RESUMEN
Kinetochores direct chromosome segregation in mitosis and meiosis. Faithful gamete formation through meiosis requires that kinetochores take on new functions that impact homolog pairing, recombination, and the orientation of kinetochore attachment to microtubules in meiosis I. Using an unbiased proteomics pipeline, we determined the composition of centromeric chromatin and kinetochores at distinct cell-cycle stages, revealing extensive reorganization of kinetochores during meiosis. The data uncover a network of meiotic chromosome axis and recombination proteins that bind to centromeres in the absence of the microtubule-binding outer kinetochore sub-complexes during meiotic prophase. We show that the Ctf19cCCAN inner kinetochore complex is essential for kinetochore organization in meiosis. Our functional analyses identify a Ctf19cCCAN-dependent kinetochore assembly pathway that is dispensable for mitotic growth but becomes critical upon meiotic entry. Therefore, changes in kinetochore composition and a distinct assembly pathway specialize meiotic kinetochores for successful gametogenesis.
Asunto(s)
Centrómero/metabolismo , Cromatina/metabolismo , Proteínas del Citoesqueleto/metabolismo , Cinetocoros/metabolismo , Meiosis , Proteínas de Saccharomyces cerevisiae/metabolismo , Segregación Cromosómica , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/aislamiento & purificación , Mitosis , Proteómica , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/aislamiento & purificaciónRESUMEN
In meiosis, crossover (CO) formation between homologous chromosomes is essential for faithful segregation. However, misplaced meiotic recombination can have catastrophic consequences on genome stability. Within pericentromeres, COs are associated with meiotic chromosome missegregation. In organisms ranging from yeast to humans, pericentromeric COs are repressed. We previously identified a role for the kinetochore-associated Ctf19 complex (Ctf19c) in pericentromeric CO suppression. Here, we develop a dCas9/CRISPR-based system that allows ectopic targeting of Ctf19c-subunits. Using this approach, we query sufficiency in meiotic CO suppression, and identify Ctf19 as a mediator of kinetochore-associated CO control. The effect of Ctf19 is encoded in its NH2-terminal tail, and depends on residues important for the recruitment of the Scc2-Scc4 cohesin regulator. This work provides insight into kinetochore-derived control of meiotic recombination. We establish an experimental platform to investigate and manipulate meiotic CO control. This platform can easily be adapted in order to investigate other aspects of chromosome biology.
Asunto(s)
Intercambio Genético , Proteínas del Citoesqueleto/metabolismo , Cinetocoros/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Supresión Genética , Proteínas Cromosómicas no Histona/metabolismo , Proteínas del Citoesqueleto/química , Proteínas del Citoesqueleto/genética , Cinetocoros/química , Meiosis , Dominios Proteicos , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
The role of proteins often changes during evolution, but we do not know how cells adapt when a protein is asked to participate in a different biological function. We forced the budding yeast, Saccharomyces cerevisiae, to use the meiosis-specific kleisin, recombination 8 (Rec8), during the mitotic cell cycle, instead of its paralog, Scc1. This perturbation impairs sister chromosome linkage, advances the timing of genome replication, and reduces reproductive fitness by 45%. We evolved 15 parallel populations for 1,750 generations, substantially increasing their fitness, and analyzed the genotypes and phenotypes of the evolved cells. Only one population contained a mutation in Rec8, but many populations had mutations in the transcriptional mediator complex, cohesin-related genes, and cell cycle regulators that induce S phase. These mutations improve sister chromosome cohesion and delay genome replication in Rec8-expressing cells. We conclude that changes in known and novel partners allow cells to use an existing protein to participate in new biological functions.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Cromosomas Fúngicos/metabolismo , Mitosis , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Adaptación Biológica/genética , Proteínas de Ciclo Celular/genética , Proteínas Cromosómicas no Histona/genética , Cromosomas Fúngicos/genética , Evolución Molecular Dirigida , Evolución Molecular , Genoma Fúngico , Meiosis , Mutación , Origen de Réplica , Proteínas de Saccharomyces cerevisiae/genética , Intercambio de Cromátides Hermanas , CohesinasRESUMEN
A plethora of biological processes like gene transcription, DNA replication, DNA recombination, and chromosome segregation are mediated through protein-DNA interactions. A powerful method for investigating proteins within a native chromatin environment in the cell is chromatin immunoprecipitation (ChIP). Combined with the recent technological advancement in next generation sequencing, the ChIP assay can map the exact binding sites of a protein of interest across the entire genome. Here we describe a-step-by step protocol for ChIP followed by library preparation for ChIP-seq from yeast cells.
Asunto(s)
Inmunoprecipitación de Cromatina/métodos , Cromosomas Fúngicos/genética , Levaduras/genética , Sitios de Unión/genética , Cromatina/genética , Segregación Cromosómica/genética , Unión Proteica/genéticaRESUMEN
Cohesin is a ring-shaped protein complex that organises the genome, enabling its condensation, expression, repair and transmission. Cohesin is best known for its role in chromosome segregation, where it provides the cohesion that is established between the two newly duplicated sister chromatids during S phase. This cohesion enables the proper attachment of sister chromatids to microtubules of the spindle by resisting their opposing pulling forces. Once all chromosomes are correctly attached, cohesin is abruptly destroyed, triggering the equal segregation of sister chromatids to opposite poles in anaphase. Here we summarise the molecular functions and regulation of cohesin that underlie its central role in chromosome segregation during mitosis.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Cromátides/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Segregación Cromosómica/fisiología , Mitosis/fisiología , Saccharomyces cerevisiae/fisiología , Segregación Cromosómica/genética , Mitosis/genética , Saccharomyces cerevisiae/genética , CohesinasRESUMEN
Meiosis is a specialized cell division that generates gametes, such as eggs and sperm. Errors in meiosis result in miscarriages and are the leading cause of birth defects; however, the molecular origins of these defects remain unknown. Studies in model organisms are beginning to identify the genes and pathways important for meiosis, but the parts list is still poorly defined. Here we present a comprehensive catalog of genes important for meiosis in the fission yeast, Schizosaccharomyces pombe Our genome-wide functional screen surveyed all nonessential genes for roles in chromosome segregation and spore formation. Novel genes important at distinct stages of the meiotic chromosome segregation and differentiation program were identified. Preliminary characterization implicated three of these genes in centrosome/spindle pole body, centromere, and cohesion function. Our findings represent a near-complete parts list of genes important for meiosis in fission yeast, providing a valuable resource to advance our molecular understanding of meiosis.
Asunto(s)
Genoma Fúngico , Genómica , Meiosis/genética , Proteínas de Schizosaccharomyces pombe/genética , Schizosaccharomyces/genética , Segregación Cromosómica , Genómica/métodos , Viabilidad Microbiana/genética , Recombinación Genética , Intercambio de Cromátides Hermanas , Esporas FúngicasRESUMEN
The ring-shaped cohesin complex brings together distant DNA domains to maintain, express, and segregate the genome. Establishing specific chromosomal linkages depends on cohesin recruitment to defined loci. One such locus is the budding yeast centromere, which is a paradigm for targeted cohesin loading. The kinetochore, a multiprotein complex that connects centromeres to microtubules, drives the recruitment of high levels of cohesin to link sister chromatids together. We have exploited this system to determine the mechanism of specific cohesin recruitment. We show that phosphorylation of the Ctf19 kinetochore protein by a conserved kinase, DDK, provides a binding site for the Scc2/4 cohesin loading complex, thereby directing cohesin loading to centromeres. A similar mechanism targets cohesin to chromosomes in vertebrates. These findings represent a complete molecular description of targeted cohesin loading, a phenomenon with wide-ranging importance in chromosome segregation and, in multicellular organisms, transcription regulation.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Cinetocoros/metabolismo , Saccharomyces cerevisiae/metabolismo , Centrómero/metabolismo , Proteínas del Citoesqueleto/metabolismo , Complejos Multiproteicos/metabolismo , Fosforilación , Filogenia , Saccharomyces cerevisiae/citología , Proteínas de Saccharomyces cerevisiae/metabolismo , Difracción de Rayos X , CohesinasRESUMEN
The chromosomal passenger complex (CPC) is a key regulator of eukaryotic cell division, consisting of the protein kinase Aurora B/Ipl1 in association with its activator (INCENP/Sli15) and two additional proteins (Survivin/Bir1 and Borealin/Nbl1). Here, we report a genome-wide genetic interaction screen in Saccharomyces cerevisiae using the bir1-17 mutant, identifying through quantitative fitness analysis deletion mutations that act as enhancers and suppressors. Gene knockouts affecting the Ctf19 kinetochore complex were identified as the strongest enhancers of bir1-17, while mutations affecting the large ribosomal subunit or the mRNA nonsense-mediated decay pathway caused strong phenotypic suppression. Thus, cells lacking a functional Ctf19 complex become highly dependent on Bir1 function and vice versa. The negative genetic interaction profiles of bir1-17 and the cohesin mutant mcd1-1 showed considerable overlap, underlining the strong functional connection between sister chromatid cohesion and chromosome biorientation. Loss of some Ctf19 components, such as Iml3 or Chl4, impacted differentially on bir1-17 compared with mutations affecting other CPC components: despite the synthetic lethality shown by either iml3∆ or chl4∆ in combination with bir1-17, neither gene knockout showed any genetic interaction with either ipl1-321 or sli15-3 Our data therefore imply a specific functional connection between the Ctf19 complex and Bir1 that is not shared with Ipl1.
Asunto(s)
Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Aptitud Genética , Cinetocoros/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiología , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Epistasis Genética , Eliminación de Gen , Expresión Génica , Estudios de Asociación Genética , Viabilidad Microbiana/genética , Mutación , Fenotipo , Unión Proteica , CohesinasRESUMEN
The cohesin ring holds newly replicated sister chromatids together until their separation at anaphase. Initiation of sister chromatid cohesion depends on a separate complex, Scc2(NIPBL)/Scc4(Mau2) (Scc2/4), which loads cohesin onto DNA and determines its localization across the genome. Proper cohesin loading is essential for cell division, and partial defects cause chromosome missegregation and aberrant transcriptional regulation, leading to severe developmental defects in multicellular organisms. We present here a crystal structure showing the interaction between Scc2 and Scc4. Scc4 is a TPR array that envelops an extended Scc2 peptide. Using budding yeast, we demonstrate that a conserved patch on the surface of Scc4 is required to recruit Scc2/4 to centromeres and to build pericentromeric cohesion. These findings reveal the role of Scc4 in determining the localization of cohesin loading and establish a molecular basis for Scc2/4 recruitment to centromeres.
Asunto(s)
Proteínas Cromosómicas no Histona/química , Proteínas Cromosómicas no Histona/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Ciclo Celular/metabolismo , División Celular , Cromátides/metabolismo , Cristalografía por Rayos X , ADN de Hongos/metabolismo , Unión Proteica , Conformación Proteica , Saccharomyces cerevisiae/fisiología , CohesinasRESUMEN
The chromosomal passenger complex (CPC) is a key regulator of eukaryotic cell division, consisting of the protein kinase Aurora B/Ipl1 in association with its activator (INCENP/Sli15) and two additional proteins (Survivin/Bir1 and Borealin/Nbl1). Here we have identified multiple sites of CPC autophosphorylation on yeast Sli15 that are located within its central microtubule-binding domain and examined the functional significance of their phosphorylation by Ipl1 through mutation of these sites, either to non-phosphorylatable alanine (sli15-20A) or to acidic residues to mimic constitutive phosphorylation (sli15-20D). Both mutant sli15 alleles confer chromosome instability, but this is mediated neither by changes in the capacity of Sli15 to activate Ipl1 kinase nor by decreased efficiency of chromosome biorientation, a key process in cell division that requires CPC function. Instead, we find that mimicking constitutive phosphorylation of Sli15 on the Ipl1 phosphorylation sites causes delocalization of the CPC in metaphase, whereas blocking phosphorylation of Sli15 on the Ipl1 sites drives excessive localization of Sli15 to the mitotic spindle in pre-anaphase cells. Consistent with these results, direct interaction of Sli15 with microtubules in vitro is greatly reduced either following phosphorylation by Ipl1 or when constitutive phosphorylation at the Ipl1-dependent phosphorylation sites is mimicked by aspartate or glutamate substitutions. Furthermore, we find that mimicking Ipl1 phosphorylation of Sli15 interferes with the 'tension checkpoint'--the CPC-dependent mechanism through which cells activate the spindle assembly checkpoint to delay anaphase in the absence of tension on kinetochore-microtubule attachments. Ipl1-dependent phosphorylation of Sli15 therefore inhibits its association with microtubules both in vivo and in vitro and may negatively regulate the tension checkpoint mechanism.
Asunto(s)
División Celular/fisiología , Inestabilidad Cromosómica/fisiología , Complejos Multiproteicos/metabolismo , Saccharomyces cerevisiae/genética , Aurora Quinasas/metabolismo , Proteínas Portadoras/metabolismo , Microscopía Fluorescente , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Fosforilación , Saccharomyces cerevisiae/fisiología , Proteínas de Saccharomyces cerevisiae/metabolismo , Imagen de Lapso de TiempoRESUMEN
Ipl1/Aurora B is the catalytic subunit of a complex that is required for chromosome segregation and nuclear division. Before anaphase, Ipl1 localizes to kinetochores, where it is required to establish proper kinetochore-microtubule associations and regulate the spindle assembly checkpoint. The protein phosphatase Glc7/PP1 opposes Ipl1 for some of these activities. To more thoroughly characterize the Glc7 phosphatase that opposes Ipl1, we have identified mutations that suppress the thermosensitivity of an ipl1-2 mutant. In addition to mutations in genes previously associated with ipl1 suppression, we recovered a null mutant in TCO89, which encodes a subunit of the TOR complex 1 (TORC1), the conserved rapamycin-sensitive kinase activity that regulates cell growth in response to nutritional status. The temperature sensitivity of ipl1-2 can also be suppressed by null mutation of TOR1 or by administration of pharmacological TORC1 inhibitors, indicating that reduced TORC1 activity is responsible for the suppression. Suppression of the ipl1-2 growth defect is accompanied by increased fidelity of chromosome segregation and increased phosphorylation of the Ipl1 substrates histone H3 and Dam1. Nuclear Glc7 levels are reduced in a tco89 mutant, suggesting that TORC1 activity is required for the nuclear accumulation of Glc7. In addition, several mutant GLC7 alleles that suppress the temperature sensitivity of ipl1-2 exhibit negative synthetic genetic interactions with TORC1 mutants. Together, our results suggest that TORC1 positively regulates the Glc7 activity that opposes Ipl1 and provide a mechanism to tie nutritional status with mitotic regulation.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/genética , Mutación , Fosfatidilinositol 3-Quinasas/genética , Proteína Fosfatasa 1/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Aurora Quinasas , Núcleo Celular/metabolismo , Deleción Cromosómica , Cromosomas Fúngicos , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMEN
Accurate chromosome segregation requires the capture of sister kinetochores by microtubules from opposite spindle poles prior to the initiation of anaphase, a state termed chromosome biorientation. In the budding yeast Saccharomyces cerevisiae, the conserved protein kinase Ipl1 (Aurora B in metazoans) is critical for ensuring correct chromosomal alignment. Ipl1 associates with its activators Sli15 (INCENP), Nbl1 (Borealin), and Bir1 (Survivin), but while Sli15 clearly functions with Ipl1 to promote chromosome biorientation, the role of Bir1 has been uncertain. Using a temperature-sensitive bir1 mutant (bir1-17), we show that Bir1 is needed to permit efficient chromosome biorientation. However, once established, chromosome biorientation is maintained in bir1-17 cells at the restrictive temperature. Ipl1 is partially delocalized in bir1-17 cells, and its protein kinase activity is markedly reduced under nonpermissive conditions. bir1-17 cells arrest normally in response to microtubule depolymerization but fail to delay anaphase when sister kinetochore tension is reduced. Thus, Bir1 is required for the tension checkpoint. Despite their robust mitotic arrest in response to nocodazole, bir1-17 cells are hypersensitive to microtubule-depolymerizing drugs and show a more severe biorientation defect on recovery from nocodazole treatment. The role of Bir1 therefore may become more critical when spindle formation is delayed.
Asunto(s)
Cromosomas Fúngicos , Proteínas Inhibidoras de la Apoptosis/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/genética , Aurora Quinasas , Centrómero/metabolismo , Proteínas Inhibidoras de la Apoptosis/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Metafase/fisiología , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Nocodazol/farmacología , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Saccharomyces cerevisiae/efectos de los fármacos , Proteínas de Saccharomyces cerevisiae/genética , Moduladores de Tubulina/farmacologíaRESUMEN
In Saccharomyces cerevisiae, the serine-threonine protein kinase activity of Dbf2p is required for tolerance to the weak organic acid sorbic acid. Here we show that Dbf2p is required for normal phosphorylation of the vacuolar H(+)-ATPase (V-ATPase) A and B subunits Vma1p and Vma2p. Loss of V-ATPase activity due to bafilomycin treatment or deletion of either VMA1 or VMA2 resulted in sorbic acid hypersensitivity and impaired vacuolar acidification, phenotypes also observed in both a kinase-inactive dbf2 mutant and cells completely lacking DBF2 (dbf2Delta). Crucially, VMA2 is a multicopy suppressor of both the sorbic acid-sensitive phenotype and the impaired vacuolar-acidification defect of dbf2Delta cells, confirming a functional interaction between Dbf2p and Vma2p. The yeast V-ATPase is therefore involved in mediating sorbic acid stress tolerance, and we have shown a novel and unexpected role for the cell cycle-regulated protein kinase Dbf2p in promoting V-ATPase function.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Respuesta al Choque Térmico , Proteínas Quinasas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimología , Ácido Sórbico/farmacología , ATPasas de Translocación de Protón Vacuolares/metabolismo , Adaptación Fisiológica , Proteínas de Ciclo Celular/genética , Regulación Fúngica de la Expresión Génica , Proteínas Quinasas/genética , Proteínas Serina-Treonina Quinasas , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Proteínas de Saccharomyces cerevisiae/genética , ATPasas de Translocación de Protón Vacuolares/genéticaRESUMEN
DNA helicases are essential components of the cellular machinery for DNA replication, recombination, repair, and transcription. The XPD and FancJ proteins are related helicases involved in the nucleotide excision repair (NER) and Fanconi anemia repair pathways, respectively. We demonstrate that both proteins have a conserved domain near the N terminus that includes an iron-sulfur (Fe-S) cluster. Three absolutely conserved cysteine residues provide ligands for the Fe-S cluster, which is essential for the helicase activity of XPD. Yeast strains harboring mutations in the Fe-S domain of Rad3 (yeast XPD) are defective in excision repair of UV photoproducts. Clinically relevant mutations in patients with trichothiodystrophy (TTD) and Fanconi anemia disrupt the Fe-S clusters of XPD and FancJ and thereby abolish helicase activity.
Asunto(s)
Proteínas Arqueales/química , Enzimas Reparadoras del ADN/química , Proteínas del Grupo de Complementación de la Anemia de Fanconi/química , Proteínas Hierro-Azufre/química , Proteína de la Xerodermia Pigmentosa del Grupo D/química , Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Secuencia de Aminoácidos , Proteínas Arqueales/genética , Proteínas Arqueales/metabolismo , Secuencia Conservada , ADN Helicasas/química , ADN Helicasas/genética , ADN Helicasas/metabolismo , Reparación del ADN , Enzimas Reparadoras del ADN/genética , Enzimas Reparadoras del ADN/metabolismo , Escherichia coli/genética , Proteínas del Grupo de Complementación de la Anemia de Fanconi/genética , Proteínas del Grupo de Complementación de la Anemia de Fanconi/metabolismo , Proteínas Hierro-Azufre/genética , Proteínas Hierro-Azufre/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/efectos de la radiación , Proteínas de Saccharomyces cerevisiae , Alineación de Secuencia , Sulfolobus acidocaldarius/enzimología , Sulfolobus acidocaldarius/genética , Rayos Ultravioleta , Proteína de la Xerodermia Pigmentosa del Grupo D/genética , Proteína de la Xerodermia Pigmentosa del Grupo D/metabolismoRESUMEN
A combination of affinity purification, 2D-PAGE and peptide mass fingerprinting was employed to study the phosphoprotein complement of Saccharomyces cerevisiae. Protein extracts were first passed through a phosphoprotein affinity column, and the phosphoprotein-enriched eluate fractions were then separated on 2D gels and visualized by staining with SYPRO Ruby. Proteins were excised from the gels and identified by peptide mass fingerprinting; 11/13 protein spots identified from a gel of the phosphoprotein-enriched fraction had prior published evidence indicating that they were phosphoproteins. Additional experiments using a specific stain for phosphoproteins, prior incubation of the protein extract with alkaline phosphatase and blotting with monoclonal antibodies to phosphothreonine, phosphoserine and phosphotyrosine demonstrated that the phosphoprotein affinity column was an effective method for enriching phosphoproteins. Further validating the method, growth of yeast in the presence of sorbic acid resulted in altered phosphorylation of 17 proteins, 13 of which had prior published evidence that they were phosphoproteins or had ATP binding activity.