RESUMEN
Eukaryotic SMC complexes, cohesin, condensin, and Smc5/6, use ATP hydrolysis to power a plethora of functions requiring organization and restructuring of eukaryotic chromosomes in interphase and during mitosis. The Smc5/6 mechanism of action and its activity on DNA are largely unknown. Here we purified the budding yeast Smc5/6 holocomplex and characterized its core biochemical and biophysical activities. Purified Smc5/6 exhibits DNA-dependent ATP hydrolysis and SUMO E3 ligase activity. We show that Smc5/6 binds DNA topologically with affinity for supercoiled and catenated DNA templates. Employing single-molecule assays to analyze the functional and dynamic characteristics of Smc5/6 bound to DNA, we show that Smc5/6 locks DNA plectonemes and can compact DNA in an ATP-dependent manner. These results demonstrate that the Smc5/6 complex recognizes DNA tertiary structures involving juxtaposed helices and might modulate DNA topology by plectoneme stabilization and local compaction.
Asunto(s)
Proteínas de Ciclo Celular/genética , Complejos Multiproteicos/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Adenosina Trifosfatasas/genética , Fenómenos Biofísicos , Proteínas de Ciclo Celular/ultraestructura , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/ultraestructura , Proteínas de Unión al ADN/genética , Humanos , Interfase/genética , Mitosis/genética , Complejos Multiproteicos/ultraestructura , Saccharomyces cerevisiae/ultraestructura , Proteínas de Saccharomyces cerevisiae/ultraestructura , Sumoilación/genética , CohesinasRESUMEN
The Smc5/6 complex is a highly conserved molecular machine involved in the maintenance of genome integrity. While its functions largely depend on restraining the fork remodeling activity of Mph1 in yeast, the presence of an analogous Smc5/6-FANCM regulation in humans remains unknown. We generated human cell lines harboring mutations in the NSE1 subunit of the Smc5/6 complex. Point mutations or truncations in the RING domain of NSE1 result in drastically reduced Smc5/6 protein levels, with differential contribution of the two zinc-coordinating centers in the RING. In addition, nse1-RING mutant cells display cell growth defects, reduced replication fork rates, and increased genomic instability. Notably, our findings uncover a synthetic sick interaction between Smc5/6 and FANCM and show that Smc5/6 controls fork progression and chromosome disjunction in a FANCM-independent manner. Overall, our study demonstrates that the NSE1 RING domain plays vital roles in Smc5/6 complex stability and fork progression through pathways that are not evolutionary conserved.
Asunto(s)
Proteínas de Ciclo Celular , Replicación del ADN , Inestabilidad Genómica , Humanos , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas Cromosómicas no Histona/metabolismo , Proteínas Cromosómicas no Histona/genética , Dominios Proteicos , Estabilidad Proteica , Mutación , Línea Celular , ADN HelicasasRESUMEN
The Smc5/6 complex plays essential roles in chromosome segregation and repair, by promoting disjunction of sister chromatids. The core of the complex is constituted by an heterodimer of Structural Maintenance of Chromosomes (SMC) proteins that use ATP hydrolysis to dynamically associate with and organize chromosomes. In addition, the Smc5/6 complex contains six non-SMC subunits. Remarkably, and differently to other SMC complexes, the Nse1 and Nse2 subunits contain RING-type domains typically found in E3 ligases, pointing to the capacity to regulate other proteins and complexes through ubiquitin-like modifiers. Nse2 codes for a C-terminal SP-RING domain with SUMO ligase activity, assisting Smc5/6 functions in chromosome segregation through sumoylation of several chromosome-associated proteins. Nse1 codes for a C-terminal NH-RING domain and, although it has been proposed to have ubiquitin ligase activity, no Smc5/6-dependent ubiquitylation target has been described to date. Here, we review the function of the two RING domains of the Smc5/6 complex in the broader context of SMC complexes as global chromosome organizers of the genome.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Cromosomas/metabolismo , Ligasas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Ubiquitina/química , Adenosina Trifosfato/química , Cromosomas/ultraestructura , ADN/química , Daño del ADN , Reparación del ADN , Humanos , Hidrólisis , Conformación Proteica , Dominios Proteicos , Estructura Secundaria de Proteína , Recombinación Genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Schizosaccharomyces pombe , SumoilaciónRESUMEN
Ubiquitination controls numerous cellular processes, and its deregulation is associated with many pathologies. The Nse1 subunit in the Smc5/6 complex contains a RING domain with ubiquitin E3 ligase activity and essential functions in genome integrity. However, Nse1-dependent ubiquitin targets remain elusive. Here, we use label-free quantitative proteomics to analyze the nuclear ubiquitinome of nse1-C274A RING mutant cells. Our results show that Nse1 impacts the ubiquitination of several proteins involved in ribosome biogenesis and metabolism that, importantly, extend beyond canonical functions of Smc5/6. In addition, our analysis suggests a connection between Nse1 and RNA polymerase I (RNA Pol I) ubiquitination. Specifically, Nse1 and the Smc5/6 complex promote ubiquitination of K408 and K410 in the clamp domain of Rpa190, a modification that induces its degradation in response to blocks in transcriptional elongation. We propose that this mechanism contributes to Smc5/6-dependent segregation of the rDNA array, the locus transcribed by RNA Pol I.
Asunto(s)
ARN Polimerasa I , Ubiquitina , Secuencia de Aminoácidos , ARN Polimerasa I/metabolismo , Proteómica , Proteínas de Ciclo Celular/metabolismo , ARN , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Replication of a damaged DNA template can threaten the integrity of the genome, requiring the use of various mechanisms to tolerate DNA lesions. The Smc5/6 complex, together with the Nse2/Mms21 SUMO ligase, plays essential roles in genome stability through undefined tasks at damaged replication forks. Various subunits within the Smc5/6 complex are substrates of Nse2, but we currently do not know the role of these modifications. Here we show that sumoylation of Smc5 is targeted to its coiled-coil domain, is upregulated by replication fork damage, and participates in bypass of DNA lesions. smc5-KR mutant cells display defects in formation of sister chromatid junctions and higher translesion synthesis. Also, we provide evidence indicating that Smc5 sumoylation modulates Mph1-dependent fork regression, acting synergistically with other pathways to promote chromosome disjunction. We propose that sumoylation of Smc5 enhances physical remodeling of damaged forks, avoiding the use of a more mutagenic tolerance pathway.
Asunto(s)
Proteínas de Ciclo Celular/genética , Replicación del ADN/genética , Proteínas de Saccharomyces cerevisiae/genética , Sumoilación/genética , Cromátides/genética , Cromosomas/genética , ADN/genética , Daño del ADN/genética , Reparación del ADN/genética , Saccharomyces cerevisiae/genéticaRESUMEN
The immobilization of phosphine-free perfluoro-tagged palladium nanoparticles Pd-1 on fluorous silica gel (FSG) and their utilization in the Heck reaction have been investigated. High yields of vinylic substitution products have been obtained. Recycling studies have shown that the solid-supported palladium catalyst can be readily recovered and reused several times without significant loss of activity. Reactions and recovery of the solid-supported palladium catalyst system can be carried out in the presence of air, without any particular precaution.
RESUMEN
Artificial hydrophobic surfaces have a great potential in a wide range of industrial applications owing to their self-cleaning, anti-fogging and anti-biofouling properties. A family of polyfluorinated reactive azo dyes has been prepared and some of them easily modified and grafted on cotton fabric and glass surfaces obtaining new coloured hydrophobic materials.
RESUMEN
Objetivo: evaluar la estabilidad en anaquel de un gel elaborado a partir del extracto acuoso de la corteza de Rhizophora mangle L. (mangle rojo).Métodos: los tres lotes pilotos del gel (GM01, GM02 y GM03) se almacenaron a temperatura de refrigeración (5 ± 3 °C) durante 12 meses. Se realizó una evaluación físico-química y microbiológica a tiempo inicial y a los 3, 6, 9 y 12 meses.Resultados: todos los lotes mantuvieron una apariencia de geles homogéneos, viscosos, libres de grumo, brillantes y de un color pardo-rojizo oscuro y mostraron amplias áreas de extensibilidad. El pH estuvo entre 6 y 7 y la reología fue característica de un fluido no newtoniano del tipo Herschel Bulkley con potencial modificado en todos los tiempos evaluados. Los tres lotes cumplieron el límite microbiano establecido, así como la concentración mínima inhibitoria que estuvo entre 8 y 10 mg/mL y la concentración de taninos entre 13 a 30 mg/g.Conclusiones: se demostró que todos los lotes del gel fueron estables durante el período de estabilidad en anaquel, por lo que se propone que se almacene de 2-8 ºC durante 1 año
Asunto(s)
Estabilidad de Medicamentos , Rhizophoraceae/microbiología , Rhizophoraceae/químicaRESUMEN
El objetivo del presente trabajo fue evaluar la estabilidad acelerada de un gel de Rhizophora mangle L. (mangle rojo) en dos condiciones de almacenamiento. Los 3 lotes pilotos producidos (GM01, GM02 y GM03) se almacenaron a dos temperaturas: 40 ± 2 °C durante 3 meses y 25 ± 2 °C durante 6 meses. Se realizó una evaluación de indicadores de estabilidad físico-química y microbiológica a tiempo 0, 1, 2 y 3 meses y a tiempo 0, 1, 2, 3 y 6 meses para cada una de las dos condiciones ensayadas respectivamente. Todos los lotes almacenados en ambas temperaturas mostraron estables las características organolépticas y la extensibilidad, el pH estuvo entre 6 y 7 y la reología confirmó un fluido no newtoniano del tipo Herschel Bulkley en los tiempos evaluados. La concentración mínima inhibitoria permaneció entre 8 y 10 mg/mL y la concentración de taninos entre 13 a 30 mg/g; todos los lotes se mantuvieron dentro del límite microbiano. El gel demostró tener buena estabilidad en condiciones aceleradas de temperatura, aspecto que es necesario confirmar en un próximo estudio de estabilidad en anaquel.
The objective of the present paper was to evaluate the accelerated stability of a Rhizophora mangle L. (red mangrove) gel under 2 storage conditions. The three pilot batches (GM01, GM02 and GM03) were stored at two temperature settings: 40 ± 2 °C for three months and 25 ± 2 °C for 6 months. One physical-chemical and microbiological evaluation was performed in two periods of time: at the months 0, 1, 2 and 3 for the first and at the months 0, 1, 2, 3 y 6 for the second tested storage condition. All the batches stored at both temperatures showed stable organoleptic characteristics and extensibility, the pH ranged from 6 to 7 and rheology confirmed a non-Newtonian fluid of Herschel Bulkley-type in the evaluated periods of time. The minimum inhibitory concentration remained 8 to 10 mg/mL whereas the tannin concentration ranged 13 to 30 mg/g. All the batches were within the allowable microbial limits. The red mangrove gel showed good stability under accelerated temperatures, but this is an aspect that requires to be confirmed in a future on-shelf stability study.
RESUMEN
RESUMEN El Centro Nacional de Sanidad Agropecuaria empleó un irradiador Gammacell 500 con 12 fuentes selladas de 60Co para la esterilización y descontaminación de dispositivos médicos, productos farmacéuticos y diversas materias primas. Debido a los 25 años de explotación de la instalación se decide desmantelar el equipo y clausurar la instalación. Para la clausura se tomó como base las regulaciones nacionales vigentes, las recomendaciones del Organismo Internacional de la Energía Atómica, la consulta al Manual de operación y mantenimiento del equipo y la historia operacional. En el trabajo se describe el diseño e implementación del procedimiento para el desmantelamiento del irradiador. El Centro Nacional de Seguridad Nuclear fue la institución encargada de realizar la evaluación de la documentación y emitió la Licencia Institucional de Cierre Definitivo de la instalación. Las operaciones de desmantelamiento consistieron en la separación y extracción del contenedor con las fuentes radiactivas y la conformación del bulto para el transporte. Todas las operaciones de desmantelamiento, así como del transporte del bulto se realizaron en condiciones radiológicas adecuadas, controlándose la tasa de dosis y contaminación superficial durante el proceso. Al finalizar todos los trabajos se recibió la autorización para la liberación de la instalación del control regulador.
ABSTRACT The National Center for Animal and Plant Health used a Gammacell 500 irradiator, with twelve cobalt 60 sealed sources, for sterilization and decontamination of medical devices, pharmaceuticals and several raw materials. As the equipmenty had been in operation for 25 years, the decision was taken to dismantle the equipment and decommission the facility. For dismantling the equipment, a methodology was developed taking into account the current national regulations in force, the recommendations of the Atomic International Energy Agency, the instructions manual and equipment maintenance as well as its operational history. The design and implementation of the procedure for dismantling the irradiator are described. The Institutional Irradiator Decommissioning License was obtained, awarded by the National Center for Nuclear Safety. The dismantling operations involved the extraction and separation of radioactive sources in the container and packaging to be transported. All dismantling as well as transport operations were performed under radiological security conditions, keeping control of the dose and superfi cial contamination rate during the whole process. After completing the works, permission for the no-longer inclusion of the facility in the nuclear safety control programme was granted by the national regulatory body.
RESUMEN
En el presente trabajo se aplicó una de las técnicas más representativas que conforman el análisis térmico, la calorimetría diferencial de barrido, para estudiar la compatibilidad entre el extracto seco de Rhizophora mangle L y distintos excipientes preseleccionados para la obtención de formulaciones a partir de esta especie vegetal. Se obtuvo como resultado la no interacción química entre el extracto vegetal seco y los excipientes
In present paper authors applied one of the more representative techniques conforming the thermal analysis, the differential scanning clometry to study the compatibility among the Rhizophora mangle L dry extract and different excipients selected to obtain the formulae from this vegetal species. As result, it was possible to obtain the non-chemical interaction between the dry vegeral extract and the excipients ones