RESUMEN
Cohesins establish sister chromatid cohesion during S phase and are removed when cohesin Scc1 is cleaved by separase at anaphase onset. During this process, cohesin Smc3 undergoes a cycle of acetylation: Smc3 acetylation by Eco1 in S phase stabilizes cohesin association with chromosomes, and its deacetylation by Hos1 in anaphase allows re-use of Smc3 in the next cell cycle. Here we find that Smc3 deacetylation by Hos1 has a more immediate effect in the early anaphase of budding yeast. Hos1 depletion significantly delayed sister chromatid separation and segregation. Smc3 deacetylation facilitated removal of cohesins from chromosomes without changing Scc1 cleavage efficiency, promoting dissolution of cohesion. This action is probably due to disengagement of Smc1-Smc3 heads prompted by de-repression of their ATPase activity. We suggest Scc1 cleavage per se is insufficient for efficient dissolution of cohesion in early anaphase; subsequent Smc3 deacetylation, triggered by Scc1 cleavage, is also required.
Asunto(s)
Anafase , Proteínas de Ciclo Celular/metabolismo , Cromátides/enzimología , Proteínas Cromosómicas no Histona/metabolismo , Segregación Cromosómica , Histona Desacetilasas/metabolismo , Histona Demetilasas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimología , Acetilación , Proteínas de Ciclo Celular/genética , Cromátides/genética , Proteínas Cromosómicas no Histona/genética , Histona Desacetilasas/genética , Histona Demetilasas/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Proteínas de Saccharomyces cerevisiae/genética , Separasa/genética , Separasa/metabolismo , Transducción de Señal , Factores de Tiempo , CohesinasRESUMEN
Eukaryotes contain three essential Structural Maintenance of Chromosomes (SMC) complexes: cohesin, condensin, and Smc5/6. Cohesin forms a ring-shaped structure that embraces sister chromatids to promote their cohesion. The cohesiveness of cohesin is promoted by acetylation of N-terminal lysines of the Smc3 subunit by the acetyltransferases Eco1 in Saccharomyces cerevisiae and the homologue, Eso1, in Schizosaccharomyces pombe. In both yeasts, these acetyltransferases are essential for cell viability. However, whereas nonacetylatable Smc3 mutants are lethal in S. cerevisiae, they are not in S. pombe We show that the lethality of a temperature-sensitive allele of eso1 (eso1-H17) is due to activation of the spindle assembly checkpoint (SAC) and is associated with premature centromere separation. The lack of cohesion at the centromeres does not correlate with Psm3 acetylation or cohesin levels at the centromeres, but is associated ith significantly reduced recruitment of the cohesin regulator Pds5. The SAC activation in this context is dependent on Smc5/6 function, which is required to remove cohesin from chromosome arms but not centromeres. The mitotic defects caused by Smc5/6 and Eso1 dysfunction are cosuppressed in double mutants. This identifies a novel function (or functions) for Eso1 and Smc5/6 at centromeres and extends the functional relationships between these SMC complexes.
Asunto(s)
Acetiltransferasas/metabolismo , Proteínas de Ciclo Celular/metabolismo , Centrómero/enzimología , Proteínas Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Acetiltransferasas/genética , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfatasas/fisiología , Proteínas de Ciclo Celular/genética , Cromátides/enzimología , Proteínas Cromosómicas no Histona/metabolismo , Segregación Cromosómica , Cromosomas Fúngicos/enzimología , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/fisiología , Mitosis/fisiología , Complejos Multiproteicos/metabolismo , Complejos Multiproteicos/fisiología , Proteínas Nucleares/genética , Fase S , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Schizosaccharomyces/citología , Schizosaccharomyces/enzimología , Proteínas de Schizosaccharomyces pombe/genética , CohesinasRESUMEN
DNA replication during S phase is accompanied by establishment of sister chromatid cohesion to ensure faithful chromosome segregation. The Eco1 acetyltransferase, helped by factors including Ctf4 and Chl1, concomitantly acetylates the chromosomal cohesin complex to stabilize its cohesive links. Here we show that Ctf4 recruits the Chl1 helicase to the replisome via a conserved interaction motif that Chl1 shares with GINS and polymerase α. We visualize recruitment by EM analysis of a reconstituted Chl1-Ctf4-GINS assembly. The Chl1 helicase facilitates replication fork progression under conditions of nucleotide depletion, partly independently of Ctf4 interaction. Conversely, Ctf4 interaction, but not helicase activity, is required for Chl1's role in sister chromatid cohesion. A physical interaction between Chl1 and the cohesin complex during S phase suggests that Chl1 contacts cohesin to facilitate its acetylation. Our results reveal how Ctf4 forms a replisomal interaction hub that coordinates replication fork progression and sister chromatid cohesion establishment.
Asunto(s)
Cromátides/enzimología , Proteínas Cromosómicas no Histona/metabolismo , Cromosomas Fúngicos/enzimología , ADN de Hongos/biosíntesis , Proteínas de Unión al ADN/metabolismo , Fase S , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimología , Acetiltransferasas/metabolismo , Acilación , Proteínas de Ciclo Celular/metabolismo , Cromátides/genética , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/ultraestructura , Cromosomas Fúngicos/genética , ADN de Hongos/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/ultraestructura , Microscopía Electrónica de Transmisión , Modelos Moleculares , Complejos Multiproteicos , Proteínas Nucleares/metabolismo , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/ultraestructura , Relación Estructura-Actividad , Factores de Tiempo , CohesinasRESUMEN
During the cell cycle, sister-chromatid cohesion tethers sister chromatids together from S phase to the metaphase-anaphase transition and ensures accurate segregation of chromatids into daughter cells. N-terminal acetylation is one of the most prevalent protein covalent modifications in eukaryotes and is mediated by a family of N-terminal acetyltransferases (NAT). Naa50 (also called San) has previously been shown to play a role in sister-chromatid cohesion in metazoans. The mechanism by which Naa50 contributes to cohesion is not understood however. Here, we show that depletion of Naa50 in HeLa cells weakens the interaction between cohesin and its positive regulator sororin and causes cohesion defects in S phase, consistent with a role of Naa50 in cohesion establishment. Strikingly, co-depletion of NatA, a heterodimeric NAT complex that physically interacts with Naa50, rescues the sister-chromatid cohesion defects and the resulting mitotic arrest caused by Naa50 depletion, indicating that NatA and Naa50 play antagonistic roles in cohesion. Purified recombinant NatA and Naa50 do not affect each other's NAT activity in vitro Because NatA and Naa50 exhibit distinct substrate specificity, we propose that they modify different effectors and regulate sister-chromatid cohesion in opposing ways.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Cromátides/enzimología , Proteínas Cromosómicas no Histona/metabolismo , Cromosomas Humanos/enzimología , Mitosis/fisiología , Acetiltransferasa A N-Terminal/metabolismo , Acetiltransferasa E N-Terminal/metabolismo , Fase S/fisiología , Proteínas de Ciclo Celular/genética , Cromátides/genética , Proteínas Cromosómicas no Histona/genética , Cromosomas Humanos/genética , Células HeLa , Humanos , Acetiltransferasa A N-Terminal/genética , Acetiltransferasa E N-Terminal/genética , CohesinasRESUMEN
Mitotic chromosome condensation has fascinated biologists since Flemming's early illustrations of mitosis in the late nineteenth century. Now--130 years later--chromatid condensation is reconstituted in vitro with the minimum components. The results are remarkably and beautifully simple, requiring only core histones, three histone chaperones, topoisomerase II and condensin I.
Asunto(s)
Cromátides/enzimología , Ensamble y Desensamble de Cromatina , Histonas/metabolismo , Mitosis , Chaperonas Moleculares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Espermatozoides/enzimología , Proteínas de Xenopus/metabolismo , Animales , Humanos , MasculinoRESUMEN
The compaction of diffuse interphase chromatin into stable mitotic chromosomes enables the segregation of replicated DNA to daughter cells. Two new studies characterise, both in vivo and in vitro, the essential contribution of the vertebrate condensin complex to chromosome organisation.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Proteínas de Ciclo Celular/genética , Cromátides/enzimología , Ensamble y Desensamble de Cromatina , Proteínas Cromosómicas no Histona/genética , Segregación Cromosómica/fisiología , Proteínas de Unión al ADN/metabolismo , Histonas/metabolismo , Meiosis/fisiología , Mitosis , Chaperonas Moleculares/metabolismo , Complejos Multiproteicos/metabolismo , Proteínas Nucleares/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Espermatozoides/enzimología , Proteínas de Xenopus/metabolismo , Animales , Humanos , MasculinoRESUMEN
The assembly of mitotic chromosomes, each composed of a pair of rod-shaped chromatids, is an essential prerequisite for accurate transmission of the genome during cell division. It remains poorly understood, however, how this fundamental process might be achieved and regulated in the cell. Here we report an in vitro system in which mitotic chromatids can be reconstituted by mixing a simple substrate with only six purified factors: core histones, three histone chaperones (nucleoplasmin, Nap1 and FACT), topoisomerase II (topo II) and condensin I. We find that octameric nucleosomes containing the embryonic variant H2A.X-F are highly susceptible to FACT and function as the most productive substrate for subsequent actions of topo II and condensin I. Cdk1 phosphorylation of condensin I is the sole mitosis-specific modification required for chromatid reconstitution. This experimental system will enhance our understanding of the mechanisms of action of individual factors and their cooperation during this process.
Asunto(s)
Cromátides/enzimología , Ensamble y Desensamble de Cromatina , Histonas/metabolismo , Mitosis , Chaperonas Moleculares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Espermatozoides/enzimología , Proteínas de Xenopus/metabolismo , Adenosina Trifosfatasas/metabolismo , Animales , Proteína Quinasa CDC2/metabolismo , ADN-Topoisomerasas de Tipo II/metabolismo , Proteínas de Unión al ADN/metabolismo , Células HeLa , Proteínas del Grupo de Alta Movilidad/metabolismo , Histonas/genética , Humanos , Masculino , Chaperonas Moleculares/genética , Complejos Multiproteicos/metabolismo , Nucleoplasminas/metabolismo , Nucleosomas/enzimología , Fosforilación , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/genética , Factores de Elongación Transcripcional/metabolismo , Transfección , Proteínas de Xenopus/genética , Xenopus laevisRESUMEN
Aurora B kinase regulates the proper biorientation of sister chromatids during mitosis. Lack of Aurora B kinase function results in the inability to correct erroneous kinetochore-microtubule attachments and gives rise to aneuploidy. Interestingly, increased Aurora B activity also leads to problems with chromosome segregation, and overexpression of this kinase has been observed in various types of cancer. However, little is known about the mechanisms by which an increase in Aurora B kinase activity can impair mitotic progression and cell viability. Here, using a yeast model, we demonstrate that increased Aurora B activity as a result of the overexpression of the Aurora B and inner centromere protein homologs triggers defects in chromosome segregation by promoting the continuous disruption of chromosome-microtubule attachments even when sister chromatids are correctly bioriented. This disruption leads to a constitutive activation of the spindle-assembly checkpoint, which therefore causes a lack of cytokinesis even though spindle elongation and chromosome segregation take place. Finally, we demonstrate that this increase in Aurora B activity causes premature collapse of the mitotic spindle by promoting instability of the spindle midzone.
Asunto(s)
Aurora Quinasa B/metabolismo , Cromosomas Fúngicos/metabolismo , Cinetocoros/enzimología , Microtúbulos/enzimología , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimología , Huso Acromático/enzimología , Aurora Quinasa B/genética , Cromátides/enzimología , Cromátides/genética , Cromosomas Fúngicos/genética , Microtúbulos/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Huso Acromático/genéticaRESUMEN
The condensin complex is a key determinant of mitotic chromosome architecture. In addition, condensin promotes resolution of sister chromatids during anaphase, a function that is conserved from prokaryotes to human. Anaphase bridges observed in cells lacking condensin are reminiscent of chromosome segregation failure after inactivation of topoisomerase II (topo II), the enzyme that removes catenanes persisting between sister chromatids following DNA replication. Circumstantial evidence has linked condensin to sister chromatid decatenation but, because of the difficulty of observing chromosome catenation, this link has remained indirect. Alternative models for how condensin facilitates chromosome resolution have been put forward. Here, we follow the catenation status of circular minichromosomes of three sizes during the Saccharomyeces cerevisiae cell cycle. Catenanes are produced during DNA replication and are for the most part swiftly resolved during and following S-phase, aided by sister chromatid separation. Complete resolution, however, requires the condensin complex, a dependency that becomes more pronounced with increasing chromosome size. Our results provide evidence that condensin prevents deleterious anaphase bridges during chromosome segregation by promoting sister chromatid decatenation.
Asunto(s)
Adenosina Trifosfatasas/fisiología , Cromátides/enzimología , ADN-Topoisomerasas de Tipo II/metabolismo , ADN Encadenado/metabolismo , Proteínas de Unión al ADN/fisiología , Complejos Multiproteicos/fisiología , Adenosina Trifosfatasas/metabolismo , Ciclo Celular/genética , Cromátides/química , Segregación Cromosómica , Proteínas de Unión al ADN/metabolismo , Complejos Multiproteicos/metabolismo , Saccharomyces cerevisiae/genéticaRESUMEN
Chromatoid body (CB) was identified as granules stained by basic dye 130 years ago and called by various names. Electron microscopy revealed that the CB belonged to nuage (cloud in French) specific for germ cells. We described the localization of several proteins, including RNA helicases, in the nuage compartments classified into six types and in several spermatogenic cell-specific structures. All the proteins examined were detected in the nuage, including the CB with different staining intensities. Several proteins were localized to non-nuage structures, suggesting that these nuage proteins structures are related to nuage function.
Asunto(s)
Cromátides/química , ARN Helicasas/análisis , Animales , Cromátides/enzimología , Técnica del Anticuerpo Fluorescente , Células Germinativas/citología , Células Germinativas/metabolismo , Humanos , Masculino , Microscopía Electrónica , Microscopía Inmunoelectrónica , EspermatogénesisRESUMEN
Tumor cells are commonly aneuploid, a condition contributing to cancer progression and drug resistance. Understanding how chromatids are linked and separated at the appropriate time will help uncover the basis of aneuploidy and will shed light on the behavior of tumor cells. Cohesion of sister chromatids is maintained by the multi-protein complex cohesin, consisting of Smc1, Smc3, Scc1 and Scc3. Sororin associates with the cohesin complex and regulates the segregation of sister chromatids. Sororin is phosphorylated in mitosis; however, the role of this modification is unclear. Here we show that mutation of potential cyclin-dependent kinase 1 (Cdk1) phosphorylation sites leaves sororin stranded on chromosomes and bound to cohesin throughout mitosis. Sororin can be precipitated from cell lysates with DNA-cellulose, and only the hypophosphorylated form of sororin shows this association. These results suggest that phosphorylation of sororin causes its release from chromatin in mitosis. Also, the hypophosphorylated form of sororin increases cohesion between sister chromatids, suggesting that phosphorylation of sororin by Cdk1 influences sister chromatid cohesion. Finally, phosphorylation-deficient sororin can alleviate the mitotic block that occurs upon knockdown of endogenous sororin. This mitotic block is abolished by ZM447439, an Aurora kinase inhibitor, suggesting that prematurely separated sister chromatids activate the spindle assembly checkpoint through an Aurora kinase-dependent pathway.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteína Quinasa CDC2/metabolismo , Proteínas de Ciclo Celular/metabolismo , Cromátides/metabolismo , Proteína Quinasa CDC2/genética , Cromátides/enzimología , Cromátides/genética , Proteínas Cromosómicas no Histona/metabolismo , Células HeLa , Humanos , Puntos de Control de la Fase M del Ciclo Celular/fisiología , Mitosis/fisiología , Mutagénesis Sitio-Dirigida , Fosforilación , Intercambio de Cromátides Hermanas , Transfección , CohesinasRESUMEN
Gonadotropin-regulated testicular RNA helicase (GRTH/DDX25), a multifunctional protein and a component of ribonucleoprotein complexes, is essential for the completion of spermatogenesis. We investigated the nuclear/cytoplasmic shuttling of GRTH in germ cells and its impact on the chromatoid body (CB)-a perinuclear organelle viewed as a storage/processing site of mRNAs. GRTH resides in the nucleus, cytoplasm and CB of round spermatids. Treatment of these cells with inhibitors of nuclear export or RNA synthesis caused nuclear retention of GRTH and its absence in the cytoplasm and CB. The nuclear levels of GRTH bound RNA messages were significantly enhanced and major reduction was observed in the cytoplasm. This indicated GRTH main transport function of mRNAs to the cytoplasm and CB. MVH, a germ cell helicase, and MIWI, a component of the RNA-induced-silencing complex (RISC), confined to the CB/cytoplasm, were absent in the CB and accumulated in the cytoplasm upon treatment. This also occurred in spermatids of GRTH-KO mice. The CB changed from lobular-filamentous to a small condensed structure after treatment resembling the CB of GRTH-KO. No interaction of GRTH with MVH or RISC members in both protein and RNA were observed. Besides of participating in the transport of messages of relevant spermatogenic genes, GRTH was found to transport its own message to cytoplasmic sites. Our studies suggest that GRTH through its export/transport function as a component of mRNP is essential to govern the CB structure in spermatids and to maintain systems that may participate in mRNA storage and their processing during spermatogenesis.
Asunto(s)
Cromátides/enzimología , ARN Helicasas DEAD-box/fisiología , ARN Mensajero/metabolismo , Espermátides/enzimología , Espermatogénesis/fisiología , Animales , Western Blotting , Núcleo Celular/enzimología , Cromátides/ultraestructura , Citoplasma/enzimología , Técnica del Anticuerpo Fluorescente , Inmunoprecipitación , Masculino , Ratones , Ratones Noqueados , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Espermátides/ultraestructura , TestículoRESUMEN
Eco1p/Ctf7p is an essential acetyltransferase required for the establishment of sister chromatid cohesion. Eco1p acetylates Smc3p and Mcd1p (Scc1p or Rad21p) to establish cohesion during S phase and in response to DNA damage, respectively. In addition to its acetyltransferase domain, Eco1p harbors a conserved zinc finger domain. The zinc finger has been implicated in the establishment of sister chromatid cohesion in S phase, yet its function on the molecular level and its contribution to damage-induced cohesion are unknown. Here, we show that the zinc finger is essential for the establishment of cohesion in both S phase and in response to DNA damage. Our results suggest that the zinc finger augments the acetylation of Eco1p itself, Smc3p and likely Mcd1p. We propose that the zinc finger is a general enhancer of substrate recognition, thereby enhances the ability of Eco1p to acetylate its substrates above a threshold needed to generate cohesion during DNA replication and repair. Finally our studies of the zinc finger led to the discovery that Eco1 is a multimer, a property that could be exploited to coordinate acetylation of substrates either spatially or temporally for establishment of sister chromatid cohesion.
Asunto(s)
Acetiltransferasas/química , Cromátides/enzimología , Proteínas Nucleares/química , Proteínas de Saccharomyces cerevisiae/química , Dedos de Zinc , Acetiltransferasas/genética , Acetiltransferasas/metabolismo , Secuencia de Aminoácidos , Daño del ADN , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Multimerización de Proteína , Fase S , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Separase is an endopeptidase that separates sister chromatids by cleaving cohesin Rad21 during the metaphase-to-anaphase transition. Conditional expression of Separase in tetracycline-inducible diploid FSK3 mouse mammary epithelial cells with both p53 WT and mutant (Ser-233-234) alleles of unknown physiological significance develops aneuploidy within 5 days of Separase induction in vitro. Overexpression of Separase induces premature separation of chromatids, lagging chromosomes, and anaphase bridges. In an in vivo mouse mammary transplant model, induction of Separase expression in the transplanted FSK3 cells for 3-4 weeks results in the formation of aneuploid tumors in the mammary gland. Xenograft studies combined with histological and cytogenetic analysis reveal that Separase-induced tumors are clonal in their genomic complements and have a mesenchymal phenotype suggestive of an epithelial-mesenchymal transition. Induction of Separase resulted in trisomies for chromosomes 8, 15, and 17; monosomy for chromosome 10; and amplification of the distal region of chromosomes 8 and 11. Separase protein is found to be significantly overexpressed in human breast tumors compared with matched normal tissue. These results collectively suggest that Separase is an oncogene, whose overexpression alone in mammary epithelial cells is sufficient to induce aneuploidy and tumorigenesis in a p53 mutant background.
Asunto(s)
Aneuploidia , Neoplasias de la Mama/enzimología , Proteínas de Ciclo Celular/metabolismo , Endopeptidasas/metabolismo , Neoplasias Mamarias Experimentales/enzimología , Anafase , Animales , Western Blotting , Línea Celular Tumoral , Cromátides/enzimología , Inestabilidad Cromosómica , Células Epiteliales/enzimología , Células Epiteliales/patología , Femenino , Humanos , Metafase , Ratones , Hibridación de Ácido Nucleico , Separasa , TetraciclinaRESUMEN
Polo-like kinase 1 (Plk1) is a key regulator of mitotic progression and cell division in eukaryotes. It is highly expressed in tumor cells and considered a potential target for cancer therapy. Here, we report the discovery and application of a novel potent small-molecule inhibitor of mammalian Plk1, ZK-Thiazolidinone (TAL). We have extensively characterized TAL in vitro and addressed TAL specificity within cells by studying Plk1 functions in sister chromatid separation, centrosome maturation, and spindle assembly. Moreover, we have used TAL for a detailed analysis of Plk1 in relation to PICH and PRC1, two prominent interaction partners implicated in spindle assembly checkpoint function and cytokinesis, respectively. Specifically, we show that Plk1, when inactivated by TAL, spreads over the arms of chromosomes, resembling the localization of its binding partner PICH, and that both proteins are mutually dependent on each other for correct localization. Finally, we show that Plk1 activity is essential for cleavage furrow formation and ingression, leading to successful cytokinesis.
Asunto(s)
Compuestos de Anilina/farmacología , Proteínas de Ciclo Celular/antagonistas & inhibidores , Proteínas de Ciclo Celular/metabolismo , Mitosis/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/metabolismo , Tiazolidinas/farmacología , Anafase/efectos de los fármacos , Compuestos de Anilina/química , Animales , Línea Celular Tumoral , Centrosoma/efectos de los fármacos , Centrosoma/enzimología , Cromátides/efectos de los fármacos , Cromátides/enzimología , Citocinesis/efectos de los fármacos , ADN Helicasas , Activación Enzimática/efectos de los fármacos , Humanos , Ratones , Índice Mitótico , Inhibidores de Proteínas Quinasas/química , Huso Acromático/efectos de los fármacos , Huso Acromático/enzimología , Tiazolidinas/química , Quinasa Tipo Polo 1RESUMEN
Faithful chromosome transmission requires establishment of sister chromatid cohesion during S phase, followed by its removal at anaphase onset. Sister chromatids are tethered together by cohesin, which is displaced from chromosomes through cleavage of its Mcd1 subunit by the separase protease. Separase is in turn inhibited, up to this moment, by securin. Budding yeast cells respond to morphogenetic defects by a transient arrest in G2 with high securin levels and unseparated chromatids. We show that neither securin elimination nor forced cohesin cleavage is sufficient for anaphase in these conditions, suggesting that other factors contribute to cohesion maintainance in G2. We find that the protein phosphatase PP2A bound to its regulatory subunit Cdc55 plays a key role in this process, uncovering a new function for PP2A(Cdc55) in controlling a noncanonical pathway of chromatid cohesion removal.
Asunto(s)
Anafase/fisiología , Proteínas de Ciclo Celular/fisiología , Fosfoproteínas Fosfatasas/fisiología , Proteínas de Saccharomyces cerevisiae/fisiología , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/enzimología , Cromátides/enzimología , Cromátides/fisiología , Proteínas Nucleares/fisiología , Proteína Fosfatasa 2 , SecurinaRESUMEN
Bloom's syndrome (BS) is an autosomal disorder characterized by predisposition to a wide variety of cancers. The gene product whose mutation leads to BS is the RecQ family helicase BLM, which forms a complex with DNA topoisomerase IIIalpha (Top3alpha). However, the physiological relevance of the interaction between BLM and Top3alpha within the cell remains unclear. We show here that Top3alpha depletion causes accumulation of cells in G2 phase, enlargement of nuclei, and chromosome gaps and breaks that occur at the same position in sister chromatids. The transition from metaphase to anaphase is also inhibited. All of these phenomena except cell lethality are suppressed by BLM gene disruption. Taken together with the biochemical properties of BLM and Top3alpha, these data indicate that BLM and Top3alpha execute the dissolution of sister chromatids.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Cromátides/enzimología , Cromátides/genética , ADN Helicasas/metabolismo , ADN-Topoisomerasas de Tipo I/metabolismo , 2-Aminopurina/farmacología , Anafase/efectos de los fármacos , Animales , Apoptosis , Pollos , Cromátides/efectos de los fármacos , Aberraciones Cromosómicas , ADN-Topoisomerasas de Tipo I/deficiencia , Fase G2/efectos de los fármacos , Marcación de Gen , Humanos , Isoenzimas/metabolismo , Metafase/efectos de los fármacos , Ratones , Modelos Genéticos , Mutación/genética , Fenotipo , RecQ HelicasasRESUMEN
Accurate chromosome segregation in mitosis and meiosis requires that the cohesin complex be protected at the centromere by the Shugoshin/MEI-S332 protein family. Recent studies show that Sgo directly binds the phosphatase PP2A, tethering it to the centromere where it can protect cohesin subunits from phosphorylation, and that localization of Sgo/MEI-S332 itself is regulated by phosphorylation.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Centrómero/metabolismo , Cromátides/enzimología , Proteínas Cromosómicas no Histona/metabolismo , Proteínas Nucleares/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Fosfotransferasas/metabolismo , Animales , Centrómero/enzimología , Cromátides/metabolismo , Drosophila/genética , Regulación Enzimológica de la Expresión Génica , Humanos , CohesinasRESUMEN
Current models of mitotic chromosome structure are based largely on the examination of maximally condensed metaphase chromosomes. Here, we test these models by correlating the distribution of two scaffold components with the appearance of prophase chromosome folding intermediates. We confirm an axial distribution of topoisomerase IIalpha and the condensin subunit, structural maintenance of chromosomes 2 (SMC2), in unextracted metaphase chromosomes, with SMC2 localizing to a 150-200-nm-diameter central core. In contrast to predictions of radial loop/scaffold models, this axial distribution does not appear until late prophase, after formation of uniformly condensed middle prophase chromosomes. Instead, SMC2 associates throughout early and middle prophase chromatids, frequently forming foci over the chromosome exterior. Early prophase condensation occurs through folding of large-scale chromatin fibers into condensed masses. These resolve into linear, 200-300-nm-diameter middle prophase chromatids that double in diameter by late prophase. We propose a unified model of chromosome structure in which hierarchical levels of chromatin folding are stabilized late in mitosis by an axial "glue."
Asunto(s)
Cromátides/fisiología , Proteínas Cromosómicas no Histona/metabolismo , Cromosomas/metabolismo , Adenosina Trifosfatasas/metabolismo , Animales , Antígenos de Neoplasias , Células CHO , Línea Celular Tumoral , Núcleo Celular/química , Núcleo Celular/metabolismo , Núcleo Celular/ultraestructura , Cromátides/enzimología , Cromátides/ultraestructura , Cromatina/metabolismo , Cromatina/ultraestructura , Proteínas Cromosómicas no Histona/ultraestructura , Cromosomas/química , Cromosomas/fisiología , Cromosomas/ultraestructura , Cricetinae , Cricetulus , Reactivos de Enlaces Cruzados , ADN-Topoisomerasas de Tipo II/metabolismo , Proteínas de Unión al ADN/metabolismo , Células HeLa , Humanos , Inmunohistoquímica , Modelos Estructurales , Complejos Multiproteicos , ProfaseRESUMEN
Anaphase A chromatid-to-pole motion is fundamental for proper chromosome segregation in most systems. During the past several decades, two models for the mechanism of anaphase A have come to prominence. The Pacman model posits that chromatids induce the depolymerization of microtubule plus-ends embedded in kinetochores, thereby 'chewing' their way poleward. Alternatively, the Poleward-flux model posits that chromatids are 'reeled-in' to poles by the continual depolymerization of the minus-ends of kinetochore-associated microtubules, which are focused at spindle poles. In a recent study, we reported that anaphase A in Drosophila requires the depolymerization of both ends of kinetochore-associated microtubules, simultaneously. This is driven by two members of the Kin I subfamily of kinesins, termed KLP59C and KLP10A, which target specifically to chromatids and spindle poles, respectively. We have termed this hybrid of Pacman and Poleward flux the Kin I-dependent Pacman-flux mechanism for anaphase A. Here, we discuss the implications of these findings and explore potential additional components required to drive chromatid-to-pole motion by such a mechanism.