RESUMO
All organisms have evolved pathways to respond to different forms of cellular stress. The Gcn2 kinase is best known as a regulator of translation initiation in response to starvation for amino acids. Work in budding yeast has showed that the molecular mechanism of GCN2 activation involves the binding of uncharged tRNAs, which results in a conformational change and GCN2 activation. This pathway requires GCN1, which ensures delivery of the uncharged tRNA onto GCN2. However, Gcn2 is activated by a number of other stresses which do not obviously involve accumulation of uncharged tRNAs, raising the question how Gcn2 is activated under these conditions. Here we investigate the requirement for ongoing translation and tRNA binding for Gcn2 activation after different stresses in fission yeast. We find that mutating the tRNA-binding site on Gcn2 or deleting Gcn1 abolishes Gcn2 activation under all the investigated conditions. These results suggest that tRNA binding to Gcn2 is required for Gcn2 activation not only in response to starvation but also after UV irradiation and oxidative stress.
Assuntos
Proteínas Serina-Treonina Quinases/metabolismo , RNA de Transferência/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Estresse Fisiológico/genética , Sequência de Aminoácidos , Cicloeximida/farmacologia , Fator de Iniciação 2 em Eucariotos/metabolismo , Peróxido de Hidrogênio/toxicidade , Mutagênese , Estresse Oxidativo/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Fosforilação/efeitos da radiação , Biossíntese de Proteínas/efeitos da radiação , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genética , Inibidores da Síntese de Proteínas/farmacologia , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Schizosaccharomyces/efeitos da radiação , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/genética , Alinhamento de Sequência , Raios UltravioletaRESUMO
Here, we describe how the rapid reversibility of the nda3-KM311 cold-sensitive ß-tubulin mutation was optimized by Mitsuhiro Yanagida's laboratory to synchronize mitotic progression in an entire cell population. The inability to form microtubules following the loss of ß-tubulin function at 20°C triggers the spindle assembly checkpoint, which arrests mitotic progression. Restoration of ß-tubulin function by rewarming to 30°C (or higher) releases the arrest, generating a highly synchronous progression through mitosis. The viability of nda3-KM311 strains at 30°C makes it feasible to generate double mutants between nda3-KM311 and any temperature-sensitive mutant that can also grow at 30°C. These double mutants can be used in reciprocal shift analyses, in which cold-induced early mitotic arrest is relieved by a shift to 36°C, which then inactivates the product of the second mutant gene. The addition of microtubule depolymerizing drugs before the return to 36°C will maintain checkpoint signaling at 36°C transiently, permitting analysis of the impact of temperature-sensitive mutations on checkpoint function. Silencing the checkpoint of nda3-KM311-arrested cells at 20°C through chemical inhibition of aurora kinase is a powerful way to study checkpoint recovery pathways and mitotic exit without anaphase.
Assuntos
Temperatura Baixa , Proteínas Fúngicas/metabolismo , Prófase , Fase S , Schizosaccharomyces/fisiologia , Schizosaccharomyces/efeitos da radiação , Proteínas Fúngicas/genética , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Schizosaccharomyces/genética , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismoRESUMO
Transient inactivation of the cdc25(+) gene product by manipulation of the culture temperature for cdc25-22 cells is the most commonly exploited approach to mitotic synchronization in fission yeast. Because Cdc25 removes the inhibitory phosphate placed on Cdk1 by Wee1, inactivation of Cdc25 arrests cells at the G2/M boundary. Incubation at the restrictive temperature of 36°C for just over one generation time forces all cells in the culture to accumulate at the G2/M boundary. Restoration of Cdc25 function via a return to the permissive temperature or chemical inhibition of Wee1 activity at 36°C can then promote a highly synchronous wave of cell division throughout the culture. These approaches can be performed on any scale and thus support simultaneous assessment of numerous events within a single culture. After describing this simple and widely applicable procedure, we discuss frequently overlooked issues that can have a considerable impact on the interpretation of data from cdc25-22 induction-synchronized cultures.
Assuntos
Proteínas Fúngicas/metabolismo , Fase G2 , Fase S , Schizosaccharomyces/fisiologia , Schizosaccharomyces/efeitos da radiação , Temperatura , Fosfatases cdc25/metabolismo , Proteínas Fúngicas/genética , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Schizosaccharomyces/genética , Fosfatases cdc25/genéticaRESUMO
The formation of healthy gametes requires pairing of homologous chromosomes (homologs) as a prerequisite for their correct segregation during meiosis. Initially, homolog alignment is promoted by meiotic chromosome movements feeding into intimate homolog pairing by homologous recombination and/or synaptonemal complex formation. Meiotic chromosome movements in the fission yeast, Schizosaccharomyces pombe, depend on astral microtubule dynamics that drag the nucleus through the zygote; known as horsetail movement. The response of microtubule-led meiotic chromosome movements to environmental stresses such as ionizing irradiation (IR) and associated reactive oxygen species (ROS) is not known. Here, we show that, in contrast to budding yeast, the horsetail movement is largely radiation-resistant, which is likely mediated by a potent antioxidant defense. IR exposure of sporulating S. pombe cells induced misrepair and irreparable DNA double strand breaks causing chromosome fragmentation, missegregation and gamete death. Comparing radiation outcome in fission and budding yeast, and studying meiosis with poisoned microtubules indicates that the increased gamete death after IR is innate to fission yeast. Inhibition of meiotic chromosome mobility in the face of IR failed to influence the course of DSB repair, indicating that paralysis of meiotic chromosome mobility in a genotoxic environment is not a universal response among species.
Assuntos
Segregação de Cromossomos , Meiose , Radiação Ionizante , Schizosaccharomyces/crescimento & desenvolvimento , Schizosaccharomyces/efeitos da radiação , Exposição Ambiental , Espécies Reativas de Oxigênio/metabolismo , Schizosaccharomyces/efeitos dos fármacosRESUMO
DNA double strand breaks (DSBs) are the most critical types of DNA damage that can leads to chromosomal aberrations, genomic instability and cancer. Several genetic disorders such as Xeroderma pigmentosum are linked with defects in DNA repair. Human Rint1, a TIP1 domain containing protein is involved in membrane trafficking but its role in DNA damage response is elusive. In this study we characterized the role of Drp1 (damage responsive protein 1), a Rint1 family protein during DNA damage response in fission yeast. We identified that Drp1 is an essential protein and indispensable for survival and growth. Using in vitro random mutagenesis approach we isolated a temperature sensitive mutant allele of drp1 gene (drp1-654) that exhibits sensitivity to DNA damaging agents, in particular to alkylation damage and UV associated DNA damage. The drp1-654 mutant cells are also sensitive to double strand break inducing agent bleomycin. Genetic interaction studies identified that Rad50 and Drp1 act in the same pathway during DNA damage response and the physical interaction of Drp1 with Rad50 was unaffected in drp1-654 mutant at permissive as well as non permissive temperature. Furthermore Drp1 was found to be required for the recovery from MMS induced DNA damage. We also demonstrated that the Drp1 protein localized to nucleus and was required to maintain the chromosome stability.
Assuntos
Segregação de Cromossomos , Dano ao DNA , Proteínas de Saccharomyces cerevisiae/metabolismo , Schizosaccharomyces/genética , Sequência de Aminoácidos , Bleomicina/farmacologia , Instabilidade Cromossômica , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Metanossulfonato de Metila/farmacologia , Dados de Sequência Molecular , Proteínas de Saccharomyces cerevisiae/genética , Schizosaccharomyces/efeitos dos fármacos , Schizosaccharomyces/efeitos da radiação , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Temperatura , Raios UltravioletaRESUMO
The clade of Schizosaccharomyces includes 4 species: S. pombe, S. octosporus, S. cryophilus, and S. japonicus. Although all 4 species exhibit unicellular growth with a binary fission mode of cell division, S. japonicus alone is dimorphic yeast, which can transit from unicellular yeast to long filamentous hyphae. Recently it was found that the hyphal cells response to light and then synchronously activate cytokinesis of hyphae. In addition to hyphal growth, S. japonicas has many properties that aren't shared with other fission yeast. Mitosis of S. japonicas is referred to as semi-open mitosis because dynamics of nuclear membrane is an intermediate mode between open mitosis and closed mitosis. Novel genetic tools and the whole genomic sequencing of S. japonicas now provide us with an opportunity for revealing unique characters of the dimorphic yeast.
Assuntos
Divisão Celular , Hifas/fisiologia , Schizosaccharomyces/fisiologia , Ritmo Circadiano , Genes Fúngicos Tipo Acasalamento , Hifas/citologia , Hifas/genética , Hifas/efeitos da radiação , Luz , Meiose , Mitose , Filogenia , Schizosaccharomyces/citologia , Schizosaccharomyces/genética , Schizosaccharomyces/efeitos da radiaçãoRESUMO
The pathogenic fungus Cryptococcus neoformans uses the Bwc1-Bwc2 photoreceptor complex to regulate mating in response to light, virulence and ultraviolet radiation tolerance. How the complex controls these functions is unclear. Here, we identify and characterize a gene in Cryptococcus, UVE1, whose mutation leads to a UV hypersensitive phenotype. The homologous gene in fission yeast Schizosaccharomyces pombe encodes an apurinic/apyrimidinic endonuclease acting in the UVDE-dependent excision repair (UVER) pathway. C. neoformans UVE1 complements a S. pombe uvde knockout strain. UVE1 is photoregulated in a Bwc1-dependent manner in Cryptococcus, and in Neurospora crassa and Phycomyces blakesleeanus that are species that represent two other major lineages in the fungi. Overexpression of UVE1 in bwc1 mutants rescues their UV sensitivity phenotype and gel mobility shift experiments show binding of Bwc2 to the UVE1 promoter, indicating that UVE1 is a direct downstream target for the Bwc1-Bwc2 complex. Uve1-GFP fusions localize to the mitochondria. Repair of UV-induced damage to the mitochondria is delayed in the uve1 mutant strain. Thus, in C. neoformans UVE1 is a key gene regulated in response to light that is responsible for tolerance to UV stress for protection of the mitochondrial genome.
Assuntos
Cryptococcus neoformans/efeitos dos fármacos , Endodesoxirribonucleases/genética , Genoma Mitocondrial/genética , Hipersensibilidade/genética , Proteínas de Schizosaccharomyces pombe/genética , Cryptococcus neoformans/genética , Cryptococcus neoformans/efeitos da radiação , Dano ao DNA/efeitos da radiação , DNA Fúngico/genética , DNA Fúngico/efeitos da radiação , Endodesoxirribonucleases/metabolismo , Técnicas de Inativação de Genes , Genoma Mitocondrial/efeitos da radiação , Mutação , Neurospora crassa/genética , Neurospora crassa/efeitos da radiação , Phycomyces/genética , Phycomyces/efeitos da radiação , Schizosaccharomyces/genética , Schizosaccharomyces/efeitos da radiação , Proteínas de Schizosaccharomyces pombe/metabolismo , Raios UltravioletaRESUMO
Rad52 is a key player in homologous recombination (HR), a DNA repair pathway that is dedicated to double strand breaks repair and recovery of perturbed replication forks. Here we show that fission yeast Rad52 homologue is phosphorylated when S phase cells are exposed to ROS inducers such as ultraviolet A radiation or hydrogen peroxide, but not to ultraviolet C or camptothecin. Phosphorylation does not depend on kinases Chk1, Rad3, Tel1 or Cdc2, but depends on a functional stress activated protein kinase (SAPK) pathway and can be partially prevented by anti-oxidant treatment. Indeed, cells lacking Sty1, the major fission yeast MAP kinase of the SAPK pathway, do not display Rad52 phosphorylation and have UVA induced Rad52 foci that persist longer if compared to wild type cells. In addition, spontaneous intrachromosomal HR is diminished in cells lacking Sty1 and, more precisely, gene conversion is affected. Moreover, HR induced by site-specific arrest of replication forks is twice less efficient in cells that do not express Sty1. Importantly, impairing HR by deletion of the gene encoding the recombinase Rhp51 leads to Sty1 dependent Rad52 phosphorylation. Thus, SAPK pathway impinges on early step of HR through phosphorylation of Rad52 in cells challenged by oxidative stress or lacking Rhp51 and is required to promote spontaneous gene conversion and recovery from blocked replication forks.
Assuntos
Recombinação Homóloga/efeitos da radiação , Sistema de Sinalização das MAP Quinases/efeitos da radiação , Schizosaccharomyces/metabolismo , Camptotecina/farmacologia , Replicação do DNA , Conversão Gênica , Proteínas de Choque Térmico/metabolismo , Peróxido de Hidrogênio/farmacologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Oxidantes/farmacologia , Estresse Oxidativo , Fosforilação , Proteínas Quinases/metabolismo , Processamento de Proteína Pós-Traducional/efeitos da radiação , Rad51 Recombinase/metabolismo , Proteína Rad52 de Recombinação e Reparo de DNA/metabolismo , Fase S , Schizosaccharomyces/genética , Schizosaccharomyces/efeitos da radiação , Proteínas de Schizosaccharomyces pombe/metabolismo , Inibidores da Topoisomerase I/farmacologia , Raios UltravioletaRESUMO
Biosynthesis of coenzyme A (CoA) requires a five-step process using pantothenate and cysteine in the fission yeast Schizosaccharomyces pombe. CoA contains a thiol (SH) group, which reacts with carboxylic acid to form thioesters, giving rise to acyl-activated CoAs such as acetyl-CoA. Acetyl-CoA is essential for energy metabolism and protein acetylation, and, in higher eukaryotes, for the production of neurotransmitters. We isolated a novel S. pombe temperature-sensitive strain ppc1-537 mutated in the catalytic region of phosphopantothenoylcysteine synthetase (designated Ppc1), which is essential for CoA synthesis. The mutant becomes auxotrophic to pantothenate at permissive temperature, displaying greatly decreased levels of CoA, acetyl-CoA and histone acetylation. Moreover, ppc1-537 mutant cells failed to restore proliferation from quiescence. Ppc1 is thus the product of a super-housekeeping gene. The ppc1-537 mutant showed combined synthetic lethal defects with five of six histone deacetylase mutants, whereas sir2 deletion exceptionally rescued the ppc1-537 phenotype. In synchronous cultures, ppc1-537 cells can proceed to the S phase, but lose viability during mitosis failing in sister centromere/kinetochore segregation and nuclear division. Additionally, double-strand break repair is defective in the ppc1-537 mutant, producing fragile broken DNA, probably owing to diminished histone acetylation. The CoA-supported metabolism thus controls the state of chromosome DNA.
Assuntos
Coenzima A/biossíntese , Histonas/metabolismo , Schizosaccharomyces/citologia , Schizosaccharomyces/metabolismo , Acetilação , Sequência de Aminoácidos , Pontos de Checagem do Ciclo Celular , Quebras de DNA de Cadeia Dupla , Reparo do DNA , DNA Fúngico/metabolismo , Genes Fúngicos , Histona Acetiltransferases/genética , Histona Acetiltransferases/metabolismo , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Mitose , Dados de Sequência Molecular , Mutação , Peptídeo Sintases/química , Peptídeo Sintases/genética , Peptídeo Sintases/metabolismo , Tolerância a Radiação , Schizosaccharomyces/genética , Schizosaccharomyces/efeitos da radiação , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Raios UltravioletaRESUMO
Although the G2/M DNA damage checkpoint is currently viewed as a set of coordinated cellular responses affecting both cell cycle progression and non-cell cycle targets, the relative contributions of the two target categories to DNA repair and cell survival after exposure to ionizing radiation have not been clearly addressed. We investigated how rad3 (ATR ortholog) or chk1/cds1 (CHK1/CHK2 orthologs) null mutations change the kinetics of double-strand break (DSB) repair in Schizosaccharomyces pombe cells under conditions of forced G2 arrest. After 200-Gy γ-ray irradiation, DSBs were repaired in rad3Δ cdc25-22 or chk1Δ cds1Δ cdc25-22 cells, almost as efficiently as in cdc25-22 cells at the restrictive temperature. In contrast, little repair was observed in the checkpoint-deficient cells up to 4h after higher-dose (500Gy) irradiation, whereas repair was still efficient in the control cdc25-22 cells. Immediate loss of viability appeared not be responsible for the repair defect after the higher dose, since both checkpoint-proficient and deficient cells with cdc25-22 allele synchronously resumed cycling with a similar time course when released to the permissive temperature 4h after irradiation. Recruitment of repair proteins Rad11 (Rpa1 ortholog), Rad22 (Rad52 ortholog), and Rhp54 (Rad54 ortholog) to the damage sites was not significantly impaired in the checkpoint-deficient cells, whereas their release was profoundly delayed. Our results suggest that sensor and effector kinases in the damage checkpoint machinery affect the efficiency of repair downstream of, or in parallel with the core repair reaction.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Dano ao DNA/fisiologia , Pontos de Checagem da Fase G2 do Ciclo Celular/fisiologia , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Reparo de DNA por Recombinação/fisiologia , Proteínas de Schizosaccharomyces pombe/metabolismo , Proteínas de Ciclo Celular/genética , Quinase 1 do Ponto de Checagem , Quinase do Ponto de Checagem 2 , Quebras de DNA de Cadeia Dupla/efeitos da radiação , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Raios gama/efeitos adversos , Deleção de Genes , Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases/genética , Schizosaccharomyces/fisiologia , Schizosaccharomyces/efeitos da radiação , Proteínas de Schizosaccharomyces pombe/genéticaRESUMO
Cdt1 plays a critical role in DNA replication regulation by controlling licensing. In Metazoa, Cdt1 is regulated by CRL4(Cdt2)-mediated ubiquitylation, which is triggered by DNA binding of proliferating cell nuclear antigen (PCNA). We show here that fission yeast Cdt1 interacts with PCNA in vivo and that DNA loading of PCNA is needed for Cdt1 proteolysis after DNA damage and in S phase. Activation of this pathway by ultraviolet (UV)-induced DNA damage requires upstream involvement of nucleotide excision repair or UVDE repair enzymes. Unexpectedly, two non-canonical PCNA-interacting peptide (PIP) motifs, which both have basic residues downstream, function redundantly in Cdt1 proteolysis. Finally, we show that poly-ubiquitylation of PCNA, which occurs after DNA damage, reduces Cdt1 proteolysis. This provides a mechanism for fine-tuning the activity of the CRL4(Cdt2) pathway towards Cdt1, allowing Cdt1 proteolysis to be more efficient in S phase than after DNA damage.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Dano ao DNA , Proteínas de Ligação a DNA/metabolismo , Antígeno Nuclear de Célula em Proliferação/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Ubiquitinação , Motivos de Aminoácidos , Sequência de Aminoácidos , Proteínas de Ciclo Celular/química , Cromatina/metabolismo , DNA Fúngico/metabolismo , Proteínas de Ligação a DNA/química , Dados de Sequência Molecular , Fase S/genética , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Schizosaccharomyces/efeitos da radiação , Proteínas de Schizosaccharomyces pombe/química , Raios UltravioletaRESUMO
The progression of meiosis is controlled by a number of gene-expression systems in the fission yeast Schizosaccharomyces pombe. A forkhead-type transcription factor Mei4 activates a number of genes essential for progression from the middle to late stages of meiosis, which include meiosis I, meiosis II and sporulation. The mei4-deletion mutant (mei4Δ) arrests after meiotic prophase and does not enter meiosis I. To further analyse the Mei4 function, we isolated novel temperature-sensitive mei4 alleles. The two alleles isolated in the initial screen turned out to contain a substitution at N136 in the forkhead DNA-binding domain. Among site-directed mutants that carried a point mutation at this position, the mei4-N136A mutant showed the most severe temperature sensitivity. The mei4-N136A mutant arrested before meiosis I at the restrictive temperature, as did the mei4Δ mutant. In fission yeast, the telomeres are clustered at the spindle pole body (SPB) in meiotic prophase and disperse from it at the onset of meiosis I. The mei4Δ mutant was found to arrest with its telomeres clustered at the SPB, demonstrating a role for Mei4 in telomere dispersion. The mei4-N136A mutant also arrested with clustered telomeres at the restrictive temperature, and the clustering was synchronously resolved after a temperature down-shift, indicating that mei4-N136A is a reversible allele. Hence, the mei4-N136A mutant will be a unique tool to synchronize the meiotic cell cycle from meiosis I onwards and may facilitate analyses of cellular activities occurring during meiosis I.
Assuntos
Meiose , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/crescimento & desenvolvimento , Schizosaccharomyces/genética , Telômero/metabolismo , Sequência de Aminoácidos , Deleção de Genes , Dados de Sequência Molecular , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutação de Sentido Incorreto , Schizosaccharomyces/efeitos da radiação , TemperaturaRESUMO
Cellular responses to DNA damage can prevent mutations and death. In this study, we have used high throughput screens and developed a comparative genomic approach, termed Functionome mapping, to discover conserved responses to UVC-damage. Functionome mapping uses gene ontology (GO) information to link proteins with similar biological functions from different organisms, and we have used it to compare 303, 311 and 288 UVC-toxicity modulating proteins from Escherichia coli, Schizosaccharomyces pombe and Saccharomyces cerevisiae, respectively. We have demonstrated that all three organisms use DNA repair, translation and aerobic respiration associated processes to modulate the toxicity of UVC, with these last two categories highlighting the importance of ribosomal proteins and electron transport machinery. Our study has demonstrated that comparative genomic approaches can be used to identify conserved responses to damage, and suggest roles for translational machinery and components of energy metabolism in optimizing the DNA damage response.
Assuntos
Respiração Celular/genética , Dano ao DNA/genética , Reparo do DNA/genética , Biossíntese de Proteínas/genética , Proteínas/genética , Tolerância a Radiação/genética , Raios Ultravioleta , Escherichia coli/genética , Escherichia coli/efeitos da radiação , Genômica/métodos , Ensaios de Triagem em Larga Escala , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/efeitos da radiação , Schizosaccharomyces/genética , Schizosaccharomyces/efeitos da radiação , Deleção de SequênciaRESUMO
In both Schizosaccharomyces pombe and Saccharomyces cerevisiae, Mms22 and Mms1 form a complex with important functions in the response to DNA damage, loss of which leads to perturbations during replication. Furthermore, in S. cerevisiae, Mms1 has been suggested to function in concert with a Cullin-like protein, Rtt101/Cul8, a potential paralog of Cullin 4. We performed epistasis analysis between Δmms1 and mutants of pathways with known functions in genome integrity, and measured the recruitment of homologous recombination proteins to blocked replication forks and recombination frequencies. We show that, in S. pombe, the functions of Mms1 and the conserved components of the Cullin 4 ubiquitin ligase, Pcu4 and Ddb1, do not significantly overlap. Furthermore, unlike in S. cerevisiae, the function of the H3K56 acetylase Rtt109 is not essential for Mms1 function. We provide evidence that Mms1 function is particularly important when a single strand break is converted into a double strand break during replication. Genetic data connect Mms1 to a Mus81 and Rad22(Rad52) dependent, but Rhp51 independent, branch of homologous recombination. This is supported by results demonstrating that Mms1 is recruited to a site-specific replication fork barrier and that, in a Δmms1 strain, Rad22(Rad52) and RPA recruitment to blocked forks are reduced, whereas Rhp51 recruitment is unaffected. In addition, Mms1 appears to specifically promote chromosomal rearrangements in a recombination assay. These observations suggest that Mms1 acts to channel repair of perturbed replication into a particular sub-pathway of homologous recombination.
Assuntos
Replicação do DNA/genética , Proteínas de Ligação a DNA/metabolismo , Recombinação Genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Quebras de DNA de Cadeia Dupla/efeitos da radiação , Reparo do DNA/genética , Replicação do DNA/efeitos da radiação , Proteínas de Ligação a DNA/genética , Genes Fúngicos Tipo Acasalamento/genética , Mutação , Rad51 Recombinase/metabolismo , Recombinação Genética/efeitos da radiação , Schizosaccharomyces/citologia , Schizosaccharomyces/efeitos da radiação , Proteínas de Schizosaccharomyces pombe/genética , Raios UltravioletaRESUMO
The DNA damage and stress response pathways interact to regulate cellular responses to genotoxins and environmental stresses. How these pathways interact in Schizosaccharomyces pombe is not well understood. We demonstrate that osmotic stress suppresses the DNA damage sensitivity of checkpoint mutants, and that this occurs through three distinct cell cycle delays. A delay in G2/M is dependent on Srk1. Progression through mitosis is halted by the Mad2-dependent spindle checkpoint. Finally, cytokinesis is impaired by modulating Cdc25 expression. These three delays, imposed by osmotic stress, together compensate for the loss of checkpoint signalling.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Citocinese/efeitos dos fármacos , Citocinese/efeitos da radiação , Hidroxiureia/toxicidade , Pressão Osmótica , Schizosaccharomyces/fisiologia , Raios Ultravioleta , Proteínas Mad2 , Proteínas Quinases Ativadas por Mitógeno , Proteínas Nucleares , Schizosaccharomyces/efeitos dos fármacos , Schizosaccharomyces/crescimento & desenvolvimento , Schizosaccharomyces/efeitos da radiação , Proteínas de Schizosaccharomyces pombe/metabolismo , Fosfatases cdc25RESUMO
Translesion synthesis is a major mechanism with which eukaryotic cells deal with DNA damage during replication. Mono-ubiquitinated PCNA is a key regulator of this process. We have investigated whether a ubiquitin-PCNA fusion can mimic ubiquitinated PCNA, by transforming plasmids expressing this fusion protein into different mutants of Schizosaccharomyces pombe. We show that the fusion protein is able to form PCNA trimers and that it can reduce the UV sensitivity and increase translesion synthesis in mutants in which PCNA cannot be ubiquitinated (pcn1-K164R and rhp18), but not of the rad8 mutant in which PCNA can be mono-ubiquitinated but not poly-ubiquitinated. We conclude that the fusion protein is a mimic of mono-ubiquitinated PCNA but it cannot be poly-ubiquitinated. Expression of the fusion protein at levels similar to that of endogenous unmodified protein has little effect on the spontaneous mutation rate of S. pombe. Replacement of the pcn1 locus with PCNA N-terminally tagged with different epitopes resulted in lethality, probably because the tagged proteins were expressed at substantially reduced levels.
Assuntos
Reparo do DNA , Antígeno Nuclear de Célula em Proliferação/metabolismo , Tolerância a Radiação , Schizosaccharomyces/metabolismo , Ubiquitina/metabolismo , Mutação , Proteínas Nucleares/genética , Antígeno Nuclear de Célula em Proliferação/genética , Proteínas de Ligação a RNA , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/efeitos da radiação , Proteínas de Schizosaccharomyces pombe/genética , Ubiquitina/genética , Raios UltravioletaRESUMO
Exposure of eukaryotic cells to UV light induces a checkpoint response that delays cell-cycle progression after cells enter S phase. It has been hypothesized that this checkpoint response provides time for repair by signaling the presence of structures generated when the replication fork encounters UV-induced DNA damage. To gain insight into the nature of the signaling structures, we used time-lapse microscopy to determine the effects of deficiencies in translesion DNA polymerases on the checkpoint response of the fission yeast Schizosaccharomyces pombe. We found that disruption of the genes encoding translesion DNA polymerases Polkappa and Poleta significantly prolonged the checkpoint response, indicating that the substrates of these enzymes are signals for checkpoint activation. Surprisingly, we found no evidence that the translesion polymerases Rev1 and Polzeta repair structures that are recognized by the checkpoint despite their role in maintaining viability after UV irradiation. Quantitative flow cytometry revealed that cells lacking translesion polymerases replicate UV-damaged DNA at the same rate at WT cells, indicating that the enhanced checkpoint response of cells lacking Polkappa and Poleta is not the result of stalled replication forks. These observations support a model in which postreplication DNA gaps with unrepaired UV lesions in the template strand act both as substrates for translesion polymerases and as signals for checkpoint activation.
Assuntos
Ciclo Celular , Replicação do DNA , Schizosaccharomyces/citologia , Schizosaccharomyces/metabolismo , Raios Ultravioleta , Dano ao DNA , DNA Fúngico/genética , DNA Fúngico/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Pirimidinas/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/efeitos da radiaçãoRESUMO
Absorption of electromagnetic irradiation results in significant heating of metallic nanoparticles, an effect which can be advantageously used in biomedical contexts. Also, metallic nanoparticles are presently finding widespread use as handles, contacts, or markers in nanometer scale systems, and for these purposes it is essential that the temperature increase associated with electromagnetic irradiation is not harmful to the environment. Regardless of whether the heating of metallic nanoparticles is desired or not, it is crucial for nanobio assays to know the exact temperature increase associated with electromagnetic irradiation of metallic nanoparticles. We performed direct measurements of the temperature surrounding single gold nanoparticles optically trapped on a lipid bilayer, a biologically relevant matrix. The lipid bilayer had incorporated fluorescent molecules which have a preference for either fluid or gel phases. The heating associated with electromagnetic radiation was measured by visualizing the melted footprint around the irradiated particle. The effect was measured for individual gold nanoparticles of a variety of sizes and for a variety of laser powers. The temperatures were highly dependent on particle size and laser power, with surface temperature increments ranging from a few to hundreds of degrees Celsius. Our results show that by a careful choice of gold nanoparticle size and strength of irradiating electromagnetic field, one can control the exact particle temperature. The method is easily applicable to any type of nanoparticle for which the photothermal effect is sought to be quantified.
Assuntos
Ouro/química , Temperatura Alta , Bicamadas Lipídicas/química , Nanopartículas Metálicas/química , Radiação , Bioensaio , Raios Infravermelhos , Lasers , Pinças Ópticas , Schizosaccharomyces/citologia , Schizosaccharomyces/efeitos da radiaçãoRESUMO
BACKGROUND: In many cell types, including the fission yeast Schizosaccharomyces pombe, a set of checkpoints are induced by perturbations of the cell cycle or by DNA damage. Many of the checkpoint responses include a substantial change of the transcriptional pattern. As part of characterising a novel G1/S checkpoint in fission yeast we have investigated whether a transcriptional response is induced after irradiation with ultraviolet light. RESULTS: Microarray analyses were used to measure the global transcription levels of all open reading frames of fission yeast after 254 nm ultraviolet irradiation, which is known to induce a G1/S checkpoint. We discovered a surprisingly weak transcriptional response, which is quite unlike the marked changes detected after some other types of treatment and in several other checkpoints. Interestingly, the alterations in gene expression after ultraviolet irradiation were not similar to those observed after ionising radiation or oxidative stress. Pathway analysis suggests that there is little systematic transcriptional response to the irradiation by ultraviolet light, but a marked, coordinated transcriptional response was noted on progression of the cells from G1 to S phase. CONCLUSION: There is little response in fission yeast to ultraviolet light at the transcriptional level. Amongst the genes induced or repressed after ultraviolet irradiation we found none that are likely to be involved in the G1/S checkpoint mechanism, suggesting that the checkpoint is not dependent upon transcriptional regulation.
Assuntos
Regulação Fúngica da Expressão Gênica/efeitos da radiação , Schizosaccharomyces/efeitos da radiação , Transcrição Gênica/efeitos da radiação , Ciclo Celular/efeitos da radiação , Análise de Sequência com Séries de Oligonucleotídeos , Schizosaccharomyces/citologia , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Raios UltravioletaRESUMO
Histone deacetylase (HDAC) inhibitors potently inhibit tumor growth and are currently being evaluated for their efficacy as chemosensitizers and radiosensitizers. This efficacy is likely to be limited by the fact that HDAC inhibitors also induce cell cycle arrest. Deletion of the class I HDAC Rpd3 has been shown to specifically suppress the sensitivity of Saccharomyces cerevisiae DNA damage checkpoint mutants to UV and hydroxyurea. We show that in the fission yeast Schizosaccharomyces pombe, inhibition of the homologous class I HDAC specifically suppresses the DNA damage sensitivity of checkpoint mutants. Importantly, the prototype HDAC inhibitor Trichostatin A also suppressed the sensitivity of DNA damage checkpoint but not of DNA repair mutants to UV and HU. TSA suppressed DNA damage activity independently of the mitogen-activated protein kinase-dependent and spindle checkpoint pathways. We show that TSA delays progression into mitosis and propose that this is the main mechanism for suppression of the DNA damage sensitivity of S. pombe checkpoint mutants, partially compensating for the loss of the G(2) checkpoint pathway. Our studies also show that the ability of HDAC inhibitors to suppress DNA damage sensitivity is not species specific. Class I HDACs are the major target of HDAC inhibitors and cancer cells are often defective in checkpoint activation. Effective use of these agents as chemosensitizers and radiosensitizers may require specific treatment schedules that circumvent their inhibition of cell cycle progression.