RESUMEN

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.

Asunto(s)

Proteínas de Ciclo Celular/metabolismo , Pollos/genética , Reparación del ADN/genética , Intercambio de Cromátides Hermanas/genética , Animales , Línea Celular , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , Cromosomas/metabolismo , Reparación del ADN/efectos de los fármacos , Reparación del ADN/efectos de la radiación , Replicación del ADN/efectos de los fármacos , Replicación del ADN/efectos de la radiación , Técnicas de Inactivación de Genes , Cinética , Metilmetanosulfonato/farmacología , Mitosis/efectos de los fármacos , Mitosis/efectos de la radiación , Radiación Ionizante , Reproducibilidad de los Resultados , Intercambio de Cromátides Hermanas/efectos de los fármacos , Intercambio de Cromátides Hermanas/efectos de la radiación

RESUMEN

DNA damage can induce centrosome overduplication in a manner that requires G2-to-M checkpoint function, suggesting that genotoxic stress can decouple the centrosome and chromosome cycles. How this happens is unclear. Using live-cell imaging of cells that express fluorescently tagged NEDD1/GCP-WD and proliferating cell nuclear antigen, we found that ionizing radiation (IR)-induced centrosome amplification can occur outside S phase. Analysis of synchronized populations showed that significantly more centrosome amplification occurred after irradiation of G2-enriched populations compared with G1-enriched or asynchronous cells, consistent with G2 phase centrosome amplification. Irradiated and control populations of G2 cells were then fused to test whether centrosome overduplication is allowed through a diffusible stimulatory signal, or the loss of a duplication-inhibiting signal. Irradiated G2/irradiated G2 cell fusions showed significantly higher centrosome amplification levels than irradiated G2/unirradiated G2 fusions. Chicken-human cell fusions demonstrated that centrosome amplification was limited to the irradiated partner. Our finding that only the irradiated centrosome can duplicate supports a model where a centrosome-autonomous inhibitory signal is lost upon irradiation of G2 cells. We observed centriole disengagement after irradiation. Although overexpression of dominant-negative securin did not affect IR-induced centrosome amplification, Plk1 inhibition reduced radiation-induced amplification. Together, our data support centriole disengagement as a licensing signal for DNA damage-induced centrosome amplification.

Asunto(s)

Centrosoma/metabolismo , Daño del ADN , Fase G2/fisiología , Transducción de Señal/fisiología , Animales , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Ciclo Celular/fisiología , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Centriolos/metabolismo , Centriolos/efectos de la radiación , Centrosoma/efectos de la radiación , Humanos , Immunoblotting , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Microscopía Fluorescente , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Antígeno Nuclear de Célula en Proliferación/genética , Antígeno Nuclear de Célula en Proliferación/metabolismo , Fase S/fisiología , Factores de Tiempo

RESUMEN

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

Asunto(s)

Cromátides/fisiología , Roturas del ADN de Doble Cadena , Reparación del ADN , Animales , Antígenos Nucleares/metabolismo , Proteínas de la Ataxia Telangiectasia Mutada , Proteínas de Ciclo Celular/metabolismo , Línea Celular , Pollos , Proteínas de Unión al ADN/metabolismo , Desoxirribonucleasas de Localización Especificada Tipo II , Histonas/análisis , Histonas/metabolismo , Humanos , Autoantígeno Ku , Ovalbúmina/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Recombinasa Rad51/análisis , Proteínas de Saccharomyces cerevisiae , Proteínas Supresoras de Tumor/metabolismo

RESUMEN

Centrosomal abnormalities are frequently observed in cancers and in cells with defective DNA repair. Here, we used light and electron microscopy to show that DNA damage induces centrosome amplification, not fragmentation, in human cells. Caffeine abrogated this amplification in both ATM (ataxia telangiectasia, mutated)- and ATR (ATM and Rad3-related)-defective cells, indicating a complementary role for these DNA-damage-responsive kinases in promoting centrosome amplification. Inhibition of checkpoint kinase 1 (Chk1) by RNA-mediated interference or drug treatment suppressed DNA-damage-induced centrosome amplification. Radiation-induced centrosome amplification was abrogated in Chk1(-/-) DT40 cells, but occurred at normal levels in Chk1(-/-) cells transgenically expressing Chk1. Expression of kinase-dead Chk1, or Chk1S345A, through which the phosphatidylinositol-3-kinase cannot signal, failed to restore centrosome amplification, showing that signalling to Chk1 and Chk1 catalytic activity are necessary to promote centrosome overduplication after DNA damage.

Asunto(s)

Centrosoma/metabolismo , Daño del ADN , Proteínas Quinasas/metabolismo , Animales , Ciclo Celular/efectos de la radiación , Línea Celular , Centrosoma/efectos de la radiación , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Pollos , Humanos , Radiación Ionizante

RESUMEN

Cells exposed to ionizing radiation die via different mechanisms, including apoptosis and mitotic catastrophe. To determine the frequency of mitotic catastrophe in tumor cells after irradiation, we used time-lapse imaging to track centrin-1 and histone H2B in U2OS osteosarcoma cells. We observed a dose-dependent increase in the frequency of mitotic catastrophe after irradiation, although a consistent 30% of cell death occurred through mitotic failure at doses from 2-10 Gy. One potential cause of mitotic catastrophe is centrosome amplification, which is induced by irradiation, and which can result in the formation of multipolar mitotic spindles. Up to 60% of mitotic catastrophes occurred in cells with >2 centrosomes after irradiation. We observed multipolar mitoses in p53(+) and p53(-) tumor cells after irradiation and found that the spindle assembly checkpoint is active in multipolar mitotic cells. However, we did not detect active caspase-3 in multipolar mitoses. These data demonstrate that a significant proportion of cell death induced by ionizing irradiation is through an apoptosis-independent mechanism involving centrosome amplification and mitotic catastrophe.

Asunto(s)

Apoptosis/efectos de la radiación , Centrosoma/metabolismo , Mitosis/efectos de la radiación , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Caspasa 3/metabolismo , Línea Celular Tumoral , Centrosoma/efectos de la radiación , Relación Dosis-Respuesta en la Radiación , Células HCT116 , Humanos , Immunoblotting , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Microscopía por Video/métodos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Huso Acromático/metabolismo , Huso Acromático/efectos de la radiación , Factores de Tiempo , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo

RESUMEN

Caspases are cysteine proteases that are essential for cytokine maturation and apoptosis. To facilitate the dissection of caspase function in vitro and in vivo, we have synthesized irreversible caspase inhibitors with biotin attached via linker arms of various lengths (12a-d) and a 2,4-dinitrophenyl labeled inhibitor (13). Affinity labeling of apoptotic extracts followed by blotting reveals that these affinity probes detect active caspases. Using the strong affinity of avidin for biotin, we have isolated affinity-labeled caspase 6 from apoptotic cytosolic extracts of cells overexpressing procaspase 6 by treatment with 12c, which contains biotin attached to the N(epsilon)-lysine of the inhibitor by a 22.5 A linker arm, followed by affinity purification on monomeric avidin-sepharose beads. Compound 13 has proven sufficiently cell permeable to rescue cells from apoptotic execution. These novel caspase inhibitors should provide powerful probes for the study of the active caspase proteome during apoptosis both in vitro and in vivo.

Asunto(s)

Inhibidores de Caspasas , Inhibidores de Cisteína Proteinasa/síntesis química , Activación Enzimática/efectos de los fármacos , Precursores Enzimáticos/química , Proteoma , Marcadores de Afinidad , Apoptosis/efectos de los fármacos , Caspasa 6/metabolismo , Cromatografía de Afinidad , Inhibidores de Cisteína Proteinasa/farmacología , Citometría de Flujo , Humanos , Immunoblotting , Técnicas In Vitro , Células Jurkat/efectos de los fármacos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción

RESUMEN

Centrosomes are the principal microtubule organising centres in somatic cells. Abnormal centrosome number is common in tumours and occurs after gamma-irradiation and in cells with mutations in DNA repair genes. To investigate how DNA damage causes centrosome amplification, we examined cells that conditionally lack the Rad51 recombinase and thereby incur high levels of spontaneous DNA damage. Rad51-deficient cells arrested in G2 phase and formed supernumerary functional centrosomes, as assessed by light and serial section electron microscopy. This centrosome amplification occurred without an additional DNA replication round and was not the result of cytokinesis failure. G2-to-M checkpoint over-ride by caffeine or wortmannin treatment strongly reduced DNA damage-induced centrosome amplification. Radiation-induced centrosome amplification was potentiated by Rad54 disruption. Gene targeting of ATM reduced, but did not abrogate, centrosome amplification induced by DNA damage in both the Rad51 and Rad54 knockout models, demonstrating ATM-dependent and -independent components of DNA damage-inducible G2-phase centrosome amplification. Our data suggest DNA damage-induced centrosome amplification as a mechanism for ensuring death of cells that evade the DNA damage or spindle assembly checkpoints.

Asunto(s)

Proteínas de Ciclo Celular/fisiología , Centrosoma/fisiología , Daño del ADN , Proteínas de Unión al ADN/fisiología , Fase G2 , Amplificación de Genes , Proteínas Nucleares/fisiología , Proteínas Serina-Treonina Quinasas/fisiología , Proteínas Supresoras de Tumor/fisiología , Androstadienos/farmacología , Animales , Proteínas de la Ataxia Telangiectasia Mutada , Proteínas Aviares , Cafeína/farmacología , Proteínas de Ciclo Celular/genética , División Celular , Estimulantes del Sistema Nervioso Central/farmacología , Pollos , Inestabilidad Cromosómica , Citocinesis , ADN Helicasas , Replicación del ADN , Proteínas de Unión al ADN/genética , Marcación de Gen , Humanos , Citometría de Barrido por Láser , Ratones , Mitosis , Micotoxinas/farmacología , Proteínas Nucleares/genética , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/genética , Recombinasa Rad51 , Proteínas Supresoras de Tumor/genética , Wortmanina

RESUMEN

Cleavage of the cohesin subunit Scc1p/Mcd1p/Rad21 permits sister chromatid separation and is considered to trigger anaphase onset. It has also been suggested that the cohesin complex is essential for chromosome condensation and for assembling fully functional kinetochores. Here, we used vertebrate cells conditionally deficient in Scc1 to probe cohesin function in mitosis. Cells lacking cohesin arrest in prometaphase, with many chromosomes failing to align at a metaphase plate and high levels of the spindle assembly checkpoint protein, BubR1, at all kinetochores. We show that the structural integrity of chromosomes is normal in the absence of Scc1. Furthermore, specific inhibition of topoisomerase II, which is required for decatenation of replicated chromosomes, can bypass the cohesin requirement for metaphase chromosome alignment and spindle checkpoint silencing. Since the kinetochore effects of Scc1 deficiency can be compensated for by topoisomerase II inhibition, we conclude that Scc1 is not absolutely required for kinetochore assembly or function, and that its principal role in allowing the onset of anaphase is the establishment of sufficient inter-sister tension to allow biorientation.

Asunto(s)

Proteínas de Ciclo Celular/genética , Cinetocoros/metabolismo , Metafase/fisiología , Proteínas Nucleares/fisiología , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Animales , Proteínas de Ciclo Celular/análisis , Proteínas de Ciclo Celular/metabolismo , Línea Celular , Pollos/genética , Pollos/metabolismo , Cromátides/genética , Cromátides/metabolismo , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Cromosomas/genética , Cromosomas/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas Fúngicas , Eliminación de Gen , Cinetocoros/ultraestructura , Metafase/genética , Microscopía Fluorescente , Mitosis/genética , Mitosis/fisiología , Complejos Multiproteicos , Proteínas Nucleares/análisis , Proteínas Nucleares/metabolismo , Fosfoproteínas , Proteínas de Saccharomyces cerevisiae , Inhibidores de Topoisomerasa II , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo , Cohesinas

RESUMEN

The chromosomal passengers, aurora-B kinase, inner centromere protein (INCENP), and survivin, are essential proteins that have been implicated in the regulation of metaphase chromosome alignment, spindle checkpoint function, and cytokinesis. All three share a common pattern of localization, and it was recently demonstrated that aurora-B, INCENP, and survivin are present in a complex in Xenopus eggs and Saccharomyces cerevisiae. The presence of aurora-B kinase in the complex and its ability to bind the other components directly suggest that INCENP and survivin could potentially be aurora-B substrates. This hypothesis was recently proven for INCENP in vitro. Here we report that human survivin is specifically phosphorylated in vitro by aurora-B kinase at threonine 117 in its carboxyl alpha-helical coil. Mutation of threonine 117 to alanine prevents survivin phosphorylation by aurora-B in vitro but does not alter its localization in HeLa cells. By contrast, a phospho-mimic, in which threonine 117 was mutated to glutamic acid, was unable to localize correctly at any stage in mitosis. Mutation at threonine 117 also prevented immunoprecipitation of INCENP with survivin in vivo. These data suggest that phosphorylation of survivin at threonine 117 by aurora-B may regulate targeting of survivin, and possibly the entire passenger complex, in mammals.

Asunto(s)

Proteínas Cromosómicas no Histona/química , Proteínas Asociadas a Microtúbulos/química , Proteínas Serina-Treonina Quinasas/fisiología , Secuencia de Aminoácidos , Animales , Aurora Quinasa B , Aurora Quinasas , Células Cultivadas , Proteínas Cromosómicas no Histona/metabolismo , Ácido Glutámico/química , Glutatión Transferasa/metabolismo , Células HeLa , Humanos , Técnicas In Vitro , Proteínas Inhibidoras de la Apoptosis , Espectrometría de Masas , Metafase , Datos de Secuencia Molecular , Mutación , Proteínas de Neoplasias , Péptidos/química , Fosforilación , Pruebas de Precipitina , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/metabolismo , Homología de Secuencia de Aminoácido , Survivin , Treonina/química , Transfección

RESUMEN

The essential Aurora B kinase is a chromosomal passenger protein that is required for mitotic chromosome alignment and segregation. Aurora B function is dependent on the chromosome passenger, INCENP. INCENP, in turn, requires sister chromatid cohesion for its appropriate behaviour. Relatively few substrates have been identified for Aurora B, so that the precise role it plays in controlling mitosis remains to be elucidated. To identify potential novel mitotic substrates of Aurora B, extracted chromosomes were prepared from mitotically-arrested HeLa S3 cells and incubated with recombinant human Aurora B in the presence of radioactive ATP. Immunoblot analysis confirmed the HeLa scaffold fraction to be enriched for known chromosomal proteins including CENP-A, CENP-B, CENP-C, ScII and INCENP. Mass spectrometry of bands excised from one-dimensional polyacrylamide gels further defined the protein composition of the extracted chromosome fraction. Cloning, fluorescent tagging and expression in HeLa cells of the putative GTP-binding protein NGB/CRFG demonstrated it to be a novel mitotic chromosome protein, with a perichromosomal localisation. Identi fication of the protein bands corresponding to those phosphorylated by Aurora B revealed topoisomerase II alpha (topo IIalpha) as a potential Aurora B substrate. Purified recombinant human topo IIalpha was phosphorylated by Aurora B in vitro, confirming this proteomic approach as a valid method for the initial definition of candidate substrates of key mitotic kinases.

Asunto(s)

Cromosomas Humanos/enzimología , ADN-Topoisomerasas de Tipo II/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Antígenos de Neoplasias , Aurora Quinasa B , Aurora Quinasas , Cromosomas Humanos/química , ADN-Topoisomerasas de Tipo II/análisis , ADN-Topoisomerasas de Tipo II/inmunología , Proteínas de Unión al ADN , Técnica del Anticuerpo Fluorescente , Proteínas de Unión al GTP/análisis , Células HeLa , Humanos , Metafase , Proteínas Nucleares/análisis , Proteínas Nucleares/clasificación , Proteínas Serina-Treonina Quinasas/análisis , Proteínas Serina-Treonina Quinasas/inmunología , Proteómica/métodos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción

RESUMEN

We identified a novel essential centromere protein, CENP-I, which shows sequence similarity with fission yeast Mis6 protein, and we showed that CENP-I is a constitutive component of the centromere that colocalizes with CENP-A, -C, and -H throughout the cell cycle in vertebrate cells. To determine the precise function of CENP-I, we examined its role in centromere function by generating a conditional loss-of-function mutant in the chicken DT40 cell line. In the absence of CENP-I, cells arrested at prometaphase with misaligned chromosomes for long periods of time. Eventually, cells exited mitosis without undergoing cytokinesis. Immunocytochemical analysis of CENP-I-deficient cells demonstrated that both CENP-I and CENP-H are necessary for localization of CENP-C but not CENP-A to the centromere.

Asunto(s)

Autoantígenos , Proteínas de Ciclo Celular , Centrómero/genética , Centrómero/metabolismo , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Proteínas de Schizosaccharomyces pombe , Animales , Muerte Celular/fisiología , Células Cultivadas , Proteína A Centromérica , Pollos , Proteínas Cromosómicas no Histona/análisis , Aberraciones Cromosómicas , Segregación Cromosómica , Proteínas Fúngicas/genética , Metafase/fisiología , Microtúbulos/metabolismo , Mitosis/fisiología , Datos de Secuencia Molecular , Mutagénesis/fisiología , Schizosaccharomyces/genética , Vertebrados