Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 32
Filtrar
1.
Nucleic Acids Res ; 52(7): 3722-3739, 2024 Apr 24.
Artigo em Inglês | MEDLINE | ID: mdl-38321948

RESUMO

Telomeres protect chromosome ends and are distinguished from DNA double-strand breaks (DSBs) by means of a specialized chromatin composed of DNA repeats bound by a multiprotein complex called shelterin. We investigated the role of telomere-associated proteins in establishing end-protection by studying viable mutants lacking these proteins. Mutants were studied using a Schizosaccharomyces pombe model system that induces cutting of a 'proto-telomere' bearing telomere repeats to rapidly form a new stable chromosomal end, in contrast to the rapid degradation of a control DSB. Cells lacking the telomere-associated proteins Taz1, Rap1, Poz1 or Rif1 formed a chromosome end that was stable. Surprisingly, cells lacking Ccq1, or impaired for recruiting Ccq1 to the telomere, converted the cleaved proto-telomere to a rapidly degraded DSB. Ccq1 recruits telomerase, establishes heterochromatin and affects DNA damage checkpoint activation; however, these functions were separable from protection of the new telomere by Ccq1. In cells lacking Ccq1, telomere degradation was greatly reduced by eliminating the nuclease activity of Mre11 (part of the Mre11-Rad50-Nbs1/Xrs2 DSB processing complex), and higher amounts of nuclease-deficient Mre11 associated with the new telomere. These results demonstrate a novel function for S. pombe Ccq1 to effect end-protection by restraining Mre11-dependent degradation of the DNA end.


Assuntos
Quebras de DNA de Cadeia Dupla , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Proteínas de Ligação a Telômeros , Telômero , Proteínas de Schizosaccharomyces pombe/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Ligação a Telômeros/metabolismo , Proteínas de Ligação a Telômeros/genética , Telômero/metabolismo , Telômero/genética , Complexo Shelterina/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Exodesoxirribonucleases/metabolismo , Exodesoxirribonucleases/genética , Telomerase/metabolismo , Telomerase/genética , Mutação , Proteína Homóloga a MRE11/metabolismo , Proteína Homóloga a MRE11/genética
2.
Blood ; 140(15): 1710-1722, 2022 10 13.
Artigo em Inglês | MEDLINE | ID: mdl-35767717

RESUMO

γ-Glutamyl carboxylase (GGCX) generates multiple carboxylated Glus (Glas) in vitamin K-dependent (VKD) proteins that are required for their functions. GGCX is processive, remaining bound to VKD proteins throughout multiple Glu carboxylations, and this study reveals the essentiality of processivity to VKD protein function. GGCX mutants (V255M and S300F) whose combined heterozygosity in a patient causes defective clotting and calcification were studied using a novel assay that mimics in vivo carboxylation. Complexes between variant carboxylases and VKD proteins important to hemostasis (factor IX [FIX]) or calcification (matrix Gla protein [MGP]) were reacted in the presence of a challenge VKD protein that could potentially interfere with carboxylation of the VKD protein in the complex. The VKD protein in the complex with wild-type carboxylase was carboxylated before challenge protein carboxylation occurred and became fully carboxylated. In contrast, the V255M mutant carboxylated both forms at the same time and did not completely carboxylate FIX in the complex. S300F carboxylation was poor with both FIX and MGP. Additional studies analyzed FIX- and MGP-derived peptides containing the Gla domain linked to sequences that mediate carboxylase binding. The total amount of carboxylated peptide generated by the V255M mutant was higher than that of wild-type GGCX; however, the individual peptides were partially carboxylated. Analysis of the V255M mutant in FIX HEK293 cells lacking endogenous GGCX revealed poor FIX clotting activity. This study shows that disrupted processivity causes disease and explains the defect in the patient. Kinetic analyses also suggest that disrupted processivity may occur in wild-type carboxylase under some conditions (eg, warfarin therapy or vitamin K deficiency).


Assuntos
Carbono-Carbono Ligases , Vitamina K , Coagulação Sanguínea , Carbono-Carbono Ligases/química , Carbono-Carbono Ligases/genética , Fator IX/metabolismo , Células HEK293 , Humanos , Peptídeos , Proteínas , Vitamina K/metabolismo , Varfarina
3.
Nucleic Acids Res ; 50(20): 11682-11695, 2022 11 11.
Artigo em Inglês | MEDLINE | ID: mdl-36330920

RESUMO

Telomere elongation is coupled with genome replication, raising the question of the repair of short telomeres in post-mitotic cells. We investigated the fate of a telomere-repeat capped end that mimics a single short telomere in quiescent fission yeast cells. We show that telomerase is able to elongate this single short telomere during quiescence despite the binding of Ku to the proto-telomere. While Taz1 and Rap1 repress telomerase in vegetative cells, both shelterin proteins are required for efficient telomere extension in quiescent cells, underscoring a distinct mode of telomerase control. We further show that Rad3ATR and Tel1ATM are redundantly required for telomere elongation in quiescence through the phosphorylation of Ccq1 and that Rif1 and its associated-PP1 phosphatases negatively regulate telomerase activity by opposing Ccq1 phosphorylation. The distinct mode of telomerase regulation in quiescent fission yeast cells may be relevant to that in human stem and progenitor cells.


Assuntos
Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Complexo Shelterina , Telomerase , Proteínas de Ligação a Telômeros , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Telomerase/genética , Telomerase/metabolismo , Telômero/genética , Telômero/metabolismo , Proteínas de Ligação a Telômeros/genética , Proteínas de Ligação a Telômeros/metabolismo
4.
Nucleic Acids Res ; 50(17): e102, 2022 09 23.
Artigo em Inglês | MEDLINE | ID: mdl-35766443

RESUMO

Arrayed libraries of defined mutants have been used to elucidate gene function in the post-genomic era. Yeast haploid gene deletion libraries have pioneered this effort, but are costly to construct, do not reveal phenotypes that may occur with partial gene function and lack essential genes required for growth. We therefore devised an efficient method to construct a library of barcoded insertion mutants with a wider range of phenotypes that can be generalized to other organisms or collections of DNA samples. We developed a novel but simple three-dimensional pooling and multiplexed sequencing approach that leveraged sequence information to reduce the number of required sequencing reactions by orders of magnitude, and were able to identify the barcode sequences and DNA insertion sites of 4391 Schizosaccharomyces pombe insertion mutations with only 40 sequencing preparations. The insertion mutations are in the genes and untranslated regions of nonessential, essential and noncoding RNA genes, and produced a wider range of phenotypes compared to the cognate deletion mutants, including novel phenotypes. This mutant library represents both a proof of principle for an efficient method to produce novel mutant libraries and a valuable resource for the S. pombe research community.


Assuntos
Schizosaccharomyces , DNA , Elementos de DNA Transponíveis/genética , Biblioteca Gênica , Genes Essenciais , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Mutagênese Insercional , RNA não Traduzido , Saccharomyces cerevisiae/genética , Schizosaccharomyces/genética , Regiões não Traduzidas
5.
PLoS Genet ; 15(8): e1008335, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31454352

RESUMO

Genomic rearrangements (gross chromosomal rearrangements, GCRs) threatens genome integrity and cause cell death or tumor formation. At the terminus of linear chromosomes, a telomere-binding protein complex, called shelterin, ensures chromosome stability by preventing chromosome end-to-end fusions and regulating telomere length homeostasis. As such, shelterin-mediated telomere functions play a pivotal role in suppressing GCR formation. However, it remains unclear whether the shelterin proteins play any direct role in inhibiting GCR at non-telomeric regions. Here, we have established a GCR assay for the first time in fission yeast and measured GCR rates in various mutants. We found that fission yeast cells lacking shelterin components Taz1 or Rap1 (mammalian TRF1/2 or RAP1 homologues, respectively) showed higher GCR rates compared to wild-type, accumulating large chromosome deletions. Genetic dissection of Rap1 revealed that Rap1 contributes to inhibiting GCRs via two independent pathways. The N-terminal BRCT-domain promotes faithful DSB repair, as determined by I-SceI-mediated DSB-induction experiments; moreover, association with Poz1 mediated by the central Poz1-binding domain regulates telomerase accessibility to DSBs, leading to suppression of de novo telomere additions. Our data highlight unappreciated functions of the shelterin components Taz1 and Rap1 in maintaining genome stability, specifically by preventing non-telomeric GCRs.


Assuntos
Reparo do DNA , Rearranjo Gênico , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/genética , Proteínas de Ligação a Telômeros/metabolismo , Quebras de DNA de Cadeia Dupla , Instabilidade Genômica , Mutação , Proteínas de Schizosaccharomyces pombe/genética , Complexo Shelterina , Homeostase do Telômero , Proteínas de Ligação a Telômeros/genética
6.
Int J Mol Sci ; 23(10)2022 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-35628569

RESUMO

Vitamin K-dependent (VKD) proteins undergo an unusual post-translational modification, which is the conversion of specific Glu residues to carboxylated Glu (Gla). Gla generation is required for the activation of VKD proteins, and occurs in the endoplasmic reticulum during their secretion to either the cell surface or from the cell. The gamma-glutamyl carboxylase produces Gla using reduced vitamin K, which becomes oxygenated to vitamin K epoxide. Reduced vitamin K is then regenerated by a vitamin K oxidoreductase (VKORC1), and this interconversion of oxygenated and reduced vitamin K is referred to as the vitamin K cycle. Many of the VKD proteins support hemostasis, which is suppressed during therapy with warfarin that inhibits VKORC1 activity. VKD proteins also impact a broad range of physiologies beyond hemostasis, which includes regulation of calcification, apoptosis, complement, growth control, signal transduction and angiogenesis. The review covers the roles of VKD proteins, how they become activated, and how disruption of carboxylation can lead to disease. VKD proteins contain clusters of Gla residues that form a calcium-binding module important for activity, and carboxylase processivity allows the generation of multiple Glas. The review discusses how impaired carboxylase processivity results in the pseudoxanthoma elasticum-like disease.


Assuntos
Processamento de Proteína Pós-Traducional , Vitamina K , Proteínas/metabolismo , Vitamina K/metabolismo , Varfarina
7.
Blood ; 131(25): 2826-2835, 2018 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-29592891

RESUMO

The anticoagulant warfarin inhibits the vitamin K oxidoreductase (VKORC1), which generates vitamin K hydroquinone (KH2) required for the carboxylation and consequent activation of vitamin K-dependent (VKD) proteins. VKORC1 produces KH2 in 2 reactions: reduction of vitamin K epoxide (KO) to quinone (K), and then KH2 Our dissection of full reduction vs the individual reactions revealed a surprising mechanism of warfarin inhibition. Warfarin inhibition of KO to K reduction and carboxylation that requires full reduction were compared in wild-type VKORC1 or mutants (Y139H, Y139F) that cause warfarin resistance. Carboxylation was much more strongly inhibited (∼400-fold) than KO reduction (two- to threefold). The K to KH2 reaction was analyzed using low K concentrations that result from inhibition of KO to K. Carboxylation that required only K to KH2 reduction was inhibited much less than observed with the KO substrate that requires full VKORC1 reduction (eg, 2.5-fold vs 70-fold, respectively, in cells expressing wild-type VKORC1 and factor IX). The results indicate that warfarin uncouples the 2 reactions that fully reduce KO. Uncoupling was revealed because a second activity, a warfarin-resistant quinone reductase, was not present. In contrast, 293 cells expressing factor IX and this reductase activity showed much less inhibition of carboxylation. This activity therefore appears to cooperate with VKORC1 to accomplish full KO reduction. Cooperation during warfarin therapy would have significant consequences, as VKD proteins function in numerous physiologies in many tissues, but may be poorly carboxylated and dysfunctional if the second activity is not ubiquitously expressed similar to VKORC1.


Assuntos
Anticoagulantes/metabolismo , Vitamina K Epóxido Redutases/metabolismo , Vitamina K/metabolismo , Varfarina/metabolismo , Animais , Anticoagulantes/farmacologia , Linhagem Celular , Cricetinae , Resistência a Medicamentos , Humanos , Oxirredução/efeitos dos fármacos , Mutação Puntual , Vitamina K 1/análogos & derivados , Vitamina K 1/metabolismo , Vitamina K Epóxido Redutases/antagonistas & inibidores , Vitamina K Epóxido Redutases/genética , Varfarina/farmacologia
8.
Curr Genet ; 64(2): 359-364, 2018 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-29018935

RESUMO

Chromosomal breaks can be healed by several repair processes, including one called non-homologous end-joining (NHEJ) where the two broken ends are ligated together with a loss of 0-5 bp of DNA. The protein requirements for NHEJ of cut DNA ends in the budding yeast Saccharomyces cerevisiae include its version of the Mre11-Rad50-Nbs1 (MRN) complex. In contrast, the fission yeast Schizosaccharomyces pombe and mammalian cells do not require MRN for this process. Recent work in S. pombe used transposon excision to generate breaks that were capped by DNA hairpins, which must be opened to produce ligatable ends. Repair in S. pombe was through an NHEJ reaction that now requires MRN. Surprisingly, wild type cells and MRN mutants that lack nuclease activity showed the same levels of excision. These genetic results suggest that MRN recruits an unknown hairpin-opening nuclease for this unusual NHEJ reaction.


Assuntos
Reparo do DNA por Junção de Extremidades/genética , Reparo do DNA/genética , DNA/genética , Schizosaccharomyces/genética , Proteínas Cromossômicas não Histona/genética , Dano ao DNA/genética , Elementos de DNA Transponíveis/genética , Exodesoxirribonucleases/genética , Saccharomyces cerevisiae/genética , Proteínas de Schizosaccharomyces pombe/genética
9.
Proc Natl Acad Sci U S A ; 110(38): 15371-6, 2013 Sep 17.
Artigo em Inglês | MEDLINE | ID: mdl-24003116

RESUMO

In Schizosaccharomyces pombe, alcohol dehydrogenase 1 (Adh1) is an abundant zinc-requiring enzyme that catalyses the conversion of acetaldehyde to ethanol during fermentation. In a zinc-replete cell, adh1 is highly expressed. However, in zinc-limited cells, adh1 gene expression is repressed, and cells induce the expression of an alternative alcohol dehydrogenase encoded by the adh4 gene. In our studies examining this zinc-dependent switch in alcohol dehydrogenase gene expression, we isolated an adh1Δ strain containing a partial loss of function mutation that resulted in higher levels of adh4 transcripts in zinc-replete cells. This mutation also led to the aberrant expression of other genes that are typically regulated by zinc. Using linkage analysis, we have mapped the position of this mutation to a single gene called Loss Of Zinc sensing 1 (loz1). Loz1 is a 55-kDa protein that contains a double C2H2-type zinc finger domain. The mapped mutation that disrupts Loz1 function leads to an arginine to glycine substitution in the second zinc finger domain, suggesting that the double zinc finger domain is important for Loz1 function. We show that loz1Δ cells hyperaccumulate zinc and that Loz1 is required for gene repression in zinc-replete cells. We also have found that Loz1 negatively autoregulates its own expression. We propose that Loz1 is a unique metalloregulatory factor that plays a central role in zinc homeostasis in S. pombe.


Assuntos
Regulação Fúngica da Expressão Gênica/genética , Homeostase/fisiologia , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/fisiologia , Fatores de Transcrição/genética , Dedos de Zinco/genética , Zinco/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Immunoblotting , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Fatores de Transcrição/metabolismo , beta-Galactosidase
10.
J Biol Chem ; 288(44): 31556-66, 2013 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-23918929

RESUMO

The vitamin K oxidoreductase (VKORC1) recycles vitamin K to support the activation of vitamin K-dependent (VKD) proteins, which have diverse functions that include hemostasis and calcification. VKD proteins are activated by Glu carboxylation, which depends upon the oxygenation of vitamin K hydroquinone (KH2). The vitamin K epoxide (KO) product is recycled by two reactions, i.e. KO reduction to vitamin K quinone (K) and then to KH2, and recent studies have called into question whether VKORC1 reduces K to KH2. Analysis in insect cells lacking endogenous carboxylation components showed that r-VKORC1 reduces KO to efficiently drive carboxylation, indicating KH2 production. Direct detection of the vitamin K reaction products is confounded by KH2 oxidation, and we therefore developed a new assay that stabilized KH2 and allowed quantitation. Purified VKORC1 analyzed in this assay showed efficient KO to KH2 reduction. Studies in 293 cells expressing tagged r-VKORC1 revealed that VKORC1 is a multimer, most likely a dimer. A monomer can only perform one reaction, and a dimer is therefore interesting in explaining how VKORC1 accomplishes both reactions. An inactive mutant (VKORC1(C132A/C135A)) was dominant negative in heterodimers with wild type VKORC1, resulting in decreased KO reduction in cells and carboxylation in vitro. The results are significant regarding human VKORC1 mutations, as warfarin-resistant patients have mutant and wild type VKORC1 alleles. A VKORC1 dimer indicates a mixed population of homodimers and heterodimers that may have different functional properties, and VKORC1 reduction may therefore be more complex in these patients than appreciated previously.


Assuntos
Hidroquinonas/metabolismo , Multimerização Proteica/fisiologia , Processamento de Proteína Pós-Traducional/fisiologia , Vitamina K Epóxido Redutases/metabolismo , Vitamina K/metabolismo , Substituição de Aminoácidos , Anticoagulantes/uso terapêutico , Resistência a Medicamentos/efeitos dos fármacos , Resistência a Medicamentos/fisiologia , Células HEK293 , Humanos , Hidroquinonas/química , Mutação de Sentido Incorreto , Oxirredução , Vitamina K/química , Vitamina K/genética , Vitamina K Epóxido Redutases/química , Vitamina K Epóxido Redutases/genética , Varfarina/uso terapêutico
11.
bioRxiv ; 2024 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-38915521

RESUMO

Cdk5 is a highly-conserved, noncanonical cell division kinase important to the terminal differentiation of mammalian cells in multiple organ systems. We previously identified Pef1, the Schizosaccharomyces pombe ortholog of cdk5, as regulator of chronological lifespan. To reveal the processes impacted by Pef1, we developed APEX2-biotin phenol-mediated proximity labeling in S. pombe. Efficient labeling required a short period of cell wall digestion and eliminating glucose and nitrogen sources from the medium. We identified 255 high-confidence Pef1 neighbors in growing cells and a novel Pef1-interacting partner, the DNA damage response protein Rad24. The Pef1-Rad24 interaction was validated by reciprocal proximity labeling and co-immunoprecipitation. Eliminating Pef1 partially rescued the DNA damage sensitivity of cells lacking Rad24. To monitor how Pef1 neighbors change under different conditions, cells induced for autophagy were labeled and 177 high-confidence Pef1 neighbors were identified. Gene ontology (GO) analysis of the Pef1 neighbors identified proteins participating in processes required for autophagosome expansion including regulation of actin dynamics and vesicle-mediated transport. Some of these proteins were identified in both exponentially growing and autophagic cells. Pef1-APEX2 proximity labeling therefore identified a new Pef1 function in modulating the DNA damage response and candidate processes that Pef1 and other cdk5 orthologs may regulate.

12.
Chromosoma ; 121(3): 277-90, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22289863

RESUMO

In many organisms, telomere DNA consists of simple sequence repeat tracts that are required to protect the chromosome end. In the yeast Saccharomyces cerevisiae, tract maintenance requires two checkpoint kinases of the ATM family, Tel1p and Mec1p. Previous work has shown that Tel1p is recruited to functional telomeres with shorter repeat tracts to promote telomerase-mediated repeat addition, but the role of Mec1p is unknown. We found that Mec1p telomere association was detected as cells senesced when telomere function was compromised by extreme shortening due to either the loss of telomerase or the double-strand break binding protein Ku. Exonuclease I effects the removal of the 5' telomeric strand, and eliminating it prevented both senescence and Mec1p telomere association. Thus, in contrast to Tel1p, Mec1p associates with short, functionally compromised telomeres.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Telômero/metabolismo , Proliferação de Células , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Exodesoxirribonucleases/genética , Exodesoxirribonucleases/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas Serina-Treonina Quinases/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
13.
Subcell Biochem ; 57: 291-314, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22094427

RESUMO

Yeasts are powerful model systems to examine the evolutionarily conserved aspects of eukaryotic aging because they maintain many of the same core cellular signaling pathways and essential organelles as human cells. We constructed a strain of the budding yeast Saccharomyces cerevisiae that could monitor the distribution of proteins involved in heterochromatic silencing and aging, and isolated mutants that alter this distribution. The largest class of such mutants cause defects in mitochondrial function, and appear to cause changes in nuclear silencing separate from the well-known Rtg2p-dependent pathway that alters nuclear transcription in response to the loss of the mitochondrial genome. Mutants that inactivate the ATP2 gene, which encodes the ATPase subunit of the mitochondrial F(1)F(0)-ATPase, were isolated twice in our screen and identify a lifespan extending pathway in a gene that is conserved in both prokaryotes and eukaryotes. The budding yeast S. cerevisiae S. cerevisiae has been used with great success to identify other lifespan-extending pathways in screens using surrogate phenotypes such as stress resistance or silencing to identify random mutants, or in high throughput screens that utilize the deletion strain set resource. However, the direct selection of long-lived mutants from a pool of random mutants is more challenging. We have established a new chronological aging assay for the evolutionarily distant fission yeast Schizosaccharomyces pombe that recapitulates aspects of aging conserved in all eukaryotes. We have constructed a novel S. pombe S. pombe DNA insertion mutant bank, and used it to show that we can directly select for a long-lived mutant. The use of both the budding and fission yeast systems should continue to facilitate the identification and validation of lifespan extending pathways that are conserved in humans.


Assuntos
Envelhecimento/genética , Saccharomyces cerevisiae/genética , Schizosaccharomyces/genética , Envelhecimento/metabolismo , Animais , Núcleo Celular/metabolismo , Montagem e Desmontagem da Cromatina , Regulação Fúngica da Expressão Gênica , Inativação Gênica , Genótipo , Humanos , Longevidade , Mitocôndrias/metabolismo , Modelos Genéticos , Mutação , Fenótipo , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Schizosaccharomyces/crescimento & desenvolvimento , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Transdução de Sinais , Fatores de Tempo
14.
Nat Genet ; 33(4): 522-6, 2003 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-12640455

RESUMO

An organism's lifespan is modulated by environmental conditions. When nutrients are abundant, the metabolism of many organisms shifts to growth or reproduction at the expense of longer lifespan, whereas a scarcity of nutrients reverses this shift. These correlations suggest that organisms respond to environmental changes by altering their metabolism to promote either reproduction and growth or long life. The only previously reported signaling mechanism involved in this response is the nutrient-responsive insulin/insulin-like growth factor-1 receptor pathway. Here we report another pathway that controls the length of yeast lifespan. Commitment to cell growth activates the Slt2p MAP kinase pathway, which phosphorylates the transcriptional silencing protein Sir3p, resulting in a shorter lifespan. Elimination of the Sir3p phosphorylation site at Ser275 extended lifespan by 38%. Lifespan extension occurs by a mechanism that is independent of suppressing rDNA recombination. Thus, Slt2p is an enzymatic regulator of silencing function that couples commitment to cell growth and shorter lifespan.


Assuntos
Inativação Gênica , Proteínas Quinases Ativadas por Mitógeno , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas Reguladoras de Informação Silenciosa de Saccharomyces cerevisiae/genética , Proteínas Reguladoras de Informação Silenciosa de Saccharomyces cerevisiae/metabolismo , Alelos , Western Blotting , Proteínas Fúngicas/metabolismo , Sistema de Sinalização das MAP Quinases , Modelos Biológicos , Modelos Genéticos , Dados de Sequência Molecular , Mutação , Fases de Leitura Aberta , Fenótipo , Fosforilação , Testes de Precipitina , Estrutura Terciária de Proteína , Recombinação Genética , Fatores de Tempo
15.
BMC Genomics ; 13: 161, 2012 May 03.
Artigo em Inglês | MEDLINE | ID: mdl-22554201

RESUMO

BACKGROUND: Barcodes are unique DNA sequence tags that can be used to specifically label individual mutants. The barcode-tagged open reading frame (ORF) haploid deletion mutant collections in the budding yeast Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe allow for high-throughput mutant phenotyping because the relative growth of mutants in a population can be determined by monitoring the proportions of their associated barcodes. While these mutant collections have greatly facilitated genome-wide studies, mutations in essential genes are not present, and the roles of these genes are not as easily studied. To further support genome-scale research in S. pombe, we generated a barcode-tagged fission yeast insertion mutant library that has the potential of generating viable mutations in both essential and non-essential genes and can be easily analyzed using standard molecular biological techniques. RESULTS: An insertion vector containing a selectable ura4+ marker and a random barcode was used to generate a collection of 10,000 fission yeast insertion mutants stored individually in 384-well plates and as six pools of mixed mutants. Individual barcodes are flanked by Sfi I recognition sites and can be oligomerized in a unique orientation to facilitate barcode sequencing. Independent genetic screens on a subset of mutants suggest that this library contains a diverse collection of single insertion mutations. We present several approaches to determine insertion sites. CONCLUSIONS: This collection of S. pombe barcode-tagged insertion mutants is well-suited for genome-wide studies. Because insertion mutations may eliminate, reduce or alter the function of essential and non-essential genes, this library will contain strains with a wide range of phenotypes that can be assayed by their associated barcodes. The design of the barcodes in this library allows for barcode sequencing using next generation or standard benchtop cloning approaches.


Assuntos
Mutagênese Insercional , Schizosaccharomyces/genética , DNA/química , DNA/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Biblioteca Gênica , Genes Fúngicos , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo
16.
Yeast ; 29(7): 275-91, 2012 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22674789

RESUMO

Double-strand DNA breaks are a serious threat to cellular viability and yeast systems have proved invaluable in helping to understand how these potentially toxic lesions are sensed and repaired. An important method to study the processing of DNA breaks in the budding yeast Saccharomyces cerevisiae is to introduce a unique double-strand break into the genome by regulating the expression of the site-specific HO endonuclease with a galactose inducible promoter. Variations of the HO site-specific DSB assay have been adapted to many organisms, but the methodology has seen only limited use in the fission yeast Schizosaccharomyces pombe because of the lack of a promoter capable of inducing endonuclease expression on a relatively short time scale (~1 h). We have overcome this limitation by developing a new assay in which expression of the homing endonuclease I-PpoI is tightly regulated with a tetracycline-inducible promoter. We show that induction of the I-PpoI endonuclease produces rapid cutting of a defined cleavage site (> 80% after 1 h), efficient cell cycle arrest and significant accumulation of the checkpoint protein Crb2 at break-adjacent regions in a manner that is analogous to published findings with DSBs produced by an acute exposure to ionizing irradiation. This assay provides an important new tool for the fission yeast community and, because many aspects of mammalian chromatin organization have been well-conserved in Sz. pombe but not in S. cerevisiae, also offers an attractive system to decipher the role of chromatin structure in modulating the repair of double-stranded DNA breaks.


Assuntos
Quebras de DNA de Cadeia Dupla , Técnicas Genéticas , Schizosaccharomyces/genética , Reparo do DNA , Endonucleases/genética , Endonucleases/metabolismo , Regulação Fúngica da Expressão Gênica , Genoma Fúngico , Schizosaccharomyces/enzimologia , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo
17.
J Thromb Haemost ; 17(7): 1053-1063, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31009158

RESUMO

Essentials A carboxylase mutation that impairs splicing to delete exon 2 sequences was previously reported. We found that the mutant was inactive for vitamin K-dependent (VKD) protein carboxylation. An incomplete splicing defect likely accounts for VKD clotting activity observed in the patient. The results indicate the importance of proper carboxylase embedment in the membrane for function. BACKGROUND: Mutations in the γ-glutamyl carboxylase (GGCX), which is required for vitamin K-dependent (VKD) protein activation, can result in vitamin K clotting factor deficiency (VKCFD1). A recent report described a VKCFD1 patient with a homozygous carboxylase mutation that altered splicing and deleted exon 2 (Δ2GGCX). Only Δ2GGCX RNA was observed in the patient. OBJECTIVES: Loss of exon 2 results in the deletion of carboxylase sequences thought to be important for membrane topology and consequent function. Carboxylase activity is required for life, and we therefore tested whether the Δ2GGCX mutant is active. METHODS: HEK 293 cells were edited by the use of CRISPR-Cas9 to eliminate endogenous carboxylase. Recombinant wild-type GGCX and recombinant Δ2GGCX were then expressed and tested for carboxylation of the VKD protein factor IX. A second approach was used to monitor carboxylation biochemically, using recombinant carboxylases expressed in insect cells that lack endogenous carboxylase. RESULTS AND CONCLUSIONS: Δ2GGCX activity was undetectable in both assays, which is strikingly different from the low levels of carboxylase activity observed with other VKCFD1 mutants. The similarity in clotting function between patients with Δ2GGCX and these mutations must therefore arise from a novel mechanism. Low levels of properly spliced carboxylase RNA that produce full-length protein would not have been observed in the previous study. The results suggest that the splicing defect is incomplete. Δ2GGCX RNA has been detected in normal human liver, and has been designated carboxylase isoform 2; however, Δ2GGCX protein was not observed in normal human liver. The lack of activity and protein expression suggest that isoform 2 is not physiologically relevant to normal VKD protein carboxylation.


Assuntos
Transtornos Herdados da Coagulação Sanguínea/sangue , Transtornos Herdados da Coagulação Sanguínea/genética , Coagulação Sanguínea/genética , Carbono-Carbono Ligases/genética , Carbono-Carbono Ligases/metabolismo , Éxons , Mutação , Splicing de RNA , Transtornos Herdados da Coagulação Sanguínea/diagnóstico , Sistemas CRISPR-Cas , Fator IX/metabolismo , Edição de Genes , Predisposição Genética para Doença , Células HEK293 , Homozigoto , Humanos , Fenótipo , Processamento de Proteína Pós-Traducional
18.
F1000Res ; 7: 1027, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30498568

RESUMO

DNA double-strand breaks (DSBs) activate the DNA damage checkpoint machinery to pause or halt the cell cycle.  Telomeres, the specific DNA-protein complexes at linear eukaryotic chromosome ends, are capped DSBs that do not activate DNA damage checkpoints.  This "checkpoint privileged" status of telomeres was previously investigated in the yeast  Schizosaccharomyces pombelacking the major double-stranded telomere DNA binding protein Taz1. Telomeric DNA repeats in cells lacking Taz1 are 10 times longer than normal and contain single-stranded DNA regions. DNA damage checkpoint proteins associate with these damaged telomeres, but the DNA damage checkpoint is not activated. This severing of the DNA damage checkpoint signaling pathway was reported to stem from exclusion of histone H4 lysine 20 dimethylation (H4K20me2) from telomeric nucleosomes in both wild type cells and cells lacking Taz1.  However, experiments to identify the mechanism of this exclusion failed, prompting our re-evaluation of H4K20me2 levels at telomeric chromatin.  In this short report, we used an extensive series of controls to identify an antibody specific for the H4K20me2 modification and show that the level of this modification is the same at telomeres and internal loci in both wild type cells and those lacking Taz1.  Consequently, telomeres must block activation of the DNA Damage Response by another mechanism that remains to be determined.


Assuntos
Pontos de Checagem do Ciclo Celular , Histonas , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Telômero , Dano ao DNA , Histonas/genética , Histonas/metabolismo , Metilação , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Telômero/genética , Telômero/metabolismo
19.
DNA Repair (Amst) ; 65: 26-33, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29544213

RESUMO

Telomeres, the ends of eukaryotic chromosomes, consist of repetitive DNA sequences and their bound proteins that protect the end from the DNA damage response. Short telomeres with fewer repeats are preferentially elongated by telomerase. Tel1, the yeast homolog of human ATM kinase, is preferentially recruited to short telomeres and Tel1 kinase activity is required for telomere elongation. Rif1, a telomere-binding protein, negatively regulates telomere length by forming a complex with two other telomere binding proteins, Rap1 and Rif2, to block telomerase recruitment. Rif1 has 14 SQ/TQ consensus phosphorylation sites for ATM kinases, including 6 in a SQ/TQ Cluster Domain (SCD) similar to other DNA damage response proteins. These 14 sites were analyzed as N-terminal, SCD and C-terminal domains. Mutating some sites to non-phosphorylatable residues increased telomere length in cells lacking Tel1 while a different set of phosphomimetic mutants increased telomere length in cells lacking Rif2, suggesting that Rif1 phosphorylation has both positive and negative effects on length regulation. While these mutations did not alter the sensitivity to DNA damaging agents, inducing telomere-specific damage by growing cells lacking YKU70 at high temperature revealed a role for the SCD. Mass spectrometry of Rif1 from wild type cells or those induced for telomere-specific DNA damage revealed increased phosphorylation in cells with telomere damage at an ATM consensus site in the SCD, S1351, and non-ATM sites S181 and S1637. A phosphomimetic rif1-S1351E mutation caused an increase in telomere length at synthetic telomeres but not natural telomeres. These results indicate that the Rif1 SCD can modulate Rif1 function. As all Rif1 orthologs have one or more SCD domains, these results for yeast Rif1 have implications for the regulation of Rif1 function in humans and other organisms.


Assuntos
Processamento de Proteína Pós-Traducional , Proteínas Repressoras/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Ligação a Telômeros/metabolismo , Telômero/metabolismo , Dano ao DNA , DNA Fúngico , Fosforilação , Saccharomyces cerevisiae/genética
20.
Mol Cell Biol ; 38(15)2018 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-29784772

RESUMO

Heterochromatin domains play important roles in chromosome biology, organismal development, and aging, including centromere function, mammalian female X chromosome inactivation, and senescence-associated heterochromatin foci. In the fission yeast Schizosaccharomyces pombe and metazoans, heterochromatin contains histone H3 that is dimethylated at lysine 9. While factors required for heterochromatin have been identified, the dynamics of heterochromatin formation are poorly understood. Telomeres convert adjacent chromatin into heterochromatin. To form a new heterochromatic region in S. pombe, an inducible DNA double-strand break (DSB) was engineered next to 48 bp of telomere repeats in euchromatin, which caused formation of a new telomere and the establishment and gradual spreading of a new heterochromatin domain. However, spreading was dynamic even after the telomere had reached its stable length, with reporter genes within the heterochromatin domain showing variegated expression. The system also revealed the presence of repeats located near the boundaries of euchromatin and heterochromatin that are oriented to allow the efficient healing of a euchromatic DSB to cap the chromosome end with a new telomere. Telomere formation in S. pombe therefore reveals novel aspects of heterochromatin dynamics and fail-safe mechanisms to repair subtelomeric breaks, with implications for similar processes in metazoan genomes.


Assuntos
Heterocromatina/metabolismo , Schizosaccharomyces/metabolismo , Telômero/metabolismo , Sequência de Bases , Quebras de DNA de Cadeia Dupla , DNA Fúngico/genética , DNA Fúngico/metabolismo , Inativação Gênica , Genoma Fúngico , Heterocromatina/genética , Código das Histonas , Cinética , Modelos Biológicos , Schizosaccharomyces/genética , Schizosaccharomyces/crescimento & desenvolvimento , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Telomerase/metabolismo , Telômero/genética , Homeostase do Telômero
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA