170 research outputs found

    The fork protection complex recruits FACT to reorganize nucleosomes during replication

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    Chromosome replication depends on efficient removal of nucleosomes by accessory factors to ensure rapid access to genomic information. Here, we show this process requires recruitment of the nucleosome reorganization activity of the histone chaperone FACT. Using single-molecule FRET, we demonstrate that reorganization of nucleosomal DNA by FACT requires coordinated engagement by the middle and C-terminal domains of Spt16 and Pob3 but does not require the N-terminus of Spt16. Using structure-guided pulldowns, we demonstrate instead that the N-terminal region is critical for recruitment by the fork protection complex subunit Tof1. Using in vitro chromatin replication assays, we confirm the importance of these interactions for robust replication. Our findings support a mechanism in which nucleosomes are removed through the coordinated engagement of multiple FACT domains positioned at the replication fork by the fork protection complex

    Druse clinopyroxene in D'Orbigny angritic meteorite studied by single-crystal X-ray diffraction, electron microprobe analysis and Mössbauer spectroscopy

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    The crystal structure of druse clinopyroxene from the D’Orbigny angrite, (Ca0.944 Fe2+ 0.042 Mg0.010Mn0.004) (Mg0.469Fe2+ 0.317Fe3+ 0.035Al0.125Cr0.010Ti0.044) (Si1.742Al0.258) O6, a = 9.7684(2), b = 8.9124(2), c = 5.2859(1) Å, β = 105.903(1)°, V = 442.58 Å3, space group C2/c, Z = 2, has been refined to an R1 index of 1.92% using single-crystal X-ray diffraction data. The unit formula, calculated from electron microprobe analysis, and the refined site scattering values were used to assign site populations. The distribution of Fe2+ and Mg over the M1 and M2 sites suggests a closure temperature of 1000 °C. Mössbauer spectroscopy measurements were done at room temperature on a single crystal and a powdered sample. The spectra are adequately fit by a Voigt-based quadrupole-splitting distribution model having two generalized sites, one for Fe2+ with two Gaussian components and one for Fe3+ with one Gaussian component. The two ferrous components are assigned to Fe2+ at the M1 site, and arise from two different next-nearest-neighbor configurations of Ca and Fe cations at the M2 site: (3Ca,0Fe) and (2Ca,1Fe). The Fe3+/Fetot ratio determined by Mössbauer spectroscopy is in agreement with that calculated from the electron microprobe analysis. The results are discussed in connection with the redox and thermal history of D’Orbigny.Fil: Abdu, Yassir A.. University of Manitoba; CanadáFil: Scorzelli, Rosa B.. Centro Brasileiro de Pesquisas Físicas; BrasilFil: Varela, Maria Eugenia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - San Juan. Complejo Astronómico "El Leoncito". Universidad Nacional de Córdoba. Complejo Astronómico "El Leoncito". Universidad Nacional de la Plata. Complejo Astronómico "El Leoncito". Universidad Nacional de San Juan. Complejo Astronómico "El Leoncito"; ArgentinaFil: Kurat, Gero. Naturhistorisches Museum; AustriaFil: Souza Azevedo, Izabel de. Centro Brasileiro de Pesquisas Físicas; BrasilFil: Stewart, Silvana Jacqueline. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Física La Plata. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Física La Plata; ArgentinaFil: Hawthorne, Frank C.. University of Manitoba; Canad

    A quantitative literature-curated gold standard for kinase-substrate pairs

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    We describe the Yeast Kinase Interaction Database (KID, http://www.moseslab.csb.utoronto.ca/KID/), which contains high- and low-throughput data relevant to phosphorylation events. KID includes 6,225 low-throughput and 21,990 high-throughput interactions, from greater than 35,000 experiments. By quantitatively integrating these data, we identified 517 high-confidence kinase-substrate pairs that we consider a gold standard. We show that this gold standard can be used to assess published high-throughput datasets, suggesting that it will enable similar rigorous assessments in the future

    A CDK-regulated chromatin segregase promoting chromosome replication

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    The replication of chromosomes during S phase is critical for cellular and organismal function. Replicative stress can result in genome instability, which is a major driver of cancer. Yet how chromatin is made accessible during eukaryotic DNA synthesis is poorly understood. Here, we report the characterization of a chromatin remodeling enzyme-Yta7-entirely distinct from classical SNF2-ATPase family remodelers. Yta7 is a AAA+ -ATPase that assembles into \~1 MDa hexameric complexes capable of segregating histones from DNA. The Yta7 chromatin segregase promotes chromosome replication both in vivo and in vitro. Biochemical reconstitution experiments using purified proteins revealed that the enzymatic activity of Yta7 is regulated by S phase-forms of Cyclin-Dependent Kinase (S-CDK). S-CDK phosphorylation stimulates ATP hydrolysis by Yta7, promoting nucleosome disassembly and chromatin replication. Our results present a mechanism for how cells orchestrate chromatin dynamics in co-ordination with the cell cycle machinery to promote genome duplication during S phase

    Druse clinopyroxene in D'Orbigny angritic meteorite studied by single-crystal X-ray diffraction, electron microprobe analysis and Mössbauer spectroscopy

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    The crystal structure of druse clinopyroxene from the D'Orbigny angrite, (Ca0.944 Fe 2+ 0.042 Mg0.010Mn0.004) (Mg0.469Fe 2+ 0.317Fe 3+ 0.035Al0.125Cr0.010Ti0.044) (Si1.742Al0.258) O6, a = 9.7684(2), b = 8.9124(2), c = 5.2859(1) A, β = 105.903(1)°, V = 442.58 A 3 , space group C2/c, Z = 2, has been refined to an R1 index of 1.92% using single-crystal X-ray diffraction data. The unit formula, calculated from electron microprobe analysis, and the refined site scattering values were used to assign site populations. The distribution of Fe 2+ and Mg over the M1 and M2 sites suggests a closure temperature of 1000 °C. Mossbauer spectroscopy measurements were done at room temperature on a single crystal and a powdered sample. The spectra are adequately fit by a Voigt-based quadrupole-splitting distribution model having two generalized sites, one for Fe 2+ with two Gaussian components and one for Fe 3+ with one Gaussian component. The two ferrous components are assigned to Fe 2+ at the M1 site, and arise from two different next-nearest-neighbor configurations of Ca and Fe cations at the M2 site: (3Ca,0Fe) and (2Ca,1Fe). The Fe 3+ /Fetot ratio determined by Mossbauer spectroscopy is in agreement with that calculated from the electron microprobe analysis. The results are discussed in connection with the redox and thermal history of D'Orbigny.Instituto de Física La Plat

    Flexibility of a Eukaryotic Lipidome – Insights from Yeast Lipidomics

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    Mass spectrometry-based shotgun lipidomics has enabled the quantitative and comprehensive assessment of cellular lipid compositions. The yeast Saccharomyces cerevisiae has proven to be a particularly valuable experimental system for studying lipid-related cellular processes. Here, by applying our shotgun lipidomics platform, we investigated the influence of a variety of commonly used growth conditions on the yeast lipidome, including glycerophospholipids, triglycerides, ergosterol as well as complex sphingolipids. This extensive dataset allowed for a quantitative description of the intrinsic flexibility of a eukaryotic lipidome, thereby providing new insights into the adjustments of lipid biosynthetic pathways. In addition, we established a baseline for future lipidomic experiments in yeast. Finally, flexibility of lipidomic features is proposed as a new parameter for the description of the physiological state of an organism

    A Role for Phosphatidic Acid in the Formation of “Supersized” Lipid Droplets

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    Lipid droplets (LDs) are important cellular organelles that govern the storage and turnover of lipids. Little is known about how the size of LDs is controlled, although LDs of diverse sizes have been observed in different tissues and under different (patho)physiological conditions. Recent studies have indicated that the size of LDs may influence adipogenesis, the rate of lipolysis and the oxidation of fatty acids. Here, a genome-wide screen identifies ten yeast mutants producing “supersized” LDs that are up to 50 times the volume of those in wild-type cells. The mutated genes include: FLD1, which encodes a homologue of mammalian seipin; five genes (CDS1, INO2, INO4, CHO2, and OPI3) that are known to regulate phospholipid metabolism; two genes (CKB1 and CKB2) encoding subunits of the casein kinase 2; and two genes (MRPS35 and RTC2) of unknown function. Biochemical and genetic analyses reveal that a common feature of these mutants is an increase in the level of cellular phosphatidic acid (PA). Results from in vivo and in vitro analyses indicate that PA may facilitate the coalescence of contacting LDs, resulting in the formation of “supersized” LDs. In summary, our results provide important insights into how the size of LDs is determined and identify novel gene products that regulate phospholipid metabolism
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