RESUMO
DNA replication initiates at many discrete loci on eukaryotic chromosomes, and individual replication origins are regulated under a spatiotemporal program. However, the underlying mechanisms of this regulation remain largely unknown. In the fission yeast Schizosaccharomyces pombe, the telomere-binding protein Taz1, ortholog of human TRF1/TRF2, regulates a subset of late replication origins by binding to the telomere-like sequence near the origins. Here, we showed using a lacO/LacI-GFP system that Taz1-dependent late origins were predominantly localized at the nuclear periphery throughout interphase, and were localized adjacent to the telomeres in the G1/S phase. The peripheral localization that depended on the nuclear membrane protein Bqt4 was not necessary for telomeric association and replication-timing control of the replication origins. Interestingly, the shelterin components Rap1 and Poz1 were required for replication-timing control and telomeric association of Taz1-dependent late origins, and this requirement was bypassed by a minishelterin Tpz1-Taz1 fusion protein. Our results suggest that Taz1 suppresses replication initiation through shelterin-mediated telomeric association of the origins at the onset of S phase.
Assuntos
Origem de Replicação/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/genética , Proteínas de Ligação a Telômeros/metabolismo , Telômero/metabolismo , Proteína 2 de Ligação a Repetições Teloméricas/metabolismo , Replicação do DNA/genética , Proteínas de Ligação a DNA/metabolismo , Fase G1/genética , Proteínas de Membrana/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Fase S/genética , Proteínas de Schizosaccharomyces pombe/genética , Complexo Shelterina , Proteínas de Ligação a Telômeros/genéticaRESUMO
In eukaryotes, the replication of chromosome DNA is coordinated by a replication timing program that temporally regulates the firing of individual replication origins. However, the molecular mechanism underlying the program remains elusive. Here, we report that the telomere-binding protein Taz1 plays a crucial role in the control of replication timing in fission yeast. A DNA element located proximal to a late origin in the chromosome arm represses initiation from the origin in early S phase. Systematic deletion and substitution experiments demonstrated that two tandem telomeric repeats are essential for this repression. The telomeric repeats recruit Taz1, a counterpart of human TRF1 and TRF2, to the locus. Genome-wide analysis revealed that Taz1 regulates about half of chromosomal late origins, including those in subtelomeres. The Taz1-mediated mechanism prevents Dbf4-dependent kinase (DDK)-dependent Sld3 loading onto the origins. Our results demonstrate that the replication timing program in fission yeast uses the internal telomeric repeats and binding of Taz1.
Assuntos
Replicação do DNA/fisiologia , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/fisiologia , Proteínas de Ligação a Telômeros/metabolismo , Sequência de Bases , DNA Fúngico/genética , DNA Fúngico/metabolismo , Proteínas de Ligação a DNA/metabolismo , Dados de Sequência Molecular , Ligação Proteica , Transporte Proteico , Origem de Replicação/fisiologia , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Ligação a Telômeros/genéticaRESUMO
Controlling the activity of the heterohexameric Mcm2-7 replicative helicase is crucial for regulation of replication origin activity in eukaryotes. Because bidirectional replication forks are generated from every replication origin, when origins are licensed for replication in the first step of DNA replication, two inactive Mcm2-7 heterohexiameric complexes are loaded around double stranded DNA as a head-to-head double hexamer. The helicases are subsequently activated via a 'firing' reaction, in which the Mcm2-7 double hexamer is converted into two active helicase units, the CMG complex, by firing factors. Dimerization of firing factors may contribute to this process by allowing simultaneous activation of two sets of helicases and thus efficient assembly of bidirectional replication forks. An example of this is dimerization of the firing factor Sld3/Treslin/Ticrr via its binding partner, Sld7/MTBP. In organisms in which no Sld7 ortholog has been identified, such as the fission yeast Schizosaccharomyces pombe, Sld3 itself has a dimerization domain, and it has been suggested that this self-interaction is crucial for the firing reaction in this organism. Dimerization induces a conformational change in Sdl3 that appears to be critical for the firing reaction. Moreover, Mcm10 also seems to be regulated by self-interaction in yeasts. Although it is not yet clear to what extent dimerization of firing factors contributes to the firing reaction in eukaryotes, we discuss the possible roles of firing factor dimerization in simultaneous helicase activation.
RESUMO
alpha-Synuclein is one of the causative proteins of the neurodegenerative disorder, Parkinson's disease. Deposits of alpha-synuclein called Lewy bodies are a hallmark of this disorder, which is implicated in its progression. In order to understand the mechanism of amyloid fibril formation of alpha-synuclein in more detail, in this study we have isolated a specific, ~20 residue peptide region of the alpha-synuclein fibril core, using a combination of Edman degradation and mass-spectroscopy analyses of protease-resistant samples. Starting from this core peptide sequence, we then synthesized a series of peptides that undergo aggregation and fibril formation under similar conditions. Interestingly, in a derivative peptide where a crucial phenylalanine residue was changed to a glycine, the ability to initiate spontaneous fibril formation was abolished, while the ability to extend from preexisting fibril seeds was conserved. This fibril extension occurred irrespective of the source of the initial fibril seed, as demonstrated in experiments using fibril seeds of insulin, lysozyme, and GroES. This interesting ability suggests that this peptide might form the basis for a possible diagnostic tool useful in detecting the presence of various fibrillogenic factors.
Assuntos
Amiloide/química , Amiloide/metabolismo , Corpos de Lewy/química , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/isolamento & purificação , alfa-Sinucleína/química , Sequência de Aminoácidos , Benzotiazóis , Dicroísmo Circular , Humanos , Insulina/química , Insulina/metabolismo , Microscopia de Força Atômica , Dados de Sequência Molecular , Fragmentos de Peptídeos/metabolismo , Tiazóis/metabolismo , alfa-Sinucleína/metabolismoRESUMO
Endothelial progenitor cell (EPC) transplantation is beneficial for ischemic diseases such as critical limb ischemia and ischemic heart disease. The scarcity of functional EPCs in adults is a limiting factor for EPC transplantation therapy. The quality and quantity culture (QQc) system is an effective ex vivo method for enhancing the number and angiogenic potential of EPCs. Further, microgravity environments have been shown to enhance the functional potential of stem cells. We therefore hypothesized that cells cultured with QQc under microgravity may have enhanced functionality. We cultured human peripheral blood mononuclear cells using QQc under normal (E), microgravity (MG), or microgravity followed by normal (ME) conditions and found that ME resulted in the most significant increase in CD34+ and double positive Dil-Ac-LDL-FITC-Ulex-Lectin cells, both EPC markers. Furthermore, angiogenic potential was determined by an EPC-colony forming assay. While numbers of primitive EPC-colony forming units (pEPC-CFU) did not change, numbers of definitive EPC-CFU colonies increased most under ME conditions. Gene-expression profiling also identified increases in angiogenic factors, including vascular endothelial growth factor, under MG and ME conditions. Thus, QQc along with ME conditions could be an efficient system for significantly enhancing the number and angiogenic potential of EPCs.
Assuntos
Células Progenitoras Endoteliais/metabolismo , Neovascularização Fisiológica/genética , Ausência de Peso , Antígenos CD34/genética , Técnicas de Cultura de Células , Diferenciação Celular/genética , Diferenciação Celular/efeitos da radiação , Proliferação de Células/efeitos da radiação , Células Progenitoras Endoteliais/fisiologia , Células Progenitoras Endoteliais/efeitos da radiação , Sangue Fetal/efeitos da radiação , Expressão Gênica/genética , Expressão Gênica/efeitos da radiação , Humanos , Leucócitos Mononucleares/fisiologia , Leucócitos Mononucleares/efeitos da radiação , Neovascularização Fisiológica/fisiologia , Neovascularização Fisiológica/efeitos da radiação , Transplante de Células-Tronco/métodos , Células-Tronco/metabolismoRESUMO
Intramolecular nonbonded S...N interactions in the crystal structures of the derivatives (7a-d) of sodium rabeprazole (1) and an intermolecular nonbonded S...N interaction between ethylmethylsulfoxide and pyridine in a solution were recognized. These results made us estimate that the intramolecular nonbonded S...N interaction existed in 1 and its derivatives in a solution, and formed the 4-membered quasi-ring in 2 (Fig. 1) followed by the increase of the reactivity of 2 to give the putative spiro sulfoxide 3, which is the key intermediate in the reaction cascade of 1 (Chart 1).
Assuntos
2-Piridinilmetilsulfinilbenzimidazóis/química , Inibidores da Bomba de Prótons/química , Cristalografia por Raios X , Cisteína/química , Indicadores e Reagentes , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Conformação Molecular , Piridinas , Rabeprazol , Soluções , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas de Bombardeamento Rápido de Átomos , Espectroscopia de Infravermelho com Transformada de Fourier , Sulfamerazina/químicaRESUMO
Tetrasubstituted (Z)-alkenes were readily prepared through the Horner-Wadsworth-Emmons reactions of methyl 2-[bis(2,2,2-trifluoroethyl)phosphono]propionate with aryl alkyl ketones by employing Sn(OSO(2)CF(3))(2) and N-ethylpiperidine.