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1.
Fujioka Y Kimata Y Nomaguchi K Watanabe K Kohno K 《The Journal of biological chemistry》2002,277(24):21585-21591
Structural maintenance of chromosomes (SMC) proteins play central roles in chromosome organization and dynamics. They have been classified into six subtypes, termed SMC1 to SMC6, and function as heterodimer components of large protein complexes that also include several non-SMC proteins. The SMC2-SMC4 and SMC1-SMC3 complexes are also known as condensin and cohesin, respectively, but the recently identified SMC5 and SMC6 complex is less well characterized. Here, we report that NSE1 from Saccharomyces cerevisiae encodes a novel non-SMC component of the SMC5(Yol034wp)-SMC6(Rhc18p) complex corresponding to the 2-3-MDa molecular mass. Nse1p is essential for cell proliferation and localizes primarily in the nucleus. nse1 mutants are highly sensitive to DNA-damaging treatments and exhibit abnormal cellular morphologies, suggesting aberrant mitosis as a terminal morphological phenotype. These results are consistent with the reported features of the Schizosaccharomyces pombe SMC6 gene, rad18, which is thought to be involved in recombinational DNA repair. We conclude that Nse1p and the SMC5-SMC6 heterodimer together form a high molecular mass complex that is conserved in eukaryotes and required for both DNA repair and proliferation. 相似文献
2.
Cohesin plays a critical role in sister chromatid cohesion, double-stranded DNA break repair and regulation of gene expression. However, the mechanism of how cohesin directly interacts with DNA remains unclear. We report single-molecule experiments analyzing the interaction of the budding yeast cohesin Structural Maintenance of Chromosome (SMC)1-SMC3 heterodimer with naked double-helix DNA. The cohesin heterodimer is able to compact DNA molecules against applied forces of 0.45 pN, via a series of extension steps of a well-defined size ≈130 nm. This reaction does not require ATP, but is dependent on DNA supercoiling: DNA with positive torsional stress is compacted more quickly than negatively supercoiled or nicked DNAs. Un-nicked torsionally relaxed DNA is a poor substrate for the compaction reaction. Experiments with mutant proteins indicate that the dimerization hinge region is crucial to the folding reaction. We conclude that the SMC1-SMC3 heterodimer is able to restructure the DNA double helix into a series of loops, with a preference for positive writhe. 相似文献
3.
Members of the evolutionary conserved Structural Maintenance of Chromosomes (SMC) protein family are involved in chromosome condensation and gene dosage compensation with the SMC2 and SMC4 subtypes, and sister chromatid cohesion with the SMC1 and SMC3 subtypes. The bovine recombination protein complex RC-1, which catalyzes DNA transfer reactions, contains two heterodimeric SMC polypeptides, the genes of which have now been cloned, sequenced, and classified as bovine (b)SMC1 and bSMC3. Both proteins display all the characteristic features of the SMC family. FISH analysis localized the mouse SMC3 gene to chromosome 19D2-D3. Mono- and polyclonal antibodies specific for either subtype detected high levels of protein expression in lymphoid tissues, lung, testis and ovary. No change in levels of bSMC1 and bSMC3 proteins occurred after X-ray or UV-light irradiation of various cell lines or primary cells, and the amounts of individual proteins and the heterodimer are roughly constant throughout the cell cycle. Immunofluorescence of mouse cells detected the SMC1 protein in foci associated with the chromatin. These foci dissolve and the SMC protein dissociates from the chromatin during M phase. 相似文献
4.
Ding C Li Y Kim BJ Malovannaya A Jung SY Wang Y Qin J 《Journal of proteome research》2011,10(8):3652-3659
Cohesin is a protein complex that plays an essential role in pairing replicated sister chromatids during cell division. The vertebrate cohesin complex consists of four core components including structure maintenance of chromosomes proteins SMC1 and SMC3, RAD21, and SA2/SA1. Extensive research suggests that cohesin traps the sister chromatids by a V-shaped SMC1/SMC3 heterodimer bound to the RAD21 protein that closes the ring. Accordingly, the single "ring" model proposes that two sister chromatids are trapped in a single ring that is composed of one molecule each of the 4 subunits. However, evidence also exists for alternative models. The hand-cuff model suggests that each sister chromatid is trapped individually by two rings that are joined through the shared SA1/SA2 subunit. We report here the determination of cohesin subunit stoichiometry of endogenous cohesin complex by quantitative mass spectrometry. Using qConCAT-based isotope labeling, we show that the cohesin core complex contains equimolar of the 4 core components, suggesting that each cohesin ring is closed by one SA1/SA2 molecule. Furthermore, we applied this strategy to quantify post-translational modification-dependent cohesin interactions. We demonstrate that quantitative mass spectrometry is a powerful tool for measuring stoichiometry of endogenous protein core complex. 相似文献
5.
Mutations in cohesin complex members SMC3 and SMC1A cause a mild variant of cornelia de Lange syndrome with predominant mental retardation
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Deardorff MA Kaur M Yaeger D Rampuria A Korolev S Pie J Gil-Rodríguez C Arnedo M Loeys B Kline AD Wilson M Lillquist K Siu V Ramos FJ Musio A Jackson LS Dorsett D Krantz ID 《American journal of human genetics》2007,80(3):485-494
Mutations in the cohesin regulators NIPBL and ESCO2 are causative of the Cornelia de Lange syndrome (CdLS) and Roberts or SC phocomelia syndrome, respectively. Recently, mutations in the cohesin complex structural component SMC1A have been identified in two probands with features of CdLS. Here, we report the identification of a mutation in the gene encoding the complementary subunit of the cohesin heterodimer, SMC3, and 14 additional SMC1A mutations. All mutations are predicted to retain an open reading frame, and no truncating mutations were identified. Structural analysis of the mutant SMC3 and SMC1A proteins indicate that all are likely to produce functional cohesin complexes, but we posit that they may alter their chromosome binding dynamics. Our data indicate that SMC3 and SMC1A mutations (1) contribute to approximately 5% of cases of CdLS, (2) result in a consistently mild phenotype with absence of major structural anomalies typically associated with CdLS, and (3) in some instances, result in a phenotype that approaches that of apparently nonsyndromic mental retardation. 相似文献
6.
Sister chromatids are held together by the multisubunit cohesin complex, which contains two SMC (Smc1 and Smc3) and two non-SMC (Scc1 and Scc3) proteins. The crystal structure of a bacterial SMC "hinge" region along with EM studies and biochemical experiments on yeast Smc1 and Smc3 proteins show that SMC protamers fold up individually into rod-shaped molecules. A 45 nm long intramolecular coiled coil separates the hinge region from the ATPase-containing "head" domain. Smc1 and Smc3 bind to each other via heterotypic interactions between their hinges to form a V-shaped heterodimer. The two heads of the V-shaped dimer are connected by different ends of the cleavable Scc1 subunit. Cohesin therefore forms a large proteinaceous loop within which sister chromatids might be entrapped after DNA replication. 相似文献
7.
Revenkova E Eijpe M Heyting C Gross B Jessberger R 《Molecular and cellular biology》2001,21(20):6984-6998
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 (SMC1beta), which-except for a unique, basic, DNA binding C-terminal motif-is highly homologous to SMC1 (which may now be called SMC1alpha) and is not present in the yeast genome. SMC1beta 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 SMC1beta along the axial elements of synaptonemal complexes in prophase I. Most SMC1beta dissociates from the chromosome arms in late-pachytene-diplotene cells. However, SMC1beta, but not SMC1alpha, remains chromatin associated at the centromeres up to metaphase II. Thus, SMC1beta and not SMC1alpha is likely involved in maintaining cohesion between sister centromeres until anaphase II. 相似文献
8.
The structural maintenance of chromosome 3 protein (SMC3) is a component of the multimeric cohesin complex that holds sister chromatids together and prevents their premature separation during mitosis. By screening a human cDNA library for interacting proteins we have established that the proto-oncogene RET finger protein (RFP) interacts with SMC3. The sites of interaction map to part of the central coiled coil region of RFP and to the C-terminal region of the SMC3 globular hinge domain. SMC3/RFP interaction was confirmed in vivo by co-immunoprecipitation studies and by performing mammalian two-hybrid interaction assays. Cytoimmunolocalization experiments showed that SMC3 and RFP co-localize in the same cell substructures. Overexpression of RFP in NIH3T3 cells significantly increased the fraction of SMC3 recovered in the nucleus supporting the idea that RFP regulates the intracellular distribution of SMC3. These studies identify a novel SMC3-interacting protein that may affect SMC3 availability to complex with its cohesin partners. 相似文献
9.
Condensin and cohesin are chromosomal protein complexes required for chromosome condensation and sister chromatid cohesion, respectively. They commonly contain the SMC (structural maintenance of chromosomes) subunits consisting of a long coiled-coil with the terminal globular domains and the central hinge. Condensin and cohesin holo-complexes contain three and two non-SMC subunits, respectively. In this study, DNA interaction with cohesin and condensin complexes purified from fission yeast was investigated. The DNA reannealing activity is strong for condensin SMC heterodimer but weak for holo-condensin, whereas no annealing activity is found for cohesin heterodimer SMC and Rad21-bound heterotrimer complexes. One set of globular domains of the same condensin SMC is essential for the DNA reannealing activity. In addition, the coiled-coil and hinge region of another SMC are needed. Atomic force microscopy discloses the molecular events of DNA reannealing. SMC assembly that occurs on reannealing DNA seems to be a necessary intermediary step. SMC is eliminated from the completed double-stranded DNA. The ability of heterodimeric SMC to reanneal DNA may be regulated in vivo possibly through the non-SMC heterotrimeric complex. 相似文献
10.
Eijpe M Offenberg H Jessberger R Revenkova E Heyting C 《The Journal of cell biology》2003,160(5):657-670
In meiotic prophase, the sister chromatids of each chromosome develop a common axial element (AE) that is integrated into the synaptonemal complex (SC). We analyzed the incorporation of sister chromatid cohesion proteins (cohesins) and other AE components into AEs. Meiotic cohesin REC8 appeared shortly before premeiotic S phase in the nucleus and formed AE-like structures (REC8-AEs) from premeiotic S phase on. Subsequently, meiotic cohesin SMC1beta, cohesin SMC3, and AE proteins SCP2 and SCP3 formed dots along REC8-AEs, which extended and fused until they lined REC8-AEs along their length. In metaphase I, SMC1beta, SMC3, SCP2, and SCP3 disappeared from the chromosome arms and accumulated around the centromeres, where they stayed until anaphase II. In striking contrast, REC8 persisted along the chromosome arms until anaphase I and near the centromeres until anaphase II. We propose that REC8 provides a basis for AE formation and that the first steps in AE assembly do not require SMC1beta, SMC3, SCP2, and SCP3. Furthermore, SMC1beta, SMC3, SCP2, and SCP3 cannot provide arm cohesion during metaphase I. We propose that REC8 then provides cohesion. RAD51 and/or DMC1 coimmunoprecipitates with REC8, suggesting that REC8 may also provide a basis for assembly of recombination complexes. 相似文献
11.
Condensin architecture and interaction with DNA: regulatory non-SMC subunits bind to the head of SMC heterodimer 总被引:8,自引:0,他引:8
Yoshimura SH Hizume K Murakami A Sutani T Takeyasu K Yanagida M 《Current biology : CB》2002,12(6):508-513
Condensin and cohesin are two protein complexes that act as the central mediators of chromosome condensation and sister chromatid cohesion, respectively. The basic underlying mechanism of action of these complexes remained enigmatic. Direct visualization of condensin and cohesin was expected to provide hints to their mechanisms. They are composed of heterodimers of distinct structural maintenance of chromosome (SMC) proteins and other non-SMC subunits. Here, we report the first observation of the architecture of condensin and its interaction with DNA by atomic force microscopy (AFM). The purified condensin SMC heterodimer shows a head-tail structure with a single head composed of globular domains and a tail with the coiled-coil region. Unexpectedly, the condensin non-SMC trimers associate with the head of SMC heterodimers, producing a larger head with the tail. The heteropentamer is bound to DNA in a distributive fashion, whereas condensin SMC heterodimers interact with DNA as aggregates within a large DNA-protein assembly. Thus, non-SMC trimers may regulate the ATPase activity of condensin by directly interacting with the globular domains of SMC heterodimer and alter the mode of DNA interaction. A model for the action of heteropentamer is presented. 相似文献
12.
The C-terminal domains of yeast structural maintenance of chromosomes (SMC) proteins were previously shown to bind double-stranded DNA, which generated the idea of the antiparallel SMC heterodimer, such as the SMC1/3 dimer, bridging two DNA molecules. Analysis of bovine SMC1 and SMC3 protein domains now reveals that not only the C-terminal domains, but also the coiled-coil region, binds DNA, while the N terminus is inactive. Duplex DNA and DNA molecules with secondary structures are highly preferred substrates for both the C-terminal and coiled-coil domains. Contrasting other cruciform DNA-binding proteins like HMG1, the SMC3 C-terminal and coiled-coil domains do not bend DNA, but rather prevent bending in ring closure assays. Phosphatase, exonuclease, and ligase assays showed that neither domain renders DNA ends inaccessible for other enzymes. These observations allow modifications of models for SMC-DNA interactions. 相似文献
13.
The structural maintenance of chromosomes (SMC) family of proteins has been implicated in the repair of DNA double-strand breaks (DSBs) by homologous recombination (HR). The SMC1/3 cohesin complex is thought to promote HR by maintaining the close proximity of sister chromatids at DSBs. The SMC5/6 complex is also required for DNA repair, but the mechanism by which it accomplishes this is unclear. Here, we show that RNAi-mediated knockdown of the SMC5/6 complex components in human cells increases the efficiency of gene targeting due to a specific requirement for hSMC5/6 in sister chromatid HR. Knockdown of the hSMC5/6 complex decreases sister chromatid HR, but does not reduce nonhomologous end-joining (NHEJ) or intra-chromatid, homologue, or extrachromosomal HR. The hSMC5/6 complex is itself recruited to nuclease-induced DSBs and is required for the recruitment of cohesin to DSBs. Our results establish a mechanism by which the hSMC5/6 complex promotes DNA repair and suggest a novel strategy to improve the efficiency of gene targeting in mammalian somatic cells. 相似文献
14.
The eukaryotic SMC1/SMC3 heterodimer is essential for sister chromatid cohesion and acts in DNA repair and recombination. Dimerization depends on the central hinge domain present in all SMC proteins, which is flanked at each side by extended coiled-coil regions that terminate in specific globular domains. Here we report on DNA interactions of the eukaryotic, heterodimeric SMC1/SMC3 hinge regions, using the two known isoforms, SMC1alpha/SMC3 and the meiotic SMC1beta/SMC3. Both dimers bind DNA with a preference for double-stranded DNA and DNA rich in potential secondary structures. Both dimers form large protein-DNA networks and promote reannealing of complementary DNA strands. DNA binding but not dimerization depends on approximately 20 amino acids of transitional sequence into the coiled-coil region. Replacement of three highly conserved glycine residues, thought to be required for dimerization, in one of the two hinge domains still allows formation of a stable dimer, but if two hinge domains are mutated dimerization fails. Single-mutant dimers bind DNA, but hinge monomers do not. Together, we show that eukaryotic hinge dimerization does not require conserved glycines in both hinge domains, that only the transition into the coiled-coil region rather than the entire coiled-coil region is necessary for DNA binding, and that dimerization is required but not sufficient for DNA binding of the eukaryotic hinge heterodimer. 相似文献
15.
Bauerschmidt C Woodcock M Stevens DL Hill MA Rothkamm K Helleday T 《Experimental cell research》2011,(3):330-337
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. 相似文献
16.
Hirano T 《Philosophical transactions of the Royal Society of London. Series B, Biological sciences》2005,360(1455):507-514
Chromosome cohesion and condensation are essential prerequisites of proper segregation of genomes during mitosis and meiosis, and are supported by two structurally related protein complexes, cohesin and condensin, respectively. At the core of the two complexes lie members of the structural maintenance of chromosomes (SMC) family of ATPases. SMC proteins are also found in most bacterial and archaeal species, implicating the existence of an evolutionarily conserved theme of higher-order chromosome organization and dynamics. SMC dimers adopt a two-armed structure with an ATP-binding cassette (ABC)-like domain at the distal end of each arm. This article reviews recent work on the bacterial and eukaryotic SMC protein complexes, and discusses current understanding of how these uniquely designed protein machines may work at a mechanistic level. It seems most likely that the action of SMC proteins is highly dynamic and plastic, possibly involving a diverse array of intramolecular and intermolecular protein-protein interactions. 相似文献
17.
Rihui Yan Sharon E. Thomas Jui-He Tsai Yukihiro Yamada Bruce D. McKee 《The Journal of cell biology》2010,188(3):335-349
Sister chromatid cohesion is essential to maintain stable connections between homologues and sister chromatids during meiosis and to establish correct centromere orientation patterns on the meiosis I and II spindles. However, the meiotic cohesion apparatus in Drosophila melanogaster remains largely uncharacterized. We describe a novel protein, sisters on the loose (SOLO), which is essential for meiotic cohesion in Drosophila. In solo mutants, sister centromeres separate before prometaphase I, disrupting meiosis I centromere orientation and causing nondisjunction of both homologous and sister chromatids. Centromeric foci of the cohesin protein SMC1 are absent in solo mutants at all meiotic stages. SOLO and SMC1 colocalize to meiotic centromeres from early prophase I until anaphase II in wild-type males, but both proteins disappear prematurely at anaphase I in mutants for mei-S332, which encodes the Drosophila homologue of the cohesin protector protein shugoshin. The solo mutant phenotypes and the localization patterns of SOLO and SMC1 indicate that they function together to maintain sister chromatid cohesion in Drosophila meiosis. 相似文献
18.
Until the onset of anaphase, sister chromatids are bound to each other by a multi-subunit protein complex called cohesin. Since chromosomes in meiosis behave differently from those in mitosis, the cohesion and separation of homologous chromosomes and sister chromatids in meiosis are thought to be regulated by meiosis-specific cohesin subunits. Actually, several meiosis-specific cohesin subunits, including Rec8, STAG3 and SMC1beta, are known to exist in mammals; however, there are no reports of meiosis-specific cohesin subunits in other vertebrates. To investigate the protein expression and localization of cohesin subunits during meiosis in non-mammalian species, we isolated cDNA clones encoding SMC1alpha, SMC1beta, SMC3 and Rad21 in the medaka and produced antibodies against recombinant proteins. Medaka SMC1beta was expressed solely in gonads, while SMC1alpha, SMC3 and Rad21 were also expressed in other organs and in cultured cells. SMC1beta forms a complex with SMC3 but not with Rad21, in contrast to SMC1alpha, which forms complexes with both SMC3 and Rad21. SMC1alpha and Rad21 were mainly expressed in mitotically dividing cells in the testis (somatic cells and spermatogonia), although their weak expression was detected in pre-leptotene spermatocytes. SMC1beta was expressed in spermatogonia and spermatocytes. SMC1beta was localized along the chromosomal arms as well as on the centromeres in meiotic prophase I, and its existence on the chromosomes persisted up to metaphase II, a situation different from that reported in the mouse, in which SMC1beta is lost from the chromosome arms in late pachytene despite its universal presence in vertebrates. 相似文献
19.
Ying Yang Zhenxing Zhang Renzhi Wang Wenbin Ma Junji Wei Guilin Li 《Molecular and cellular biochemistry》2013,381(1-2):209-215
SMC1A is a member of cohesin complex which has essential functions in cell cycle progression and DNA repair. Therefore, we choose SMC1A as a target gene therapy of glioblastoma. It is well known that glioblastoma has very low survival rate because of ineffectiveness of conventional treatments. This study was designed to explore the possibilities of small interfering RNA (siRNA)-mediated SMC1A silencing as alternative method of treatment. We found that the lentivirus-mediated RNAi system efficiently decreased the expression level of SMC1A. Inhibiting SMC1A expression efficiently (P < 0.001) resulted in inhibiting the proliferation and colony formation of U251 and U87MG cells. Moreover, we found that SMC1A silencing led to S cell-cycle arresting. Collectively, these results demonstrated the possibility of siRNA-mediated silencing of SMC1A as a therapeutic tool for the treatment of glioblastoma. 相似文献
20.
Minichromosomes in the nuclear genome of Trypanosoma brucei exhibit unusual patterns of mitotic segregation. To address whether differences in their mode of segregation in relation to large chromosomes are reflected at a molecular level, we characterized two different proteins that have highly conserved functions in eukaryotic chromosomes segregation: the SMC3 protein, a component of the chromatid cohesion apparatus, and the protease separase that resolves the cohesin complex at the onset of anaphase and has, in other organisms, additional functions during mitosis. Using in situ hybridization we show that RNA interference-mediated depletion of SMC3 has no visible effect on the segregation of the minichromosomal population but interferes with the faithful mitotic separation of large chromosomes. In contrast, separase depletion causes missegregation of both mini- and large chromosomes. We also show that SMC3 persists as a soluble protein throughout the cell cycle and only associates with chromatin between G1 and metaphase. Separase is present in the cell during the entire cell cycle, but is excluded from the nucleus until the metaphase–anaphase transition, thereby providing a potential control mechanism to prevent the untimely cleavage of the cohesin complex. 相似文献