid cohesion, with a later association of both RAD21 and SA1 subunits at zygotene to reinforce and stabilize the bivalent structure. Therefore, we speculate that more than one cohesin complex participates in the sister chromatid cohesion at prophase I.
Meiosis is a specialized cell division by which sexually reproducing organisms prompt the formation of specialized cells presenting a half of the species chromosomal number. These cells, the so-called gametes, are able to fertilize or be fertilized, depending on the sex in which they are produced and thus restore the species chromosomal number after fertilization. The reduction in the chromosome number is achieved by two successive rounds of chromosome segregations preceded by a single replication of the genetic material. Different proteins, mainly referred to as cohesins, are implied in the correct establishment and maintenance of an intimate association between homologous chromosomes by ensuring their close association until their separation in the first meiotic division. Grasshoppers have been considered as a gorgeous model for meiotic studies for decades due to their low chromosomal number, the large size of their chromosomes, and the well-defined meiotic stages at cytological level. On these grounds, we have combined classical grasshopper chromosome knowledge with protein immunolocalization tools in order to precisely analyze the presence of cohesins throughout the prophase of the first meiotic division. The results not only describe the dynamic loading pattern of several cohesin subunits in two grasshopper species, but they also surprisingly bring into light that different cohesins are sequentially loaded onto meiotic chromosomes throughout the first meiotic prophase. Finally, we discuss the possible roles for this sequential protein loading in relation to the processes that operate during meiosis, proposing a model for meiotic chromosome structure. Besides the novel scientific contributions for a better understanding of the meiotic process, this study clearly points out that classical cytogenetic models can be used to solve modern biological problems.
The cohesin complex, which is essential for sister chromatid cohesion and chromosome segregation, also inhibits resolution of sister chromatid intertwinings (SCIs) by the topoisomerase Top2. The cohesin-related Smc5/6 complex (Smc5/6) instead accumulates on chromosomes after Top2 inactivation, known to lead to a buildup of unresolved SCIs. This suggests that cohesin can influence the chromosomal association of Smc5/6 via its role in SCI protection. Using high-resolution ChIP-sequencing, we show that the localization of budding yeast Smc5/6 to duplicated chromosomes indeed depends on sister chromatid cohesion in wild-type and top2-4 cells. Smc5/6 is found to be enriched at cohesin binding sites in the centromere-proximal regions in both cell types, but also along chromosome arms when replication has occurred under Top2-inhibiting conditions. Reactivation of Top2 after replication causes Smc5/6 to dissociate from chromosome arms, supporting the assumption that Smc5/6 associates with a Top2 substrate. It is also demonstrated that the amount of Smc5/6 on chromosomes positively correlates with the level of missegregation in top2-4, and that Smc5/6 promotes segregation of short chromosomes in the mutant. Altogether, this shows that the chromosomal localization of Smc5/6 predicts the presence of the chromatid segregation-inhibiting entities which accumulate in top2-4 mutated cells. These are most likely SCIs, and our results thus indicate that, at least when Top2 is inhibited, Smc5/6 facilitates their resolution.
When cells divide, sister chromatids have to be segregated away from each other for the daughter cells to obtain a correct set of chromosomes. Using yeast as model organism, we have analyzed the function of the cohesin and the Smc5/6 complexes, which are essential for chromosome segregation. Cohesin is known to hold sister chromatid together until segregation occurs, and our results show that cohesin also controls Smc5/6, which is found to associate to linked chromatids specifically. In line with this, our analysis points to that the chromosomal localization of Smc5/6 is an indicator of the level of entanglement between sister chromatids. When Smc5/6 is non-functional, the resolution of these entanglements is shown to be inhibited, thereby preventing segregation of chromatids. Our results also indicate that DNA entanglements are maintained on chromosomes at specific sites until segregation. In summary, we uncover new functions for cohesin, in regulating when and where Smc5/6 binds to chromosomes, and for the Smc5/6 complex in facilitating the resolution of sister chromatid entanglements.
The cohesin protein complex holds sister chromatids together after synthesis until mitosis. It also contributes to post-replicative DNA repair in yeast and higher eukaryotes and accumulates at sites of laser-induced damage in human cells. Our goal was to determine whether the cohesin subunits SMC1 and Rad21 contribute to DNA double-strand break repair in X-irradiated human cells in the G2 phase of the cell cycle. RNA interference-mediated depletion of SMC1 sensitized HeLa cells to X-rays. Repair of radiation-induced DNA double-strand breaks, measured by γH2AX/53BP1 foci analysis, was slower in SMC1- or Rad21-depleted cells than in controls in G2 but not in G1. Inhibition of the DNA damage kinase DNA-PK, but not ATM, further inhibited foci loss in cohesin-depleted cells in G2. SMC1 depletion had no effect on DNA single-strand break repair in either G1 or late S/G2. Rad21 and SMC1 were recruited to sites of X-ray-induced DNA damage in G2-phase cells, but not in G1, and only when DNA damage was concentrated in subnuclear stripes, generated by partially shielded ultrasoft X-rays. Our results suggest that the cohesin complex contributes to cell survival by promoting the repair of radiation-induced DNA double-strand breaks in G2-phase cells in an ATM-dependent pathway.
Cornelia de Lange syndrome (CdLS) is a clinically heterogeneous developmental disorder characterized by facial dysmorphia, upper limb malformations, growth and cognitive retardation. Mutations in the sister chromatid cohesion factor genes NIPBL, SMC1A and SMC3 are present in ∼65% of CdLS patients. In addition to their canonical roles in chromosome segregation, the cohesin proteins are involved in other biological processes such as regulation of gene expression, DNA repair and maintenance of genome stability. To gain insights into the molecular basis of CdLS, we analyzed the affinity of mutated SMC1A and SMC3 hinge domains for DNA. Mutated hinge dimers bind DNA with higher affinity than wild-type proteins. SMC1A- and SMC3-mutated CdLS cell lines display genomic instability and sensitivity to ionizing radiation and interstrand crosslinking agents. We propose that SMC1A and SMC3 CdLS mutations affect the dynamic association between SMC proteins and DNA, providing new clues to the underlying molecular cause of CdLS.
Cohesin is a macromolecular complex that links sister chromatids together at the metaphase plate during mitosis. The links are formed during DNA replication and destroyed during the metaphase-to-anaphase transition. In budding yeast, the 14S cohesin complex comprises at least two classes of SMC (structural maintenance of chromosomes) proteins - Smc1 and Smc3 - and two SCC (sister-chromatid cohesion) proteins - Scc1 and Scc3. The exact function of these proteins is unknown.
Searches of protein sequence databases have revealed new homologs of cohesin proteins. In mouse, Mmip1 (Mad member interacting protein 1) and Smc3 share 99% sequence identity and are products of the same gene. A phylogenetic tree of SMC homologs reveals five families: Smc1, Smc2, Smc3, Smc4 and an ancestral family that includes the sequences from the Archaea and Eubacteria. This ancestral family also includes sequences from eukaryotes. A cohesion interaction network, comprising 17 proteins, has been constructed using two proteomic databases. Genes encoding six proteins in the cohesion network share a common upstream region that includes the MluI cell-cycle box (MCB) element. Pairs of the proteins in this network share common sequence motifs that could represent common structural features such as binding sites. Scc2 shares a motif with Chk1 (kinase checkpoint protein), that comprises part of the serine/threonine protein kinase motif, including the active-site residue.
We have combined genomic and proteomic data into a comprehensive network of information to reach a better understanding of the function of the cohesin complex. We have identified new SMC homologs, created a new SMC phylogeny and identified shared DNA and protein motifs. The potential for Scc2 to function as a kinase - a hypothesis that needs to be verified experimentally - could provide further evidence for the regulation of sister-chromatid cohesion by phosphorylation mechanisms, which are currently poorly understood.
The structural maintenance of chromosome proteins SMC1 and SMC3 play an important role in the maintenance of chromosomal integrity by preventing the premature separation of the sister chromatids at the onset of anaphase. The two proteins are constitutive components of the multimeric complex cohesin and form dimers by interacting at their central globular regions.
In order to identify proteins that by binding to SMC3 may interfere with the protein dimerization process, a human cDNA library was screened by the yeast two-hybrid system by using the hinge region of SMC3 as bait. This has lead to the identification of Hinderin, a novel five domains protein including two coiled-coil motifs and sharing a strikingly structural similarity to the SMC family of proteins. Hinderin is ubiquitously expressed in human tissues. Orthologue forms of the protein are present in other vertebrates but not in lower organisms. A mapping of the interaction sites revealed that the N- and C-terminal globular domains mediate the binding of Hinderin to SMC3. Hinderin/SMC3 complexes could be recovered by immunoprecipitation from cell lysates using an anti-SMC3 antibody, thus demonstrating that the two proteins interact in vivo. On the contrary, Hinderin did not interact with SMC1. In vivo the rate of SMC1/SMC3 interaction was decreased by the ectopic expression of Hinderin.
Hinderin is a novel binding partner of SMC3. Based on its ability to modulate SMC1/SMC3 interaction we postulate that Hinderin affects the availability of SMC3 to engage in the formation of multimeric protein complexes.
The SMC1/SMC3 heterodimer acts in sister chromatid cohesion, and recent data indicate a function in DNA double-strand break repair (DSBR). Since this role of SMC proteins has remained largely elusive, we explored interactions between SMC1 and the homologous recombination (HR) or non-homologous end-joining (NHEJ) pathways for DSBR in Saccharomyces cerevisiae. Analysis of conditional single- and double mutants of smc1-2 with rad52Δ, rad54Δ, rad50Δ or dnl4Δ illustrates a significant contribution of SMC1 to the overall capacity of cells to repair DSBs. smc1 but not smc2 mutants show increased hypersensitivity of HR mutants to ionizing irradiation and to the DNA crosslinking agent cis-platin. Haploid, but not diploid smc1-2 mutants were severely affected in repairing multiple genomic DNA breaks, suggesting a selective role of SMC1 in sister chromatid recombination. smc1-2 mutants were also 15-fold less efficient and highly error-prone in plasmid end-joining through the NHEJ pathway. Strikingly, inactivation of RAD52 or RAD54 fully rescued efficiency and accuracy of NHEJ in the smc1 background. Therefore, we propose coordination of HR and NHEJ processes by Smc1p through interaction with the RAD52 pathway.
Cohesins are important for chromosome structure and chromosome segregation during mitosis and meiosis. Cohesins are composed of two structural maintenance of chromosomes (SMC1-SMC3) proteins that form a V-shaped heterodimer structure, which is bridged by a α-kleisin protein and a stromal antigen (STAG) protein. Previous studies in mouse have shown that there is one SMC1 protein (SMC1β), two α-kleisins (RAD21L and REC8) and one STAG protein (STAG3) that are meiosis-specific. During meiosis, homologous chromosomes must recombine with one another in the context of a tripartite structure known as the synaptonemal complex (SC). From interaction studies, it has been shown that there are at least four meiosis-specific forms of cohesin, which together with the mitotic cohesin complex, are lateral components of the SC. STAG3 is the only meiosis-specific subunit that is represented within all four meiosis-specific cohesin complexes. In Stag3 mutant germ cells, the protein level of other meiosis-specific cohesin subunits (SMC1β, RAD21L and REC8) is reduced, and their localization to chromosome axes is disrupted. In contrast, the mitotic cohesin complex remains intact and localizes robustly to the meiotic chromosome axes. The instability of meiosis-specific cohesins observed in Stag3 mutants results in aberrant DNA repair processes, and disruption of synapsis between homologous chromosomes. Furthermore, mutation of Stag3 results in perturbation of pericentromeric heterochromatin clustering, and disruption of centromere cohesion between sister chromatids during meiotic prophase. These defects result in early prophase I arrest and apoptosis in both male and female germ cells. The meiotic defects observed in Stag3 mutants are more severe when compared to single mutants for Smc1β, Rec8 and Rad21l, however they are not as severe as the Rec8, Rad21l double mutants. Taken together, our study demonstrates that STAG3 is required for the stability of all meiosis-specific cohesin complexes. Furthermore, our data suggests that STAG3 is required for structural changes of chromosomes that mediate chromosome pairing and synapsis, DNA repair and progression of meiosis.
Meiosis is a specialized cell division required for the formation of gametes (sperm and egg). Early in meiosis, the chromosome pairs that we inherit from our mother and father become linked and genetic material is exchanged. This is a remarkable process as every gamete that we make is unique, and the unison between a sperm and egg will create a new individual that harbors novel combinations of characteristics from each parents' family tree. Linkage and genetic exchange between chromosomes is facilitated by a linear protein scaffold structure. A group of protein complexes known as cohesins are a key component of the protein scaffold. To date, there are 4 meiosis-specific cohesin complexes identified. Only one cohesin component known as STAG3 is represented in all meiosis-specific cohesins. We mutated the gene that encodes for STAG3 in mouse and discovered that it results in meiotic failure and absence of gametes. From careful analysis we have determined that STAG3 is required for the stability of meiosis-specific cohesins, which ensure that chromosomes are paired and genetic material is exchanged. Our findings imply that abnormalities in human STAG3 will give rise to chromosome defects, infertility and gonad atrophy.
Ataxia telangiectasia mutated (ATM) and DNA-dependent protein kinase (DNA-PK) play a crucial role in the initial stages of cell response, when cells are exposed to DNA insult such as ionizing radiation (IR) and chemical agents. We previously demonstrated that ATM requires BAAT1 for its activation in response to IR. In the present study, BAAT1 was found to bind to the DNA-PK catalytic subunit (DNA-PKcs) and SMC1. Biochemical analysis indicated that several regions of BAAT1 were responsible for the interaction with these proteins, and their binding affinity was altered after treatment with the IR mimetic, neocarzinostatin (NCS). Phosphorylation of the DNA-PKcs at Ser2056 and SMC1 at Ser966 was induced by NCS, while phosphorylation was reduced when BAAT1 was depleted by siRNA. These results indicate that BAAT1 globally regulates DNA damage signaling during the early stages of apoptosis.
BAAT1; DNA-dependent protein kinase catalytic subunit; DNA damage
Cornelia de Lange syndrome (CdLS) is a dominantly inherited congenital malformation disorder caused by mutations in the cohesin-loading protein NIPBL1,2 for nearly 60% of individuals with classical CdLS3-5 and in the core cohesin components SMC1A (~5%) and SMC3 (<1%) for a smaller fraction of probands6,7. In humans, the multi-subunit complex cohesin is comprised of SMC1, SMC3, RAD21 and a STAG protein to form a ring structure proposed to encircle sister chromatids to mediate sister chromatid cohesion (SCC)8 as well as play key roles in gene regulation9. SMC3 is acetylated during S-phase to establish cohesiveness of chromatin-loaded cohesin10-13 and in yeast, HOS1, a class I histone deacetylase, deacetylates SMC3 during anaphase14-16. Here we report the identification of HDAC8 as the vertebrate SMC3 deacetylase as well as loss-of-function HDAC8 mutations in six CdLS probands. Loss of HDAC8 activity results in increased SMC3 acetylation (SMC3-ac) and inefficient dissolution of the “used” cohesin complex released from chromatin in both prophase and anaphase. While SMC3 with retained acetylation is loaded onto chromatin, ChIP-Seq analysis demonstrates decreased occupancy of cohesin localization sites that results in a consistent pattern of altered transcription seen in CdLS cell lines with either NIPBL or HDAC8 mutations.
The SMC proteins are involved in DNA repair, chromosome condensation, and sister chromatid cohesion throughout Eukaryota. Long, anti-parallel coiled coils are a prominent feature of SMC proteins, and are thought to serve as spacer rods to provide an elongated structure and to separate domains. We reported recently that the coiled coils of mammalian condensin (SMC2/4) showed moderate sequence divergence (≈10–15%) consistent with their functioning as spacer rods. The coiled coils of mammalian cohesins (SMC1/3), however, were very highly constrained, with amino acid sequence divergence typically <0.5%. These coiled coils are among the most highly conserved mammalian proteins, suggesting that they make extensive contacts over their entire surface.
Here, we broaden our initial analysis of condensin and cohesin to include additional vertebrate and invertebrate organisms and multiple species of yeast. We found that the coiled coils of SMC1/3 are highly constrained in Drosophila and other insects, and more generally across all animal species. However, in yeast they are no more constrained than the coils of SMC2/4 and Ndc80/Nuf2p, suggesting that they are serving primarily as spacer rods.
SMC1/3 functions for sister chromatid cohesion in all species. Since its coiled coils apparently serve only as spacer rods in yeast, it is likely that this is sufficient for sister chromatid cohesion in all species. This suggests an additional function in animals that constrains the sequence of the coiled coils. Several recent studies have demonstrated that cohesin has a role in gene expression in post-mitotic neurons of Drosophila, and other animal cells. Some variants of human Cornelia de Lange Syndrome involve mutations in human SMC1/3. We suggest that the role of cohesin in gene expression may involve intimate contact of the coiled coils of SMC1/3, and impose the constraint on sequence divergence.
The SMC5/6 protein complex consists of the Smc5, Smc6 and Non-Smc-Element (Nse) proteins and is important for genome stability in many species. To identify novel components in the DNA repair pathway, we carried out a genetic screen to identify mutations that confer reduced resistance to the genotoxic effects of caffeine, which inhibits the ATM and ATR DNA damage response proteins. This approach identified inactivating mutations in CG5524 and MAGE, homologs of genes encoding Smc6 and Nse3 in yeasts. The fact that Smc5 mutants are also caffeine-sensitive and that Mage physically interacts with Drosophila homologs of Nse proteins suggests that the structure of the Smc5/6 complex is conserved in Drosophila. Although Smc5/6 proteins are required for viability in S. cerevisiae, they are not essential under normal circumstances in Drosophila. However, flies carrying mutations in Smc5, Smc6 and MAGE are hypersensitive to genotoxic agents such as ionizing radiation, camptothecin, hydroxyurea and MMS, consistent with the Smc5/6 complex serving a conserved role in genome stability. We also show that mutant flies are not compromised for pre-mitotic cell cycle checkpoint responses. Rather, caffeine-induced apoptosis in these mutants is exacerbated by inhibition of ATM or ATR checkpoint kinases but suppressed by Rad51 depletion, suggesting a functional interaction involving homologous DNA repair pathways that deserves further scrutiny. Our insights into the SMC5/6 complex provide new challenges for understanding the role of this enigmatic chromatin factor in multi-cellular organisms.
The cohesin complex that mediates sister chromatid cohesion contains three core subunits: Smc1, Smc3, and Scc1. Heterotypic interactions between Smc1 and Smc3 dimerization domains create stable V-shaped Smc1/Smc3 heterodimers with a hinge at the center and nucleotide-binding domains (NBDs) at the ends of each arm. Interconnection of each NBD through their association with the N- and C-terminal domains of Scc1 creates a tripartite ring, within which sister DNAs are thought to be entrapped (the ring model). Crystal structures show that the Smc1/Smc3 hinge has a toroidal shape, with independent “north” and “south” interaction surfaces on an axis of pseudosymmetry. The ring model predicts that sister chromatid cohesion would be lost by transient hinge opening.
We find that mutations within either interface weaken heterodimerization of isolated half hinges in vitro but do not greatly compromise formation of cohesin rings in vivo. They do, however, reduce the residence time of cohesin on chromosomes and cause lethal defects in sister chromatid cohesion. This demonstrates that mere formation of rings is insufficient for cohesin function. Stable cohesion requires cohesin rings that cannot easily open.
Either the north or south hinge interaction surface is sufficient for the assembly of V-shaped Smc1/Smc3 heterodimers in vivo. Any tendency of Smc proteins with weakened hinges to dissociate will be suppressed by interconnection of their NBDs by Scc1. We suggest that transient hinge dissociation caused by the mutations described here is incompatible with stable sister chromatid cohesion because it permits chromatin fibers to escape from cohesin rings.
► Unstable Smc1/3 hinge dimerization reduces the residence time of cohesin on chromatin
Structural maintenance of chromosomes (SMC) proteins fulfill pivotal roles in chromosome dynamics. In yeast, the SMC1-SMC3 heterodimer is required for meiotic sister chromatid cohesion and DNA recombination. Little is known, however, about mammalian SMC proteins in meiotic cells. We have identified a novel SMC protein (SMC1β), which—except for a unique, basic, DNA binding C-terminal motif—is highly homologous to SMC1 (which may now be called SMC1α) and is not present in the yeast genome. SMC1β is specifically expressed in testes and coimmunoprecipitates with SMC3 from testis nuclear extracts, but not from a variety of somatic cells. This establishes for mammalian cells the concept of cell-type- and tissue-specific SMC protein isoforms. Analysis of testis sections and chromosome spreads of various stages of meiosis revealed localization of SMC1β along the axial elements of synaptonemal complexes in prophase I. Most SMC1β dissociates from the chromosome arms in late-pachytene-diplotene cells. However, SMC1β, but not SMC1α, remains chromatin associated at the centromeres up to metaphase II. Thus, SMC1β and not SMC1α is likely involved in maintaining cohesion between sister centromeres until anaphase II.
Defining the mechanisms of chromosomal cohesion and dissolution of the cohesin complex from chromatids is important for understanding the chromosomal missegregation seen in many tumor cells. Here we report the identification of a novel cohesin-resolving protease and describe its role in chromosomal segregation. Sister chromatids are held together by cohesin, a multiprotein ring-like complex comprised of Rad21, Smc1, Smc3, and SA2 (or SA1). Cohesin is known to be removed from vertebrate chromosomes by two distinct mechanisms, namely, the prophase and anaphase pathways. First, PLK1-mediated phosphorylation of SA2 in prophase leads to release of cohesin from chromosome arms, leaving behind centromeric cohesins that continue to hold the sisters together. Then, at the onset of anaphase, activated separase cleaves the centromeric cohesin Rad21, thereby opening the cohesin ring and allowing the sister chromatids to separate. We report here that the calcium-dependent cysteine endopeptidase calpain-1 is a Rad21 peptidase and normally localizes to the interphase nuclei and chromatin. Calpain-1 cleaves Rad21 at L192, in a calcium-dependent manner. We further show that Rad21 cleavage by calpain-1 promotes separation of chromosome arms, which coincides with a calcium-induced partial loss of cohesin at several chromosomal loci. Engineered cleavage of Rad21 at the calpain-cleavable site without activation of calpain-1 can lead to a loss of sister chromatid cohesion. Collectively, our work reveals a novel function of calpain-1 and describes an additional pathway for sister chromatid separation in humans.