RESUMEN
Fbh1, an F-box helicase related to bacterial UvrD, has been proposed to modulate homologous recombination in fission yeast. We provide several lines of evidence for such modulation. Fbh1, but not the related helicases Srs2 and Rqh1, suppressed the formation of crossover recombinants from single HO-induced DNA double-strand breaks. Purified Fbh1 in complex with Skp1 (Fbh1-Skp1 complex) inhibited Rad51-driven DNA strand exchange by disrupting Rad51 nucleoprotein filaments in an ATP-dependent manner; this disruption was alleviated by the Swi5-Sfr1 complex, an auxiliary activator of Rad51. In addition, the reconstituted SCFFbh1 complex, composed of purified Fbh1-Skp1 and Pcu1-Rbx1, displayed ubiquitin-ligase E3 activity toward Rad51. Furthermore, Fbh1 reduced the protein level of Rad51 in stationary phase in an F-box-dependent, but not in a helicase domain-independent manner. These results suggest that Fbh1 negatively regulates Rad51-mediated homologous recombination via its two putative, unrelated activities, namely DNA unwinding/translocation and ubiquitin ligation. In addition to its anti-recombinase activity, we tentatively suggest that Fbh1 might also have a pro-recombination role in vivo, because the Fbh1-Skp1 complex stimulated Rad51-mediated strand exchange in vitro after strand exchange had been initiated.
Asunto(s)
ADN Helicasas/genética , Recombinación Homóloga/genética , Recombinasa Rad51/biosíntesis , Proteínas de Schizosaccharomyces pombe/biosíntesis , Proteínas de Schizosaccharomyces pombe/genética , Schizosaccharomyces/genética , Citoesqueleto/genética , Roturas del ADN de Doble Cadena , ADN Helicasas/metabolismo , Mutación , Nucleoproteínas/genética , Nucleoproteínas/metabolismo , Recombinasa Rad51/genética , Proteínas Ligasas SKP Cullina F-box/genética , Proteínas Ligasas SKP Cullina F-box/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
In forward genetics, identification of mutations is a time-consuming and laborious process. Modern whole-genome sequencing, coupled with bioinformatics analysis, has enabled fast and cost-effective mutation identification. However, for many experimental researchers, bioinformatics analysis is still a difficult aspect of whole-genome sequencing. To address this issue, we developed a browser-accessible and easy-to-use bioinformatics tool called Mutation discovery (Mudi; http://naoii.nig.ac.jp/mudi_top.html), which enables 'one-click' identification of causative mutations from whole-genome sequence data. In this study, we optimized Mudi for pooled-linkage analysis aimed at identifying mutants in yeast model systems. After raw sequencing data are uploaded, Mudi performs sequential analysis, including mapping, detection of variant alleles, filtering and removal of background polymorphisms, prioritization, and annotation. In an example study of suppressor mutants of ptr1-1 in the fission yeast Schizosaccharomyces pombe, pooled-linkage analysis with Mudi identified mip1(+) , a component of Target of Rapamycin Complex 1 (TORC1), as a novel component involved in RNA interference (RNAi)-related cell-cycle control. The accessibility of Mudi will accelerate systematic mutation analysis in forward genetics.
Asunto(s)
Biología Computacional/métodos , Genoma Fúngico , Internet , Diana Mecanicista del Complejo 1 de la Rapamicina , Complejos Multiproteicos/genética , Mutación , Interferencia de ARN , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética , Supresión Genética , Serina-Treonina Quinasas TOR/genética , Interfaz Usuario-ComputadorRESUMEN
To identify the genes required to sustain aneuploid viability, we screened a deletion library of non-essential genes in the fission yeast Schizosaccharomyces pombe, in which most types of aneuploidy are eventually lethal to the cell. Aneuploids remain viable for a period of time and can form colonies by reducing the extent of the aneuploidy. We hypothesized that a reduction in colony formation efficiency could be used to screen for gene deletions that compromise aneuploid viability. Deletion mutants were used to measure the effects on the viability of spores derived from triploid meiosis and from a chromosome instability mutant. We found that the CCR4-NOT complex, an evolutionarily conserved general regulator of mRNA turnover, and other related factors, including poly(A)-specific nuclease for mRNA decay, are involved in aneuploid viability. Defective mutations in CCR4-NOT complex components in the distantly related yeast Saccharomyces cerevisiae also affected the viability of spores produced from triploid cells, suggesting that this complex has a conserved role in aneuploids. In addition, our findings suggest that the genes required for homologous recombination repair are important for aneuploid viability.
Asunto(s)
Supervivencia Celular/genética , Recombinación Homóloga , Proteínas de Unión al ARN , Saccharomyces cerevisiae , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Aneuploidia , Exorribonucleasas/genética , Exorribonucleasas/metabolismo , Regulación Fúngica de la Expresión Génica , Genoma Fúngico , Recombinación Homóloga/genética , Meiosis , Estabilidad del ARN/genética , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Schizosaccharomyces/genética , Schizosaccharomyces/crecimiento & desarrollo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Eliminación de Secuencia , Esporas/genética , Esporas/crecimiento & desarrolloRESUMEN
Ago1, an effector protein of RNA interference (RNAi), regulates heterochromatin silencing and cell cycle arrest in fission yeast. However, the mechanism by which Ago1 controls cell cycle checkpoint following hydroxyurea (HU) treatment has not been elucidated. In this study, we show that Ago1 and other RNAi factors control cell cycle checkpoint following HU treatment via a mechanism independent of silencing. While silencing requires dcr1(+), the overexpression of ago1(+) alleviated the cell cycle defect in dcr1Δ. Ago1 interacted with the mRNA export factor, Ptr1. The ptr1-1 mutation impaired cell cycle checkpoint but gene silencing was unaffected. Genetic analysis revealed that the regulation of cell cycle checkpoint by ago1(+) is dependent on ptr1(+). Nuclear accumulation of poly(A)(+) RNAs was detected in mutants of ago1(+) and ptr1(+), suggesting there is a functional link between the cell cycle checkpoint and RNAi-mediated RNA quality control.
Asunto(s)
Proteínas Argonautas/metabolismo , Puntos de Control del Ciclo Celular/genética , Proteínas Asociadas a Matriz Nuclear/metabolismo , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Interferencia de ARN , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/fisiología , Proteínas Argonautas/genética , Núcleo Celular/metabolismo , Heterocromatina/metabolismo , Hidroxiurea/farmacología , Mutación , Proteínas Asociadas a Matriz Nuclear/genética , Proteínas de Transporte Nucleocitoplasmático/genética , Edición de ARN , ARN Mensajero/genética , ARN Mensajero/metabolismo , Schizosaccharomyces/efectos de los fármacos , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genéticaRESUMEN
Cell cycle events are regulated by sequential activation and inactivation of Cdk kinases. Mitotic exit is accomplished by the inactivation of mitotic Cdk kinase, which is mainly achieved by degradation of cyclins. The ubiquitin-proteasome system is involved in this process, requiring APC/C (anaphase-promoting complex/cyclosome) as a ubiquitin ligase. In Xenopus and clam oocytes, the ubiquitin-conjugating enzymes that function with APC/C have been identified as two proteins, UBC4 and UBCx/E2-C. Previously we reported that the fission yeast ubiquitin-conjugating enzyme UbcP4/Ubc11, a homologue of UBCx/E2-C, is required for mitotic transition. Here we show that the other fission yeast ubiquitin-conjugating enzyme, UbcP1/Ubc4, which is homologous to UBC4, is also required for mitotic transition in the same manner as UbcP4/Ubc11. Both ubiquitin-conjugating enzymes are essential for cell division and directly required for the degradation of mitotic cyclin Cdc13. They function nonredundantly in the ubiquitination of CDC13 because a defect in ubcP1/ubc4+ cannot be suppressed by high expression of UbcP4/Ubc11 and a defect in ubcP4/ubc11+ cannot be suppressed by high expression of UbcP1/Ubc4. In vivo analysis of the ubiquitinated state of Cdc13 shows that the ubiquitin chains on Cdc13 were short in ubcP1/ubc4 mutant cells while ubiquitinated Cdc13 was totally reduced in ubcP4/ubc11 mutant cells. Taken together, these results indicate that the two ubiquitin-conjugating enzymes play distinct and essential roles in the degradation of mitotic cyclin Cdc13, with the UbcP4/Ubc11-pathway initiating ubiquitination of Cdc13 and the UbcP1/Ubc4-pathway elongating the short ubiquitin chains on Cdc13.
Asunto(s)
Proteínas Portadoras/fisiología , Ciclinas/metabolismo , Ligasas/fisiología , Proteínas de Saccharomyces cerevisiae/fisiología , Enzimas Ubiquitina-Conjugadoras , Secuencia de Aminoácidos , Animales , Western Blotting , Ciclina B/metabolismo , Mitosis , Datos de Secuencia Molecular , Mutación , Oocitos/metabolismo , Plásmidos/metabolismo , Unión Proteica , Homología de Secuencia de Aminoácido , Temperatura , Factores de Tiempo , Ubiquitina/metabolismo , XenopusRESUMEN
Research in the ubiquitin field requires large amounts of ubiquitin-activating enzyme (E1) for in vitro ubiquitination assays. Typically, the mammalian enzyme is either isolated from natural sources or produced recombinantly using baculovirus/insect cell protein expression systems. Escherichia coli is seldom used to produce mammalian E1 probably due to the instability and insolubility of this high-molecular mass protein. In this report, we show that 5-10 mg of histidine-tagged mouse E1 can be easily obtained from a 1 l E. coli culture. A low temperature during the protein induction step was found to be critical to obtain an active enzyme.
Asunto(s)
Escherichia coli/química , Escherichia coli/enzimología , Proteínas Recombinantes de Fusión/aislamiento & purificación , Enzimas Activadoras de Ubiquitina/aislamiento & purificación , Animales , Cromatografía Líquida de Alta Presión , Pruebas de Enzimas , Escherichia coli/genética , Histidina/análogos & derivados , Histidina/química , Histidina/genética , Hígado/química , Hígado/citología , Ratones , Receptor de la Señal 1 de Direccionamiento al Peroxisoma , Receptores Citoplasmáticos y Nucleares/química , Receptores Citoplasmáticos y Nucleares/metabolismo , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Temperatura , Enzimas Activadoras de Ubiquitina/biosíntesis , Enzimas Activadoras de Ubiquitina/genéticaRESUMEN
Specialized chromatin exists at centromeres and must be precisely transmitted during DNA replication. The mechanisms involved in the propagation of these structures remain elusive. Fission yeast centromeres are composed of two chromatin domains: the central CENP-A(Cnp1) kinetochore domain and flanking heterochromatin domains. Here we show that fission yeast Mcl1, a DNA polymerase alpha (Pol alpha) accessory protein, is critical for maintenance of centromeric chromatin. In a screen for mutants that alleviate both central domain and outer repeat silencing, we isolated several cos mutants, of which cos1 is allelic to mcl1. The mcl1-101 mutation causes reduced CENP-A(Cnp1) in the central domain and an aberrant increase in histone acetylation in both domains. These phenotypes are also observed in a mutant of swi7(+), which encodes a catalytic subunit of Pol alpha. Mcl1 forms S-phase-specific nuclear foci, which colocalize with those of PCNA and Pol alpha. These results suggest that Mcl1 and Pol alpha are required for propagation of centromere chromatin structures during DNA replication.
Asunto(s)
Centrómero , ADN Polimerasa I/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/fisiología , Schizosaccharomyces/ultraestructura , Acetilación , Inmunoprecipitación de Cromatina , Cromosomas Fúngicos , Clonación Molecular , Silenciador del Gen , Histona Desacetilasas/genética , Histonas/metabolismo , Microscopía Fluorescente , Mutación , Proteína 1 de la Secuencia de Leucemia de Células Mieloides , Schizosaccharomyces/enzimología , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismoRESUMEN
Several accessory proteins referred to as mediators are required for the full activity of the Rad51 (Rhp51 in fission yeast) recombinase. In this study, we analyzed in vivo functions of the recently discovered Swi5/Sfr1 complex from fission yeast. In normally growing cells, the Swi5-GFP protein localizes to the nucleus, where it forms a diffuse nuclear staining pattern with a few distinct foci. These spontaneous foci do not form in swi2Delta mutants. Upon UV irradiation, Swi5 focus formation is induced in swi2Delta mutants, a response that depends on Sfr1 function, and Sfr1 also forms foci that colocalize with damage-induced Rhp51 foci. The number of UV-induced Rhp51 foci is partially reduced in swi5Delta and rhp57Delta mutants and completely abolished in an swi5Delta rhp57Delta double mutant. An assay for products generated by HO endonuclease-induced DNA double-strand breaks (DSBs) reveals that Rhp51 and Rhp57, but not Swi5/Sfr1, are essential for crossover production. These results suggest that Swi5/Sfr1 functions as an Rhp51 mediator but processes DSBs in a manner different from that of the Rhp55/57 mediator.
Asunto(s)
Recombinación Genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Núcleo Celular/metabolismo , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Roturas del ADN de Doble Cadena/efectos de la radiación , Daño del ADN , Reparación del ADN/genética , ADN de Hongos/genética , ADN de Hongos/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Heterocromatina/metabolismo , Microscopía Fluorescente , Unión Proteica , Recombinasa Rad51/genética , Recombinasa Rad51/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/efectos de la radiación , Proteínas de Schizosaccharomyces pombe/genética , Rayos UltravioletaRESUMEN
Schizosaccharomyces pombe rad2 is involved in Okazaki fragments processing during lagging-strand DNA replication. Previous studies identified several slr mutants that are co-lethal with rad2Delta and sensitive to methyl methanesulfonate as single mutants. One of these mutants, slr3-1, is characterized here. Complementation and sequence analyses show that slr3-1 (mcl1-101) is allelic to mcl1(+), which is required for chromosome replication, cohesion and segregation. mcl1-101 is temperature-sensitive for growth and is highly sensitive to DNA damage. mcl1 cells arrest with 2C DNA content and chromosomal DNA double-strand breaks accumulate at the restrictive temperature. Mcl1p, which belongs to the Ctf4p/SepBp family, interacts both genetically and physically with DNA polymerase alpha. Mutations in rhp51 and dna2 enhance the growth defect of the mcl1-101 mutant. These results strongly suggest that Mcl1p is a functional homologue of Saccharomyces cerevisiae Ctf4p and plays a role in lagging-strand synthesis and Okazaki fragment processing, in addition to DNA repair.
Asunto(s)
ADN Polimerasa I/genética , Reparación del ADN/fisiología , Replicación del ADN/fisiología , Endodesoxirribonucleasas/genética , Endonucleasas de ADN Solapado/genética , Proteínas de Neoplasias/genética , Proteínas Proto-Oncogénicas c-bcl-2/genética , Schizosaccharomyces/genética , Clonación Molecular , ADN , Daño del ADN , ADN Polimerasa I/metabolismo , Electroforesis en Gel de Campo Pulsado , Endodesoxirribonucleasas/metabolismo , Endonucleasas de ADN Solapado/fisiología , Mutación , Proteína 1 de la Secuencia de Leucemia de Células Mieloides , Proteínas de Neoplasias/metabolismo , Plásmidos , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Técnicas del Sistema de Dos HíbridosRESUMEN
Bismuth compounds are known for their low levels of toxicity in mammals, and various types of bismuth salts have been used to treat medical disorders. As part of our program to probe this aspect of bismuth chemistry, cyclic organobismuth compounds 1 to 8 bearing a nitrogen or sulfur atom as an additional ring member have been synthesized, and their antimicrobial activities against five standard strains of gram-negative and gram-positive bacteria were assessed. The eight-membered-ring compounds, compounds 1 to 3, exhibited MICs of less than 0.5 microg/ml against Staphylococcus aureus and were more active than the six-membered ones, compounds 5 to 8 (MICs, 4.0 to 16 microg/ml). The gram-positive bacteria (Staphylococcus aureus, Bacillus subtilis, and Enterococcus faecalis) were more susceptible to both types of ring compounds than the gram-negative ones (Escherichia coli and Pseudomonas aeruginosa). Treatment with polymyxin B nonapeptide increased the susceptibility of E. coli to cyclic organobismuth compounds, indicating the low permeability of the outer membrane of gram-negative bacteria to the compounds. Compound 1 also had activity against methicillin-resistant S. aureus, which had an MIC for 90% of the hospital stock strains of 1.25 microg/ml. The killing curves for S. aureus treated with compound 1 or 3 revealed a static effect at a low dose (2x the MIC). However, when S. aureus was treated with 10x the MIC of compound 1 or 3, there was an approximately 3-log reduction in the viable cell number after 48 h of treatment. Electron microscopic inspection demonstrated a considerable increase in the size of S. aureus and the proportion of cells undergoing cell division after treatment with compound 1 at 0.5x the MIC.
Asunto(s)
Antibacterianos/farmacología , Bismuto/farmacología , Bacterias Gramnegativas/efectos de los fármacos , Bacterias Grampositivas/efectos de los fármacos , Compuestos Organometálicos/farmacología , Antibacterianos/síntesis química , Antibacterianos/química , Bismuto/química , Resistencia a la Meticilina , Pruebas de Sensibilidad Microbiana , Microscopía Electrónica de Transmisión , Compuestos Organometálicos/síntesis química , Compuestos Organometálicos/química , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/ultraestructura , Relación Estructura-ActividadRESUMEN
In order to elucidate the mechanism controlling the biogenesis of the Golgi complex, we have studied whether the expression of a resident membrane protein p138 of the Golgi complex is dependent upon the cell cycle. The protein level of p138 in human KB cells was increased during thymidine block to synchronize the cells in the early-S phase, but changed little from S to G2 after release from the block. On the other hand, the mRNA level of the p138 gene was constant during the block. The change in mRNA level in the cells was small with a low peak at S to G2. Both p138 protein and mRNA levels decreased after cell division and then rose rapidly to the same level as those of log-phase cells in the next G1 to S. Thus, translation of p138 protein was upregulated in the cells at the early-S phase. However, we found also that the p138 protein level increased during an arrest at G2/M caused by etoposide. The kinetics of centrosome duplication apparently differ from those of p138 protein production. The duplication occurred mainly at S to G2 after the release from thymidine block, while the ratio of cells containing duplicated centrosomes increased gradually during the block. Taken together, these results show that both the translation and transcription of p138 protein are regulated independent of the cell cycle and dissociated from the duplication of the centrosome. Rather, the expression of p138 protein seems to be coupled with a change in cell size since both thymidine block and etoposide inhibition resulted in an apparent increase in cell size.