RESUMO
The segregation of centromeres and telomeres at mitosis is coordinated at multiple levels to prevent the formation of aneuploid cells, a phenotype frequently observed in cancer. Mitotic instability arises from chromosome segregation defects, giving rise to chromatin bridges at anaphase. Most of these defects are corrected before anaphase onset by a mechanism involving Aurora B kinase, a key regulator of mitosis in a wide range of organisms. Here, we describe a new role for Aurora B in telomere dispersion and disjunction during fission yeast mitosis. Telomere dispersion initiates in metaphase, whereas disjunction takes place in anaphase. Dispersion is promoted by the dissociation of Swi6/HP1 and cohesin Rad21 from telomeres, whereas disjunction occurs at anaphase after the phosphorylation of condensin subunit Cnd2. Strikingly, we demonstrate that deletion of Ccq1, a telomeric shelterin component, rescued cell death after Aurora inhibition by promoting the loading of condensin on chromosome arms. Our findings reveal an essential role for telomeres in chromosome arm segregation.
Assuntos
Aurora Quinases/fisiologia , Cromossomos Fúngicos/fisiologia , Proteínas de Schizosaccharomyces pombe/fisiologia , Schizosaccharomyces/enzimologia , Telômero/metabolismo , Adenosina Trifosfatases/metabolismo , Proteínas de Ciclo Celular/metabolismo , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Ligação a DNA/metabolismo , Mitose , Complexos Multiproteicos/metabolismo , Não Disjunção Genética , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Fosforilação , Processamento de Proteína Pós-Traducional , Schizosaccharomyces/citologia , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Complexo Shelterina , Fuso Acromático/metabolismo , Proteínas de Ligação a Telômeros/metabolismoRESUMO
Cytokinesis is the final stage of the cell cycle, and ensures completion of both genome segregation and organelle distribution to the daughter cells. Cytokinesis requires the cell to solve a spatial problem (to divide in the correct place, orthogonally to the plane of chromosome segregation) and a temporal problem (to coordinate cytokinesis with mitosis). Defects in the spatiotemporal control of cytokinesis may cause cell death, or increase the risk of tumor formation [Fujiwara et al., 2005 (Fujiwara T, Bandi M, Nitta M, Ivanova EV, Bronson RT, Pellman D. 2005. Cytokinesis failure generating tetraploids promotes tumorigenesis in p53-null cells. Nature 437:10431047); reviewed by Ganem et al., 2007 (Ganem NJ, Storchova Z, Pellman D. 2007. Tetraploidy, aneuploidy and cancer. Curr Opin Genet Dev 17:157162.)]. Asymmetric cytokinesis, which permits the generation of two daughter cells that differ in their shape, size and properties, is important both during development, and for cellular homeostasis in multicellular organisms [reviewed by Li, 2007 (Li R. 2007. Cytokinesis in development and disease: variations on a common theme. Cell Mol Life Sci 64:30443058)]. The principal focus of this review will be the mechanisms of cytokinesis in the mitotic cycle of the yeast Schizosaccharomyces pombe. This simple model has contributed significantly to our understanding of how the cell cycle is regulated, and serves as an excellent model for studying aspects of cytokinesis. Here we will discuss the state of our knowledge of how the contractile ring is assembled and disassembled, how it contracts, and what we know of the regulatory mechanisms that control these events and assure their coordination with chromosome segregation.
Assuntos
Segregação de Cromossomos/fisiologia , Cromossomos Fúngicos/fisiologia , Citocinese/fisiologia , Mitose/fisiologia , Modelos Biológicos , Schizosaccharomyces/fisiologiaRESUMO
The ribosomal RNA (rDNA) gene repeats are essential housekeeping genes found in all organisms. A gene amplification system maintains large cluster(s) of tandemly repeated copies in the chromosome, with each species having a specific number of copies. Yeast has many untranscribed rDNA copies (extra copies), and we found that when they are lost, the cells become sensitive to DNA damage induced by mutagens. We show that this sensitivity is dependent on rDNA transcriptional activity, which interferes with cohesion between rDNA loci of sister chromatids. The extra rDNA copies facilitate condensin association and sister-chromatid cohesion, thereby facilitating recombinational repair. These results suggest that high concentrations of heavily transcribed genes are toxic to the cells, and therefore amplified genes, such as rDNA, have evolved.
Assuntos
DNA Ribossômico/genética , Dosagem de Genes , Genes de RNAr , Genoma Fúngico , Instabilidade Genômica , Saccharomyces cerevisiae/genética , Adenosina Trifosfatases/metabolismo , Proteínas de Ciclo Celular/metabolismo , Cromátides/fisiologia , Proteínas Cromossômicas não Histona/metabolismo , Cromossomos Fúngicos/genética , Cromossomos Fúngicos/fisiologia , Dano ao DNA , Reparo do DNA , Replicação do DNA , DNA Fúngico/genética , Proteínas de Ligação a DNA/metabolismo , Amplificação de Genes , Genes Fúngicos , Metanossulfonato de Metila/farmacologia , Complexos Multiproteicos/metabolismo , Mutagênicos/farmacologia , RNA Fúngico/genética , RNA Fúngico/metabolismo , Recombinação Genética , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/crescimento & desenvolvimento , Transcrição Gênica , Regulação para Cima , CoesinasRESUMO
Replication factor C (RFC) plays a key role in eukaryotic chromosome replication by acting as a loading factor for the essential sliding clamp and polymerase processivity factor, proliferating cell nuclear antigen (PCNA). RFC is a pentamer comprising a large subunit, Rfc1, and four small subunits, Rfc2-Rfc5. Each RFC subunit is a member of the AAA+ family of ATPase and ATPase-like proteins, and the loading of PCNA onto double-stranded DNA is an ATP-dependent process. Here, we describe the properties of a collection of 38 mutant forms of the Rfc2 protein generated by pentapeptide-scanning mutagenesis of the fission yeast rfc2 gene. Each insertion was tested for its ability to support growth in fission yeast rfc2Delta cells lacking endogenous Rfc2 protein and the location of each insertion was mapped onto the 3D structure of budding yeast Rfc2. This analysis revealed that the majority of the inactivating mutations mapped in or adjacent to ATP sites C and D in Rfc2 (arginine finger and P-loop, respectively) or to the five-stranded beta sheet at the heart of the Rfc2 protein. By contrast, nonlethal mutations map predominantly to loop regions or to the outer surface of the RFC complex, often in highly conserved regions of the protein. Possible explanations for the effects of the various insertions are discussed.
Assuntos
Trifosfato de Adenosina/metabolismo , Modelos Moleculares , Oligopeptídeos/genética , Proteína de Replicação C/metabolismo , Schizosaccharomyces/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Sítios de Ligação , Cromossomos Fúngicos/fisiologia , Dados de Sequência Molecular , Mutagênese Insercional , Mutação , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteína de Replicação C/genética , Schizosaccharomyces/genéticaRESUMO
The structural maintenance of chromosome (SMC) proteins constitute the cores of three protein complexes involved in chromosome metabolism; cohesin, condensin and the Smc5-Smc6 complex. While the roles of cohesin and condensin in sister chromatid cohesion and chromosome condensation respectively have been described, the cellular function of Smc5-Smc6 is as yet not understood, consequently the less descriptive name. The complex is involved in a variety of DNA repair pathways. It contains activities reminiscent of those described for cohesin and condensin, as well as several DNA helicases and endonucleases. It is required for sister chromatid recombination, and smc5-smc6 mutants suffer from the accumulation of unscheduled recombination intermediates. The complex contains a SUMO-ligase and potentially an ubiquitin-ligase; thus Smc5-Smc6 might presently have a dull name, but it seems destined to be recognized as a key player in the maintenance of chromosome stability. In this review we summarize our present understanding of this enigmatic protein complex.
Assuntos
Proteínas de Ciclo Celular/fisiologia , Cromossomos/fisiologia , Complexos Multiproteicos/fisiologia , Adenosina Trifosfatases/fisiologia , Animais , Cromátides/fisiologia , Cromátides/ultraestrutura , Proteínas Cromossômicas não Histona/fisiologia , Cromossomos/ultraestrutura , Cromossomos Fúngicos/efeitos dos fármacos , Cromossomos Fúngicos/fisiologia , Cromossomos Fúngicos/efeitos da radiação , Cromossomos Fúngicos/ultraestrutura , Reparo do DNA/fisiologia , Replicação do DNA/fisiologia , DNA Fúngico/genética , DNA Ribossômico/genética , Proteínas de Ligação a DNA/fisiologia , Humanos , Recombinação Genética/fisiologia , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/fisiologia , Schizosaccharomyces/citologia , Schizosaccharomyces/efeitos dos fármacos , Schizosaccharomyces/genética , Schizosaccharomyces/efeitos da radiação , Proteínas de Schizosaccharomyces pombe/fisiologia , Ubiquitina-Proteína Ligases/fisiologia , Proteínas de Xenopus/fisiologia , Xenopus laevis/genética , CoesinasRESUMO
The protein complex including Mre11, Rad50, and Nbs1 (MRN) functions in DNA double-strand break repair to recognize and process DNA ends as well as signal for cell cycle arrest. Amino acid sequence similarity and overall architecture make Rad50 a member of the structural maintenance of chromosome (SMC) protein family. Like SMC proteins, Rad50 function depends on ATP binding and hydrolysis. All current evidence indicates that ATP binding and hydrolysis cause architectural rearrangements in SMC protein complexes that are important for their functions in organizing DNA. In the case of the MRN complex, the functional significance of ATP binding and hydrolysis are not yet defined. Here we review the data on the ATP-dependent activities of MRN and their possible mechanistic significance. We present some speculation on the role of ATP for function of the MRN complex based on the similarities and differences in the molecular architecture of the Rad50-containing complexes and the SMC complexes condensin and cohesin.
Assuntos
Trifosfato de Adenosina/fisiologia , Cromossomos/fisiologia , Enzimas Reparadoras do DNA/fisiologia , Reparo do DNA/fisiologia , Proteínas de Ligação a DNA/fisiologia , Complexos Multiproteicos/fisiologia , Proteínas de Saccharomyces cerevisiae/fisiologia , Proteínas de Schizosaccharomyces pombe/fisiologia , Hidrolases Anidrido Ácido , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/fisiologia , Ciclo Celular , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/fisiologia , Cromossomos/ultraestrutura , Cromossomos Fúngicos/fisiologia , Cromossomos Fúngicos/ultraestrutura , Quebras de DNA , Dano ao DNA , Enzimas Reparadoras do DNA/química , DNA Fúngico/genética , Proteínas de Ligação a DNA/química , Humanos , Mamíferos , Modelos Genéticos , Modelos Moleculares , Complexos Multiproteicos/química , Estrutura Terciária de Proteína , Recombinação Genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Schizosaccharomyces pombe/química , Leveduras/genéticaRESUMO
The initiation of chromosomal DNA replication is tightly regulated to achieve genome replication just once per cell cycle and cyclin-dependent kinase (CDK) plays an important role in this process. Adenine nucleotides that bind to the origin recognition complex (ORC) are also suggested to be involved in this process. Of the six subunits of the Saccharomyces cerevisiae ORC (Orc1-6p), both Orc1p and Orc5p have ATP binding activity, and both Orc2p and Orc6p are phosphorylated by CDK in cells. In this study we constructed a series of yeast strains expressing phospho-mimetic mutants of Orc2p or Orc6p and found that expression of a Ser-188 mutant of Orc2p (Orc2-5Dp) delays G1-S transition and S phase progression and causes the accumulation of cells with 2C DNA content. Using antibody that specifically recognizes Ser-188-phosphorylated Orc2p, we showed that Ser-188 is phosphorylated by CDK in a cell cycle-regulated manner. Expression of Orc2-5Dp caused phosphorylation of Rad53p and inefficient loading of the six minichromosome maintenance proteins. These results suggest that the accumulation of cells with 2C DNA content is due to inefficient origin firing and induction of the cell cycle checkpoint response and that dephosphorylation of Ser-188 of Orc2p in late M or G1 phase may be involved in pre-RC formation. In vitro, a purified mutant ORC containing Orc2-5Dp lost Orc5p ATP binding activity. This is the first demonstration of a link between phosphorylation of the ORC and its ability to bind ATP, which may be important for the cell cycle-regulated initiation of DNA replication.
Assuntos
Trifosfato de Adenosina/metabolismo , Proteínas de Ciclo Celular/metabolismo , Replicação do DNA/fisiologia , DNA Fúngico/metabolismo , Complexo de Reconhecimento de Origem/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Trifosfato de Adenosina/genética , Proteínas de Ciclo Celular/genética , Quinase do Ponto de Checagem 2 , Cromossomos Fúngicos/fisiologia , Quinases Ciclina-Dependentes/genética , Quinases Ciclina-Dependentes/metabolismo , DNA Fúngico/genética , Fase G1/fisiologia , Complexo de Reconhecimento de Origem/genética , Fosforilação/fisiologia , Ligação Proteica/fisiologia , Proteínas Serina-Treonina Quinases/genética , Fase S/fisiologia , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Condensin is the core activity responsible for chromosome condensation in mitosis. In the yeast S. cerevisiae, condensin binding is enriched at the regions where DNA replication terminates. Therefore, we investigated whether DNA replication completion determines the condensin-binding proficiency of chromatin. In order to fulfill putative mitotic requirements for condensin activity we analyzed chromosome condensation and condensin binding to unreplicated chromosomes in mitosis. For this purpose we used pGAL:CDC6 cdc15-ts cells that are known to enter mitosis without DNA replication if CDC6 transcription is repressed prior to S-phase. Both the condensation of nucleolar chromatin and proper condensin targeting to rDNA sites failed when unreplicated chromosomes were driven in mitosis. We propose that the DNA replication results in structural and/or biochemical changes to replicated chromatin, which are required for two-phase condensin binding and proper chromosome condensation.
Assuntos
Cromossomos Fúngicos/fisiologia , DNA Fúngico/genética , Mitose/fisiologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/fisiologia , Adenosina Trifosfatases/metabolismo , Cromossomos Fúngicos/genética , Replicação do DNA , DNA Fúngico/metabolismo , DNA Ribossômico/genética , DNA Ribossômico/metabolismo , Proteínas de Ligação a DNA/metabolismo , Complexos Multiproteicos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
The Spindle Assembly Checkpoint (SAC) delays the onset of anaphase until every chromosome is properly bioriented at the spindle equator. Mutations in SAC genes have been found in tumors and compromised SAC function can increase the incidence of some carcinomas in mice, providing further links between cancer etiology, chromosome segregation defects and aneuploidy. Here we review recent developments in our understanding of SAC control with particular emphasis on the role of the kinetochore, the nature of the tension sensing mechanism and the possibility that the SAC encompasses more than just stabilization of securin and/or cyclin-B via inhibition of the APC/C to delay anaphase initiation. Our primary emphasis is on the SAC in the budding yeast Saccharomyces cerevisiae. However, relevant findings in other cells are also discussed to highlight the generally conserved nature of SAC signaling mechanisms.
Assuntos
Cromatina/fisiologia , Cinetocoros/fisiologia , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/fisiologia , Fuso Acromático/fisiologia , Ciclo Celular , Cromátides/fisiologia , Cromossomos Fúngicos/genética , Cromossomos Fúngicos/fisiologia , DNA Topoisomerases Tipo II/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Estresse MecânicoRESUMO
The spindle checkpoint delays cell cycle progression until microtubules attach each pair of sister chromosomes to opposite poles of the mitotic spindle. Following sister chromatid separation, however, the checkpoint ignores chromosomes whose kinetochores are attached to only one spindle pole, a state that activates the checkpoint prior to metaphase. We demonstrate that, in budding yeast, mutual inhibition between the anaphase-promoting complex (APC) and Mps1, an essential component of the checkpoint, leads to sustained inactivation of the spindle checkpoint. Mps1 protein abundance decreases in anaphase, and Mps1 is a target of the APC. Furthermore, expression of Mps1 in anaphase, or repression of the APC in anaphase, reactivates the spindle checkpoint. This APC-Mps1 feedback circuit allows cells to irreversibly inactivate the checkpoint during anaphase.
Assuntos
Anáfase/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/citologia , Fuso Acromático/fisiologia , Complexos Ubiquitina-Proteína Ligase/metabolismo , Sequência de Aminoácidos , Ciclossomo-Complexo Promotor de Anáfase , Proteínas Cdc20 , Proteínas de Ciclo Celular/metabolismo , Cromossomos Fúngicos/fisiologia , Retroalimentação Fisiológica , Proteínas de Ligação ao GTP/metabolismo , Cinetocoros/fisiologia , Proteínas Mad2 , Mitose , Dados de Sequência Molecular , Proteínas Nucleares/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Proteínas Tirosina Quinases/genética , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , SecurinaRESUMO
SMC proteins are key components of large ring-shaped chromosomal protein complexes, such as cohesin and condensin. New evidence supports the idea that these rings topologically encircle DNA. Hints also emerge as to what it may take for DNA to enter the ring.
Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Cromossomos Fúngicos/fisiologia , Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Modelos Biológicos , Complexos Multiproteicos/metabolismo , Proteínas Nucleares/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Schizosaccharomyces , CoesinasRESUMO
During meiosis, segregation of homologous chromosomes necessitates the coordination of sister chromatid cohesion, chromosome condensation, and recombination. Cohesion and condensation require the SMC complexes, cohesin and condensin, respectively. Here we use budding yeast Saccharomyces cerevisiae to show that condensin and Cdc5, a Polo-like kinase, facilitate the removal of cohesin from chromosomes prior to the onset of anaphase I when homologs segregate. This cohesin removal is critical for homolog segregation because it helps dissolve the recombination-dependent links between homologs that form during prophase I. Condensin enhances the association of Cdc5 with chromosomes and its phosphorylation of cohesin, which in turn likely stimulates cohesin removal. Condensin/Cdc5-dependent removal of cohesin underscores the potential importance of crosstalk between chromosome structural components in chromosome morphogenesis and provides a mechanism to couple chromosome morphogenesis with other meiotic events.
Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Ciclo Celular/metabolismo , Cromossomos Fúngicos/fisiologia , Proteínas de Ligação a DNA/metabolismo , Proteínas Fúngicas/metabolismo , Meiose , Complexos Multiproteicos/metabolismo , Proteínas Nucleares/metabolismo , Saccharomyces cerevisiae/fisiologia , Adenosina Trifosfatases/genética , Anáfase , Proteínas de Ciclo Celular/genética , Proteínas Cromossômicas não Histona/metabolismo , Segregação de Cromossomos , Proteínas de Ligação a DNA/genética , Ativação Enzimática , Proteínas Fúngicas/genética , Complexos Multiproteicos/genética , Mutação , Proteínas Nucleares/genética , Fosforilação , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Troca de Cromátide Irmã , CoesinasAssuntos
Segregação de Cromossomos , Fuso Acromático/fisiologia , Animais , Proteínas de Ciclo Celular/fisiologia , Transformação Celular Neoplásica/genética , Cromossomos Fúngicos/fisiologia , Proteínas Fúngicas , Cinetocoros/fisiologia , Modelos Genéticos , Mutação , Proteínas Nucleares , Fosforilação , Proteínas Quinases/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae , Schizosaccharomyces/citologia , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombeRESUMO
Cell division involves the inheritance of a complete set of the genome in the form of chromosomes. One of the strategies employed by eukaryotic cells is to maintain replicated sister chromatids together until the anaphase onset. A protein complex named cohesin holds sisters together following replication until anaphase when cleavage of cohesin by the protease separase initiates segregation. Recent studies in budding yeast have shown that cohesin cleavage alone is not sufficient for the segregation of the entire genome. Instead, repetitive regions, such as the ribosomal DNA (rDNA) array and telomeres, require additional mechanisms during mitotic disjunction. The segregation of such chromosome regions is delayed and needs specific cell cycle regulators such as the FEAR network and the conserved phosphatase Cdc14, all of which orchestrate the timely completion of chromosome segregation before mitotic exit. Future studies will be targeted towards unravelling the nature of the additional segregation requirements for repetitive regions and the specifics of its cell cycle control.
Assuntos
Proteínas de Ciclo Celular/fisiologia , Segregação de Cromossomos/fisiologia , Mitose/fisiologia , Proteínas Tirosina Fosfatases/fisiologia , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/citologia , Saccharomycetales/citologia , Adenosina Trifosfatases/fisiologia , Anáfase/fisiologia , Proteínas Cromossômicas não Histona , Segregação de Cromossomos/genética , Cromossomos Fúngicos/genética , Cromossomos Fúngicos/fisiologia , DNA Ribossômico/genética , DNA Ribossômico/fisiologia , Proteínas de Ligação a DNA/fisiologia , Endopeptidases/fisiologia , Ativação Enzimática/fisiologia , Proteínas Fúngicas/fisiologia , Mitose/genética , Complexos Multiproteicos/fisiologia , Proteínas Nucleares/fisiologia , Saccharomyces cerevisiae/fisiologia , Saccharomycetales/fisiologia , Separase , Telômero/fisiologia , Transcrição Gênica , CoesinasRESUMO
The Ssn6-Tup1 corepressor complex regulates many genes in Saccharomyces cerevisiae. Three mechanisms have been proposed to explain its repression functions: 1) nucleosome positioning by binding histone tails; 2) recruitment of histone deacetylases; and 3) direct interference with the general transcription machinery or activators. It is unclear if Ssn6-Tup1 utilizes each of these mechanisms at a single gene in a redundant manner or each individually at different loci. A systematic analysis of the contribution of each mechanism at a native promoter has not been reported. Here we employed a genetic strategy to analyze the contributions of nucleosome positioning, histone deacetylation, and Mediator interference in the repression of chromosomal Tup1 target genes in vivo. We exploited the fact that Ssn6-Tup1 requires the ISW2 chromatin remodeling complex to establish nucleosome positioning in vivo to disrupt chromatin structure without affecting other Tup1 repression functions. Deleting ISW2, the histone deacetylase gene HDA1, or genes encoding Mediator subunits individually caused slight or no derepression of RNR3 and HUG1. However, when Mediator mutations were combined with Deltaisw2 or Deltahda1 mutations, enhanced transcription was observed, and the strongest level of derepression was observed in triple Deltaisw2/Deltahda1/Mediator mutants. The increased transcription in the mutants was not due to the loss of Tup1 at the promoter and correlated with increased TBP cross-linking to promoters. Thus, Tup1 utilizes multiple redundant mechanisms to repress transcription of native genes, which may be important for it to act as a global corepressor at a wide variety of promoters.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Acetilação , Adenosina Trifosfatases/metabolismo , Cromatina/fisiologia , Cromossomos Fúngicos/fisiologia , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Histona Desacetilases/metabolismo , Histonas/metabolismo , Nucleossomos/fisiologia , Fatores de Transcrição/metabolismo , Transcrição Gênica/fisiologiaRESUMO
Haploid Saccharomyces can change mating type through HO-endonuclease cleavage of an expressor locus, MAT, followed by gene conversion using one of two repository loci, HML or HMR, as donor. The mating type of a cell dictates which repository locus is used as donor, with a cells using HML and alpha cells using HMR. This preference is established in part by RE, a locus on the left arm of chromosome III that activates the surrounding region, including HML, for recombination in a cells, an activity suppressed by alpha 2 protein in alpha cells. We have examined the ability of RE to stimulate different forms of interchromosomal recombination. We found that RE exerted an effect on interchromosomal mating-type switching and on intrachromosomal homologous recombination but not on interchromosomal homologous recombination. Also, even in the absence of RE, MAT alpha still influenced donor preference in interchromosomal mating-type switching, supporting a role of alpha 2 in donor preference independent of RE. These results suggest a model in which RE affects competition between productive and nonproductive recombination outcomes. In interchromosome gene conversion, RE enhances both productive and nonproductive pathways, whereas in intrachromosomal gene conversion and mating-type switching, RE enhances only the productive pathway.
Assuntos
Cromossomos Fúngicos/fisiologia , Elementos Facilitadores Genéticos , Recombinação Genética , Saccharomyces cerevisiae/genética , Conversão Gênica , Regulação Fúngica da Expressão Gênica , Genes de Troca , Haploidia , Fator de Acasalamento , Modelos Genéticos , PeptídeosRESUMO
Meiosis is a specialized cell division in which two chromosome segregation phases follow a single DNA replication phase. The budding yeast Polo-like kinase Cdc5 was found to be instrumental in establishing the meiosis I chromosome segregation program. Cdc5 was required to phosphorylate and remove meiotic cohesin from chromosomes. Furthermore, in the absence of CDC5 kinetochores were bioriented during meiosis I, and Mam1, a protein essential for coorientation, failed to associate with kinetochores. Thus, sister-kinetochore coorientation and chromosome segregation during meiosis I are coupled through their dependence on CDC5.
Assuntos
Segregação de Cromossomos , Cromossomos Fúngicos/fisiologia , Meiose , Proteínas Quinases/metabolismo , Saccharomyces cerevisiae/fisiologia , Anáfase , Proteínas Cdc20 , Proteínas de Ciclo Celular/metabolismo , Núcleo Celular/metabolismo , Centrômero/fisiologia , Cromátides/fisiologia , Cinetocoros/fisiologia , Metáfase , Proteínas Nucleares/metabolismo , Fosforilação , Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , SecurinaRESUMO
Little is known about the dynamics of chromosomes in interphase nuclei. By tagging four chromosomal regions with a green fluorescent protein fusion to lac repressor, we monitored the movement and subnuclear position of specific sites in the yeast genome, sampling at short time intervals. We found that early and late origins of replication are highly mobile in G1 phase, frequently moving at or faster than 0.5 micrometers/10 seconds, in an energy-dependent fashion. The rapid diffusive movement of chromatin detected in G1 becomes constrained in S phase through a mechanism dependent on active DNA replication. In contrast, telomeres and centromeres provide replication-independent constraint on chromatin movement in both G1 and S phases.
Assuntos
Cromatina/fisiologia , Cromossomos Fúngicos/fisiologia , Interfase , Saccharomyces cerevisiae/fisiologia , Trifosfato de Adenosina/metabolismo , Núcleo Celular/fisiologia , Centrômero/fisiologia , Replicação do DNA , DNA Fúngico/biossíntese , Fase G1 , Proteínas de Fluorescência Verde , Proteínas Luminescentes , Filmes Cinematográficos , Mutação , Membrana Nuclear/fisiologia , Origem de Replicação , Fase S , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Telômero/fisiologiaRESUMO
BACKGROUND: In mammals, proteins containing BIR domains (IAPs and survivin) are implicated in inhibiting apoptosis and sister chromatid separation. In the nematode, Bir1 is required for a proper localization of aurora kinase, which moves from the mitotic chromosome in metaphase to the spindle midzone in anaphase as a passenger. Fission yeast Bir1/Pbh1 is essential for normal mitosis. RESULTS: A temperature sensitive mutant cut17-275 exhibits the defect in condensation and spindle elongation at 36 degrees C, while securin is degraded. Gene cloning shows that the cut17+ gene is identical to bir1+/pbh1+. At 26 degrees C, cut17-275 is UV sensitive as the repair of DNA damage is severely compromised. Bir1/Cut17 is a nuclear protein in interphase, which is then required for recruiting condensin to the mitotic nucleus, and concentrates to form a discrete number of dots from prometaphase to metaphase. Once the chromatids are separated, Bir1/Cut17 no longer binds to kinetochores and instead moves to the middle of spindle. Chromatin immunoprecipitation suggested that Bir1/Cut17 associates with the outer repetitious centromere region in metaphase. Following the initiation of anaphase the protein switches from being a chromosomal protein to a spindle protein. This transit is stringently regulated by the state of sister chromatid cohesion proteins Mis4 and Rad21. Ark1, is an aurora kinase homologue whose mitotic distribution is identical to, and under the control of Bir1/Cut17. CONCLUSIONS: Bir1/Cut17 and Ark1 act as "passengers" but they may play a main role as a recruitment factor, essential for condensation, spindle elongation and DNA repair. Bir1/Cut17 should have roles both in mitotic and in interphase chromosome. The proper location of Ark1 requires Bir1/Cut17, and the mitotic localization of Bir1/Cut17 requires sister cohesion.
Assuntos
Cromossomos Fúngicos/fisiologia , Reparo do DNA/fisiologia , Proteínas Fúngicas/fisiologia , Schizosaccharomyces/genética , Fuso Acromático/fisiologia , Sequência de Aminoácidos , Animais , Aurora Quinases , Sobrevivência Celular , Células Cultivadas , DNA Fúngico/análise , Corantes Fluorescentes , Hidroxiureia/farmacologia , Hibridização in Situ Fluorescente , Indóis , Interfase/genética , Dados de Sequência Molecular , Mutação , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , Proteínas Proto-Oncogênicas c-myc/metabolismo , Homologia de Sequência de Aminoácidos , Temperatura , Tiabendazol/farmacologia , Raios UltravioletaRESUMO
CTF4 and CTF18 are required for high-fidelity chromosome segregation. Both exhibit genetic and physical ties to replication fork constituents. We find that absence of either CTF4 or CTF18 causes sister chromatid cohesion failure and leads to a preanaphase accumulation of cells that depends on the spindle assembly checkpoint. The physical and genetic interactions between CTF4, CTF18, and core components of replication fork complexes observed in this study and others suggest that both gene products act in association with the replication fork to facilitate sister chromatid cohesion. We find that Ctf18p, an RFC1-like protein, directly interacts with Rfc2p, Rfc3p, Rfc4p, and Rfc5p. However, Ctf18p is not a component of biochemically purified proliferating cell nuclear antigen loading RF-C, suggesting the presence of a discrete complex containing Ctf18p, Rfc2p, Rfc3p, Rfc4p, and Rfc5p. Recent identification and characterization of the budding yeast polymerase kappa, encoded by TRF4, strongly supports a hypothesis that the DNA replication machinery is required for proper sister chromatid cohesion. Analogous to the polymerase switching role of the bacterial and human RF-C complexes, we propose that budding yeast RF-C(CTF18) may be involved in a polymerase switch event that facilities sister chromatid cohesion. The requirement for CTF4 and CTF18 in robust cohesion identifies novel roles for replication accessory proteins in this process.