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The cellular RNA-binding protein EAP recognizes a conserved stem-loop in the Epstein-Barr virus small RNA EBER 1. 总被引:8,自引:2,他引:6
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EAP (EBER-associated protein) is an abundant, 15-kDa cellular RNA-binding protein which associates with certain herpesvirus small RNAs. We have raised polyclonal anti-EAP antibodies against a glutathione S-transferase-EAP fusion protein. Analysis of the RNA precipitated by these antibodies from Epstein-Barr virus (EBV)- or herpesvirus papio (HVP)-infected cells shows that > 95% of EBER 1 (EBV-encoded RNA 1) and the majority of HVP 1 (an HVP small RNA homologous to EBER 1) are associated with EAP. RNase protection experiments performed on native EBER 1 particles with affinity-purified anti-EAP antibodies demonstrate that EAP binds a stem-loop structure (stem-loop 3) of EBER 1. Since bacterially expressed glutathione S-transferase-EAP fusion protein binds EBER 1, we conclude that EAP binding is independent of any other cellular or viral protein. Detailed mutational analyses of stem-loop 3 suggest that EAP recognizes the majority of the nucleotides in this hairpin, interacting with both single-stranded and double-stranded regions in a sequence-specific manner. Binding studies utilizing EBER 1 deletion mutants suggest that there may also be a second, weaker EAP-binding site on stem-loop 4 of EBER 1. These data and the fact that stem-loop 3 represents the most highly conserved region between EBER 1 and HVP 1 suggest that EAP binding is a critical aspect of EBER 1 and HVP 1 function. 相似文献
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Vega LR Phillips JA Thornton BR Benanti JA Onigbanjo MT Toczyski DP Zakian VA 《PLoS genetics》2007,3(6):e105
The Saccharomyces cerevisiae Pif1p helicase is a negative regulator of telomere length that acts by removing telomerase from chromosome ends. The catalytic subunit of yeast telomerase, Est2p, is telomere associated throughout most of the cell cycle, with peaks of association in both G1 phase (when telomerase is not active) and late S/G2 phase (when telomerase is active). The G1 association of Est2p requires a specific interaction between Ku and telomerase RNA. In mutants lacking this interaction, telomeres were longer in the absence of Pif1p than in the presence of wild-type PIF1, indicating that endogenous Pif1p inhibits the active S/G2 form of telomerase. Pif1p abundance was cell cycle regulated, low in G1 and early S phase and peaking late in the cell cycle. Low Pif1p abundance in G1 phase was anaphase-promoting complex dependent. Thus, endogenous Pif1p is unlikely to act on G1 bound Est2p. Overexpression of Pif1p from a non-cell cycle-regulated promoter dramatically reduced viability in five strains with impaired end protection (cdc13–1, yku80Δ, yku70Δ, yku80–1, and yku80–4), all of which have longer single-strand G-tails than wild-type cells. This reduced viability was suppressed by deleting the EXO1 gene, which encodes a nuclease that acts at compromised telomeres, suggesting that the removal of telomerase by Pif1p exposed telomeres to further C-strand degradation. Consistent with this interpretation, depletion of Pif1p, which increases the amount of telomere-bound telomerase, suppressed the temperature sensitivity of yku70Δ and cdc13–1 cells. Furthermore, eliminating the pathway that recruits Est2p to telomeres in G1 phase in a cdc13–1 strain also reduced viability. These data suggest that wild-type levels of telomere-bound telomerase are critical for the viability of strains whose telomeres are already susceptible to degradation. 相似文献
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A unified view of the DNA-damage checkpoint 总被引:36,自引:0,他引:36
Recent investigation of the DNA-damage checkpoint in several organisms has highlighted the conservation of this pathway. The checkpoint's signal transduction pathway consists of four conserved classes of molecules: two large protein kinases having homology to phosphatidylinositol 3-kinases, three "sensor" proteins with homology to proliferating cell nuclear antigen, two serine/threonine (S/T) kinases, and two adaptors for the S/T kinases. This review compares the role of these four classes of checkpoint proteins in humans and model organisms. 相似文献
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Intrinsic protein fluorescence may interfere with the visualization of proteins after SDS-polyacrylamide electrophoresis. In an attempt to analyze tear glycoproteins in gels, we ran tear samples and stained the proteins with a glycoprotein-specific fluorescent dye. The fluorescence detected was not limited to glycoproteins. There was strong intrinsic fluorescence of proteins normally found in tears after soaking the gels in 40% methanol plus 1-10% acetic acid and, to a lesser extent, in methanol or acetic acid alone. Nanograms of proteins gave visible native fluorescence and interfere with extrinsic fluorescent dye detection. Poly-L-lysine, which does not contain intrinsically fluorescent amino acids, did not fluoresce. 相似文献
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Complicated tails: histone modifications and the DNA damage response 总被引:14,自引:0,他引:14
In recent years, several ATP-dependent chromatin-remodeling complexes and covalent histone modifications have been implicated in the response to double-stranded DNA breaks (DSBs). When a DSB occurs, cells must identify the DSB, activate the DNA damage checkpoint, and repair the break. Chromatin modification appears to be important but not essential for each of these processes, yet its precise mechanistic roles are only beginning to come into focus. Here, we discuss the role of chromatin in signaling by the DNA damage checkpoint pathway. 相似文献
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In this issue of Molecular Cell, De Piccoli et al. (2012) show that, contrary to current models of DNA replication checkpoint function, replication proteins remain associated with each other and with replicating DNA when replication is stressed in checkpoint-deficient cells. 相似文献
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A study of bacterial surface oligosaccharides were investigated among
different strains of Neisseria gonorrhoeae to correlate structural features
essential for binding to the MAb 2C7. This epitope is widely expressed and
conserved in gonococcal isolates, characteristics essential to an effective
candidate vaccine antigen. Sample lipooligosaccharides (LOS), was prepared
by a modification of the hot phenol-water method from which de-O-acetylated
LOS and oligosaccharide (OS) components were analyzed by ES-MS-CID-MS and
ES-MSnin a triple quadrupole and an ion trap mass spectrometer,
respectively. Previously documented natural heterogeneity was apparent from
both LOS and OS preparations which was admixed with fragments induced by
hydrazine and mild acid treatment. Natural heterogeneity was limited to
phosphorylation and antenni extensions to the alpha-chain. Mild acid
hydrolysis to release OS also hydrolyzed the beta(1-->6) glycosidic
linkage of lipid A. OS structures were determined by collisional and
resonance excitation combined with MS and multistep MSn which provided
sequence information from both neutral loss, and nonreducing terminal
fragments. A comparison of OS structures, with earlier knowledge of MAb
binding, enzyme treatment, and partial acid hydrolysis indicates a generic
overlapping domain for 2C7 binding. Reoccurring structural features include
a Hepalpha(1-->3)Hepbeta(1-->5)KDO trisaccharide core branched on the
nonreducing terminus (Hep-2) with an alpha(1-->2) linked GlcNAc
(gamma-chain), and an alpha-linked lactose (beta-chain) residue. From the
central heptose (Hep-1), a beta(1-->4) linked lactose (alpha-chain),
moiety is required although extensions to this residue appear unnecessary.
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