WorldWideScience

Sample records for regulates helicase access

  1. [RTEL1 (regulator of telomere elongation helicase 1), a DNA helicase essential for genome stability].

    Science.gov (United States)

    Le Guen, Tangui; Jullien, Laurent; Schertzer, Mike; Lefebvre, Axelle; Kermasson, Laetitia; de Villartay, Jean-Pierre; Londoño-Vallejo, Arturo; Revy, Patrick

    2013-12-01

    RTEL1 (regulator of telomere length helicase 1) is a DNA helicase that has been identified more than 10 years ago. Many works since, mainly in the nematode Caenorhabditis elegans and the mouse, have highlighted its role in chromosomal stability, maintenance of telomere length, and DNA repair. Recently, four laboratories have characterized RTEL1 mutations in patients with dyskeratosis congenita (DC) and Hoyeraal-Hreidarsson (HH) syndrome, a rare and severe variant of DC. We here summarize the current knowledge on RTEL1 and discuss the possible other functions that RTEL1 could play. © 2013 médecine/sciences – Inserm.

  2. A new role for FBP21 as regulator of Brr2 helicase activity.

    Science.gov (United States)

    Henning, Lisa M; Santos, Karine F; Sticht, Jana; Jehle, Stefanie; Lee, Chung-Tien; Wittwer, Malte; Urlaub, Henning; Stelzl, Ulrich; Wahl, Markus C; Freund, Christian

    2017-07-27

    Splicing of eukaryotic pre-mRNA is carried out by the spliceosome, which assembles stepwise on each splicing substrate. This requires the concerted action of snRNPs and non-snRNP accessory proteins, the functions of which are often not well understood. Of special interest are B complex factors that enter the spliceosome prior to catalytic activation and may alter splicing kinetics and splice site selection. One of these proteins is FBP21, for which we identified several spliceosomal binding partners in a yeast-two-hybrid screen, among them the RNA helicase Brr2. Biochemical and biophysical analyses revealed that an intrinsically disordered region of FBP21 binds to an extended surface of the C-terminal Sec63 unit of Brr2. Additional contacts in the C-terminal helicase cassette are required for allosteric inhibition of Brr2 helicase activity. Furthermore, the direct interaction between FBP21 and the U4/U6 di-snRNA was found to reduce the pool of unwound U4/U6 di-snRNA. Our results suggest FBP21 as a novel key player in the regulation of Brr2. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  3. Dna2 nuclease-helicase structure, mechanism and regulation by Rpa.

    Science.gov (United States)

    Zhou, Chun; Pourmal, Sergei; Pavletich, Nikola P

    2015-11-02

    The Dna2 nuclease-helicase maintains genomic integrity by processing DNA double-strand breaks, Okazaki fragments and stalled replication forks. Dna2 requires ssDNA ends, and is dependent on the ssDNA-binding protein Rpa, which controls cleavage polarity. Here we present the 2.3 Å structure of intact mouse Dna2 bound to a 15-nucleotide ssDNA. The nuclease active site is embedded in a long, narrow tunnel through which the DNA has to thread. The helicase domain is required for DNA binding but not threading. We also present the structure of a flexibly-tethered Dna2-Rpa interaction that recruits Dna2 to Rpa-coated DNA. We establish that a second Dna2-Rpa interaction is mutually exclusive with Rpa-DNA interactions and mediates the displacement of Rpa from ssDNA. This interaction occurs at the nuclease tunnel entrance and the 5' end of the Rpa-DNA complex. Hence, it only displaces Rpa from the 5' but not 3' end, explaining how Rpa regulates cleavage polarity.

  4. Association between regulator of telomere elongation helicase1 (RTEL1) gene and HAPE risk

    Science.gov (United States)

    Rong, Hao; He, Xue; Zhu, Linhao; Zhu, Xikai; Kang, Longli; Wang, Li; He, Yongjun; Yuan, Dongya; Jin, Tianbo

    2017-01-01

    Abstract High altitude pulmonary edema (HAPE) is a paradigm of pulmonary edema. Mutations in regulator of telomere elongation helicase1 (RTEL1) represent an important contributor to risk for pulmonary fibrosis. However, little information is found about the association between RTEL1 and HAPE risk. The present study was undertaken to tentatively explore the potential relation between single-nucleotide polymorphisms (SNPs) in RTEL1 and HAPE risk in Chinese Han population. A total of 265 HAPE patients and 303 healthy controls were included in our case-control study. Four SNPs in RTEL1 were selected and genotyped using the Sequenom MassARRAY method. Odds ratios (ORs) and 95% confidence intervals (95% CIs) were calculated by unconditional logistic regression with adjustment for gender and age. All P values were Bonferroni corrected, and statistical significance was set at P RTEL1 and a decreased risk HAPE in the Chinese population. The results need further confirmation. PMID:28953687

  5. Chl1 DNA helicase regulates Scc2 deposition specifically during DNA-replication in Saccharomyces cerevisiae.

    Directory of Open Access Journals (Sweden)

    Soumya Rudra

    Full Text Available The conserved family of cohesin proteins that mediate sister chromatid cohesion requires Scc2, Scc4 for chromatin-association and Eco1/Ctf7 for conversion to a tethering competent state. A popular model, based on the notion that cohesins form huge ring-like structures, is that Scc2, Scc4 function is essential only during G1 such that sister chromatid cohesion results simply from DNA replisome passage through pre-loaded cohesin rings. In such a scenario, cohesin deposition during G1 is temporally uncoupled from Eco1-dependent establishment reactions that occur during S-phase. Chl1 DNA helicase (homolog of human ChlR1/DDX11 and BACH1/BRIP1/FANCJ helicases implicated in Fanconi anemia, breast and ovarian cancer and Warsaw Breakage Syndrome plays a critical role in sister chromatid cohesion, however, the mechanism through which Chl1 promotes cohesion remains poorly understood. Here, we report that Chl1 promotes Scc2 loading unto DNA such that both Scc2 and cohesin enrichment to chromatin are defective in chl1 mutant cells. The results further show that both Chl1 expression and chromatin-recruitment are tightly regulated through the cell cycle, peaking during S-phase. Importantly, kinetic ChIP studies reveals that Chl1 is required for Scc2 chromatin-association specifically during S-phase, but not during G1. Despite normal chromatin enrichment of both Scc2 and cohesin during G1, chl1 mutant cells exhibit severe chromosome segregation and cohesion defects--revealing that G1-loaded cohesins is insufficient to promote cohesion. Based on these findings, we propose a new model wherein S-phase cohesin loading occurs during DNA replication and in concert with both cohesion establishment and chromatin assembly reactions--challenging the notion that DNA replication fork navigates through or around pre-loaded cohesin rings.

  6. Translational control by the DEAD Box RNA helicase belle regulates ecdysone-triggered transcriptional cascades.

    Directory of Open Access Journals (Sweden)

    Robert J Ihry

    Full Text Available Steroid hormones act, through their respective nuclear receptors, to regulate target gene expression. Despite their critical role in development, physiology, and disease, however, it is still unclear how these systemic cues are refined into tissue-specific responses. We identified a mutation in the evolutionarily conserved DEAD box RNA helicase belle/DDX3 that disrupts a subset of responses to the steroid hormone ecdysone during Drosophila melanogaster metamorphosis. We demonstrate that belle directly regulates translation of E74A, an ets transcription factor and critical component of the ecdysone-induced transcriptional cascade. Although E74A mRNA accumulates to abnormally high levels in belle mutant tissues, no E74A protein is detectable, resulting in misregulation of E74A-dependent ecdysone response genes. The accumulation of E74A mRNA in belle mutant salivary glands is a result of auto-regulation, fulfilling a prediction made by Ashburner nearly 40 years ago. In this model, Ashburner postulates that, in addition to regulating secondary response genes, protein products of primary response genes like E74A also inhibit their own ecdysone-induced transcription. Moreover, although ecdysone-triggered transcription of E74A appears to be ubiquitous during metamorphosis, belle-dependent translation of E74A mRNA is spatially restricted. These results demonstrate that translational control plays a critical, and previously unknown, role in refining transcriptional responses to the steroid hormone ecdysone.

  7. Human SUV3 helicase regulates growth rate of the HeLa cells and can localize in the nucleoli.

    Science.gov (United States)

    Szewczyk, Maciej; Fedoryszak-Kuśka, Natalia; Tkaczuk, Katarzyna; Dobrucki, Jurek; Waligórska, Agnieszka; Stępień, Piotr P

    2017-01-01

    The human SUV3 helicase (SUV3, hSUV3, SUPV3L1) is a DNA/RNA unwinding enzyme belonging to the class of DexH-box helicases. It localizes predominantly in the mitochondria, where it forms an RNA-degrading complex called mitochondrial degradosome with exonuclease PNP (polynucleotide phosphorylase). Association of this complex with the polyA polymerase can modulate mitochondrial polyA tails. Silencing of the SUV3 gene was shown to inhibit the cell cycle and to induce apoptosis in human cell lines. However, since small amounts of the SUV3 helicase were found in the cell nuclei, it was not clear whether the observed phenotypes of SUV3 depletion were of mitochondrial or nuclear origin. In order to answer this question we have designed gene constructs able to inhibit the SUV3 activity exclusively in the cell nuclei. The results indicate that the observed growth rate impairment upon SUV3 depletion is due to its nuclear function(s). Unexpectedly, overexpression of the nuclear-targeted wild-type copies of the SUV3 gene resulted in a higher growth rate. In addition, we demonstrate that the SUV3 helicase can be found in the HeLa cell nucleoli, but it is not detectable in the DNA-repair foci. Our results indicate that the nucleolar-associated human SUV3 protein is an important factor in regulation of the cell cycle.

  8. Human regulator of telomere elongation helicase 1 (RTEL1) is required for the nuclear and cytoplasmic trafficking of pre-U2 RNA

    OpenAIRE

    Schertzer , Michael; Jouravleva , Karina; Perderiset , Mylène; Dingli , Florent; Loew , Damarys; Le Guen , Tangui; Bardoni , Barbara; De Villartay , Jean-Pierre; Revy , Patrick; Londono-Vallejo , Arturo

    2015-01-01

    International audience; Hoyeraal-Hreidarsson syndrome (HHS) is a severe form of Dyskeratosis congenita characterized by developmental defects, bone marrow failure and im-munodeficiency and has been associated with telom-ere dysfunction. Recently, mutations in Regulator of Telomere ELongation helicase 1 (RTEL1), a helicase first identified in Mus musculus as being responsible for the maintenance of long telomeres, have been identified in several HHS patients. Here we show that RTEL1 is require...

  9. RTEL1: an essential helicase for telomere maintenance and the regulation of homologous recombination

    OpenAIRE

    Uringa, Evert-Jan; Youds, Jillian L.; Lisaingo, Kathleen; Lansdorp, Peter M.; Boulton, Simon J.

    2010-01-01

    Telomere maintenance and DNA repair are crucial processes that protect the genome against instability. RTEL1, an essential iron–sulfur cluster-containing helicase, is a dominant factor that controls telomere length in mice and is required for telomere integrity. In addition, RTEL1 promotes synthesis-dependent strand annealing to direct DNA double-strand breaks into non-crossover outcomes during mitotic repair and in meiosis. Here, we review the role of RTEL1 in telomere maintenance and homolo...

  10. RTEL1: an essential helicase for telomere maintenance and the regulation of homologous recombination.

    Science.gov (United States)

    Uringa, Evert-Jan; Youds, Jillian L; Lisaingo, Kathleen; Lansdorp, Peter M; Boulton, Simon J

    2011-03-01

    Telomere maintenance and DNA repair are crucial processes that protect the genome against instability. RTEL1, an essential iron-sulfur cluster-containing helicase, is a dominant factor that controls telomere length in mice and is required for telomere integrity. In addition, RTEL1 promotes synthesis-dependent strand annealing to direct DNA double-strand breaks into non-crossover outcomes during mitotic repair and in meiosis. Here, we review the role of RTEL1 in telomere maintenance and homologous recombination and discuss models linking RTEL1's enzymatic activity to its function in telomere maintenance and DNA repair.

  11. Mcm10 regulates DNA replication elongation by stimulating the CMG replicative helicase.

    Science.gov (United States)

    Lõoke, Marko; Maloney, Michael F; Bell, Stephen P

    2017-02-01

    Activation of the Mcm2-7 replicative DNA helicase is the committed step in eukaryotic DNA replication initiation. Although Mcm2-7 activation requires binding of the helicase-activating proteins Cdc45 and GINS (forming the CMG complex), an additional protein, Mcm10, drives initial origin DNA unwinding by an unknown mechanism. We show that Mcm10 binds a conserved motif located between the oligonucleotide/oligosaccharide fold (OB-fold) and A subdomain of Mcm2. Although buried in the interface between these domains in Mcm2-7 structures, mutations predicted to separate the domains and expose this motif restore growth to conditional-lethal MCM10 mutant cells. We found that, in addition to stimulating initial DNA unwinding, Mcm10 stabilizes Cdc45 and GINS association with Mcm2-7 and stimulates replication elongation in vivo and in vitro. Furthermore, we identified a lethal allele of MCM10 that stimulates initial DNA unwinding but is defective in replication elongation and CMG binding. Our findings expand the roles of Mcm10 during DNA replication and suggest a new model for Mcm10 function as an activator of the CMG complex throughout DNA replication. © 2017 Lõoke et al.; Published by Cold Spring Harbor Laboratory Press.

  12. Germline mutations of regulator of telomere elongation helicase 1, RTEL1, in Dyskeratosis congenita.

    Science.gov (United States)

    Ballew, Bari J; Yeager, Meredith; Jacobs, Kevin; Giri, Neelam; Boland, Joseph; Burdett, Laurie; Alter, Blanche P; Savage, Sharon A

    2013-04-01

    Dyskeratosis congenita (DC) is an inherited bone marrow failure and cancer predisposition syndrome caused by aberrant telomere biology. The classic triad of dysplastic nails, abnormal skin pigmentation, and oral leukoplakia is diagnostic of DC, but substantial clinical heterogeneity exists; the clinically severe variant Hoyeraal Hreidarsson syndrome (HH) also includes cerebellar hypoplasia, severe immunodeficiency, enteropathy, and intrauterine growth retardation. Germline mutations in telomere biology genes account for approximately one-half of known DC families. Using exome sequencing, we identified mutations in RTEL1, a helicase with critical telomeric functions, in two families with HH. In the first family, two siblings with HH and very short telomeres inherited a premature stop codon from their mother who has short telomeres. The proband from the second family has HH and inherited a premature stop codon in RTEL1 from his father and a missense mutation from his mother, who also has short telomeres. In addition, inheritance of only the missense mutation led to very short telomeres in the proband's brother. Targeted sequencing identified a different RTEL1 missense mutation in one additional DC proband who has bone marrow failure and short telomeres. Both missense mutations affect the helicase domain of RTEL1, and three in silico prediction algorithms suggest that they are likely deleterious. The nonsense mutations both cause truncation of the RTEL1 protein, resulting in loss of the PIP box; this may abrogate an important protein-protein interaction. These findings implicate a new telomere biology gene, RTEL1, in the etiology of DC.

  13. Association between regulator of telomere elongation helicase1 (RTEL1) gene and HAPE risk: A case-control study.

    Science.gov (United States)

    Rong, Hao; He, Xue; Zhu, Linhao; Zhu, Xikai; Kang, Longli; Wang, Li; He, Yongjun; Yuan, Dongya; Jin, Tianbo

    2017-09-01

    High altitude pulmonary edema (HAPE) is a paradigm of pulmonary edema. Mutations in regulator of telomere elongation helicase1 (RTEL1) represent an important contributor to risk for pulmonary fibrosis. However, little information is found about the association between RTEL1 and HAPE risk. The present study was undertaken to tentatively explore the potential relation between single-nucleotide polymorphisms (SNPs) in RTEL1 and HAPE risk in Chinese Han population. A total of 265 HAPE patients and 303 healthy controls were included in our case-control study. Four SNPs in RTEL1 were selected and genotyped using the Sequenom MassARRAY method. Odds ratios (ORs) and 95% confidence intervals (95% CIs) were calculated by unconditional logistic regression with adjustment for gender and age. All P values were Bonferroni corrected, and statistical significance was set at P RTEL1 and a decreased risk HAPE in the Chinese population. The results need further confirmation.

  14. A temperature-sensitive allele of a putative mRNA splicing helicase down-regulates many cell wall genes and causes radial swelling in Arabidopsis thaliana.

    Science.gov (United States)

    Howles, Paul A; Gebbie, Leigh K; Collings, David A; Varsani, Arvind; Broad, Ronan C; Ohms, Stephen; Birch, Rosemary J; Cork, Ann H; Arioli, Tony; Williamson, Richard E

    2016-05-01

    The putative RNA helicase encoded by the Arabidopsis gene At1g32490 is a homolog of the yeast splicing RNA helicases Prp2 and Prp22. We isolated a temperature-sensitive allele (rsw12) of the gene in a screen for root radial swelling mutants. Plants containing this allele grown at the restrictive temperature showed weak radial swelling, were stunted with reduced root elongation, and contained reduced levels of cellulose. The role of the protein was further explored by microarray analysis. By using both fold change cutoffs and a weighted gene coexpression network analysis (WGCNA) to investigate coexpression of genes, we found that the radial swelling phenotype was not linked to genes usually associated with primary cell wall biosynthesis. Instead, the mutation has strong effects on expression of secondary cell wall related genes. Many genes potentially associated with secondary walls were present in the most significant WGCNA module, as were genes coding for arabinogalactans and proteins with GPI anchors. The proportion of up-regulated genes that possess introns in rsw12 was above that expected if splicing was unrelated to the activity of the RNA helicase, suggesting that the helicase does indeed play a role in splicing in Arabidopsis. The phenotype may be due to a change in the expression of one or more genes coding for cell wall proteins.

  15. RNA Helicase DDX5 Regulates MicroRNA Expression and Contributes to Cytoskeletal Reorganization in Basal Breast Cancer Cells

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Daojing; Huang, Jing; Hu, Zhi

    2011-11-15

    RNA helicase DDX5 (also p68) is involved in all aspects of RNA metabolism and serves as a transcriptional co-regulator, but its functional role in breast cancer remains elusive. Here, we report an integrative biology study of DDX5 in breast cancer, encompassing quantitative proteomics, global MicroRNA profiling, and detailed biochemical characterization of cell lines and human tissues. We showed that protein expression of DDX5 increased progressively from the luminal to basal breast cancer cell lines, and correlated positively with that of CD44 in the basal subtypes. Through immunohistochemistry analyses of tissue microarrays containing over 200 invasive human ductal carcinomas, we observed that DDX5 was upregulated in the majority of malignant tissues, and its expression correlated strongly with those of Ki67 and EGFR in the triple-negative tumors. We demonstrated that DDX5 regulated a subset of MicroRNAs including miR-21 and miR-182 in basal breast cancer cells. Knockdown of DDX5 resulted in reorganization of actin cytoskeleton and reduction of cellular proliferation. The effects were accompanied by upregulation of tumor suppressor PDCD4 (a known miR-21 target); as well as upregulation of cofilin and profilin, two key proteins involved in actin polymerization and cytoskeleton maintenance, as a consequence of miR-182 downregulation. Treatment with miR-182 inhibitors resulted in morphologic phenotypes resembling those induced by DDX5 knockdown. Using bioinformatics tools for pathway and network analyses, we confirmed that the network for regulation of actin cytoskeleton was predominantly enriched for the predicted downstream targets of miR-182. Our results reveal a new functional role of DDX5 in breast cancer via the DDX5→miR-182→actin cytoskeleton pathway, and suggest the potential clinical utility of DDX5 and its downstream MicroRNAs in the theranostics of breast cancer.

  16. Regulator of telomere elongation helicase 1 (RTEL1) rs6010620 polymorphism contribute to increased risk of glioma.

    Science.gov (United States)

    Zhao, Wei; Bian, Yusong; Zhu, Wei; Zou, Peng; Tang, Guotai

    2014-06-01

    Regulator of telomere elongation helicase 1 (RTEL1) is critical for genome stability and tumor avoidance. Many studies have reported the associations of RTEL1 rs6010620 with glioma risk, but individually published results were inconclusive. This meta-analysis was performed to quantitatively summarize the evidence for such a relationship. The PubMed, Embase, and Web of Science were systematically searched to identify relevant studies. The odds ratio (OR) and 95 % confidence interval (95 % CI) were computed to estimate the strength of the association using a fixed or random effects model. Ten studies were eligible for meta-analysis including data on glioma with 6,490 cases and 9,288 controls. Overall, there was a significant association between RTEL1 rs6010620 polymorphism and glioma risk in all four genetic models (GG vs. AA: OR=1.87, 95 % CI=1.60-2.18, P heterogeneity=0.552; GA vs. AA: OR=1.30, 95 % CI=1.16-1.46, P heterogeneity=0.495; dominant model-GG+GA vs. AA: OR=1.46, 95 % CI=1.31-1.63, P heterogeneity=0.528; recessive model-GG vs. GA+AA: OR=1.36, 95 % CI=1.27-1.46, P heterogeneity=0.093). Subgroup analyses by ethnicity showed that RTEL1 rs6010620 polymorphism resulted in a higher risk of glioma among both Asians and Caucasians. In the stratified analysis by ethnicity and source of controls, significantly increased risk was observed for Asians and Europeans in all genetic models, population-based studies in all genetic models, and hospital-based studies in three genetic models (heterozygote comparison, homozygote comparison, and dominant model). Our meta-analysis suggested that RTEL1 rs6010620 polymorphism is likely to be associated with increased glioma risk, which lends further biological plausibility to these findings.

  17. Regulation of gene expression by the BLM helicase correlates with the presence of G-quadruplex DNA motifs

    DEFF Research Database (Denmark)

    Nguyen, Giang Huong; Tang, Weiliang; Robles, Ana I

    2014-01-01

    Bloom syndrome is a rare autosomal recessive disorder characterized by genetic instability and cancer predisposition, and caused by mutations in the gene encoding the Bloom syndrome, RecQ helicase-like (BLM) protein. To determine whether altered gene expression might be responsible for pathologic...

  18. Human regulator of telomere elongation helicase 1 (RTEL1) is required for the nuclear and cytoplasmic trafficking of pre-U2 RNA.

    Science.gov (United States)

    Schertzer, Michael; Jouravleva, Karina; Perderiset, Mylene; Dingli, Florent; Loew, Damarys; Le Guen, Tangui; Bardoni, Barbara; de Villartay, Jean-Pierre; Revy, Patrick; Londoño-Vallejo, Arturo

    2015-02-18

    Hoyeraal-Hreidarsson syndrome (HHS) is a severe form of Dyskeratosis congenita characterized by developmental defects, bone marrow failure and immunodeficiency and has been associated with telomere dysfunction. Recently, mutations in Regulator of Telomere ELongation helicase 1 (RTEL1), a helicase first identified in Mus musculus as being responsible for the maintenance of long telomeres, have been identified in several HHS patients. Here we show that RTEL1 is required for the export and the correct cytoplasmic trafficking of the small nuclear (sn) RNA pre-U2, a component of the major spliceosome complex. RTEL1-HHS cells show abnormal subcellular partitioning of pre-U2, defects in the recycling of ribonucleotide proteins (RNP) in the cytoplasm and splicing defects. While most of these phenotypes can be suppressed by re-expressing the wild-type protein in RTEL1-HHS cells, expression of RTEL1 mutated variants in immortalized cells provokes cytoplasmic mislocalizations of pre-U2 and other RNP components, as well as splicing defects, thus phenocopying RTEL1-HHS cellular defects. Strikingly, expression of a cytoplasmic form of RTEL1 is sufficient to correct RNP mislocalizations both in RTEL1-HHS cells and in cells expressing nuclear mutated forms of RTEL1. This work unravels completely unanticipated roles for RTEL1 in RNP trafficking and strongly suggests that defects in RNP biogenesis pathways contribute to the pathology of HHS. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  19. Structural basis for the function of DEAH helicases

    DEFF Research Database (Denmark)

    He, Yangzi; Andersen, Gregers Rom; Nielsen, Klaus Hvid

    2010-01-01

    DEAH helicases participate in pre‐messenger RNA splicing and ribosome biogenesis. The structure of yeast Prp43p‐ADP reveals the homology of DEAH helicases to DNA helicases and the presence of an oligonucleotide‐binding motif. A β‐hairpin from the second RecA domain is wedged between two carboxy......‐terminal domains and blocks access to the occluded RNA binding site formed by the RecA domains and a C‐terminal domain. ATP binding and hydrolysis are likely to induce conformational changes in the hairpin that are important for RNA unwinding or ribonucleoprotein remodelling. The structure of Prp43p provides...

  20. RecQ Helicases

    DEFF Research Database (Denmark)

    Larsen, Nicolai Balle; Hickson, Ian D

    2013-01-01

    The RecQ family of DNA helicases is highly conserved throughout -evolution, and is important for the maintenance of genome stability. In humans, five RecQ family members have been identified: BLM, WRN, RECQ4, RECQ1 and RECQ5. Defects in three of these give rise to Bloom's syndrome (BLM), Werner...

  1. Overcoming natural replication barriers: differential helicase requirements.

    Science.gov (United States)

    Anand, Ranjith P; Shah, Kartik A; Niu, Hengyao; Sung, Patrick; Mirkin, Sergei M; Freudenreich, Catherine H

    2012-02-01

    DNA sequences that form secondary structures or bind protein complexes are known barriers to replication and potential inducers of genome instability. In order to determine which helicases facilitate DNA replication across these barriers, we analyzed fork progression through them in wild-type and mutant yeast cells, using 2-dimensional gel-electrophoretic analysis of the replication intermediates. We show that the Srs2 protein facilitates replication of hairpin-forming CGG/CCG repeats and prevents chromosome fragility at the repeat, whereas it does not affect replication of G-quadruplex forming sequences or a protein-bound repeat. Srs2 helicase activity is required for hairpin unwinding and fork progression. Also, the PCNA binding domain of Srs2 is required for its in vivo role of replication through hairpins. In contrast, the absence of Sgs1 or Pif1 helicases did not inhibit replication through structural barriers, though Pif1 did facilitate replication of a telomeric protein barrier. Interestingly, replication through a protein barrier but not a DNA structure barrier was modulated by nucleotide pool levels, illuminating a different mechanism by which cells can regulate fork progression through protein-mediated stall sites. Our analyses reveal fundamental differences in the replication of DNA structural versus protein barriers, with Srs2 helicase activity exclusively required for fork progression through hairpin structures.

  2. The Smc5/6 complex regulates the yeast Mph1 helicase at RNA-DNA hybrid-mediated DNA damage

    DEFF Research Database (Denmark)

    Lafuente-Barquero, Juan; Luke-Glaser, Sarah; Graf, Marco

    2017-01-01

    of Fanconi anemia protein M (FANCM), is required for cell viability in the absence of RNase H enzymes. The integrity of the Mph1 helicase domain is crucial to prevent the accumulation of RNA-DNA hybrids and RNA-DNA hybrid-dependent DNA damage, as determined by Rad52 foci. Mph1 forms foci when RNA-DNA hybrids...

  3. DEAD-box helicase DDX27 regulates 3′ end formation of ribosomal 47S RNA and stably associates with the PeBoW-complex

    Energy Technology Data Exchange (ETDEWEB)

    Kellner, Markus; Rohrmoser, Michaela [Department of Molecular Epigenetics, Helmholtz Center Munich, Center for Integrated Protein Science Munich (CIPSM), Marchioninistr. 25, Munich 81377 (Germany); Forné, Ignasi [Adolf Butenandt Institute, Ludwig Maximilians University of Munich, Center for Integrated Protein Science Munich (CIPSM), Schillerstr. 44, Munich 80336 (Germany); Voss, Kirsten; Burger, Kaspar; Mühl, Bastian; Gruber-Eber, Anita [Department of Molecular Epigenetics, Helmholtz Center Munich, Center for Integrated Protein Science Munich (CIPSM), Marchioninistr. 25, Munich 81377 (Germany); Kremmer, Elisabeth [Institute of Molecular Immunology, Helmholtz Center Munich, Marchioninistr. 25, Munich 81377 (Germany); Imhof, Axel [Adolf Butenandt Institute, Ludwig Maximilians University of Munich, Center for Integrated Protein Science Munich (CIPSM), Schillerstr. 44, Munich 80336 (Germany); Eick, Dirk, E-mail: eick@helmholtz-muenchen.de [Department of Molecular Epigenetics, Helmholtz Center Munich, Center for Integrated Protein Science Munich (CIPSM), Marchioninistr. 25, Munich 81377 (Germany)

    2015-05-15

    PeBoW, a trimeric complex consisting of pescadillo (Pes1), block of proliferation (Bop1), and the WD repeat protein 12 (WDR12), is essential for processing and maturation of mammalian 5.8S and 28S ribosomal RNAs. Applying a mass spectrometric analysis, we identified the DEAD-box helicase DDX27 as stably associated factor of the PeBoW-complex. DDX27 interacts with the PeBoW-complex via an evolutionary conserved F×F motif in the N-terminal domain and is recruited to the nucleolus via its basic C-terminal domain. This recruitment is RNA-dependent and occurs independently of the PeBoW-complex. Interestingly, knockdown of DDX27, but not of Pes1, induces the accumulation of an extended form of the primary 47S rRNA. We conclude that DDX27 can interact specifically with the Pes1 and Bop1 but fulfils critical function(s) for proper 3′ end formation of 47S rRNA independently of the PeBoW-complex. - Highlights: • DEAD-box helicase DDX27 is a new constituent of the PeBoW-complex. • The N-terminal F×F motif of DDX27 interacts with the PeBoW components Pes1 and Bop1. • Nucleolar anchoring of DDX27 via its basic C-terminal domain is RNA dependent. • Knockdown of DDX27 induces a specific defect in 3′ end formation of 47S rRNA.

  4. Viral hijacking of a replicative helicase loader and its implications for helicase loading control and phage replication

    Energy Technology Data Exchange (ETDEWEB)

    Hood, Iris V.; Berger, James M.

    2016-05-31

    Replisome assembly requires the loading of replicative hexameric helicases onto origins by AAA+ ATPases. How loader activity is appropriately controlled remains unclear. Here, we use structural and biochemical analyses to establish how an antimicrobial phage protein interferes with the function of theStaphylococcus aureusreplicative helicase loader, DnaI. The viral protein binds to the loader’s AAA+ ATPase domain, allowing binding of the host replicative helicase but impeding loader self-assembly and ATPase activity. Close inspection of the complex highlights an unexpected locus for the binding of an interdomain linker element in DnaI/DnaC-family proteins. We find that the inhibitor protein is genetically coupled to a phage-encoded homolog of the bacterial helicase loader, which we show binds to the host helicase but not to the inhibitor itself. These findings establish a new approach by which viruses can hijack host replication processes and explain how loader activity is internally regulated to prevent aberrant auto-association.

  5. Mitochondrial helicases and mitochondrial genome maintenance

    DEFF Research Database (Denmark)

    de Souza-Pinto, Nadja C; Aamann, Maria Diget; Kulikowicz, Tomasz

    2010-01-01

    Helicases are essential enzymes that utilize the energy of nucleotide hydrolysis to drive unwinding of nucleic acid duplexes. Helicases play roles in all aspects of DNA metabolism including DNA repair, DNA replication and transcription. The subcellular locations and functions of several helicases...

  6. Expanding Access to Insurance by the Poor : Policy, Regulation and ...

    International Development Research Centre (IDRC) Digital Library (Canada)

    Expanding Access to Insurance by the Poor : Policy, Regulation and Supervision of Micro Insurance. This project aims to facilitate poor people's access to insurance products and services as a means of addressing their vulnerability to risk. It will do so by carrying out case studies in five countries. Potential candidates ...

  7. Yeast Srs2 Helicase Promotes Redistribution of Single-Stranded DNA-Bound RPA and Rad52 in Homologous Recombination Regulation

    Directory of Open Access Journals (Sweden)

    Luisina De Tullio

    2017-10-01

    Full Text Available Srs2 is a super-family 1 helicase that promotes genome stability by dismantling toxic DNA recombination intermediates. However, the mechanisms by which Srs2 remodels or resolves recombination intermediates remain poorly understood. Here, single-molecule imaging is used to visualize Srs2 in real time as it acts on single-stranded DNA (ssDNA bound by protein factors that function in recombination. We demonstrate that Srs2 is highly processive and translocates rapidly (∼170 nt per second in the 3′→5′ direction along ssDNA saturated with replication protein A (RPA. We show that RPA is evicted from DNA during the passage of Srs2. Remarkably, Srs2 also readily removes the recombination mediator Rad52 from RPA-ssDNA and, in doing so, promotes rapid redistribution of both Rad52 and RPA. These findings have important mechanistic implications for understanding how Srs2 and related nucleic acid motor proteins resolve potentially pathogenic nucleoprotein intermediates.

  8. Yeast Srs2 Helicase Promotes Redistribution of Single-Stranded DNA-Bound RPA and Rad52 in Homologous Recombination Regulation.

    Science.gov (United States)

    De Tullio, Luisina; Kaniecki, Kyle; Kwon, Youngho; Crickard, J Brooks; Sung, Patrick; Greene, Eric C

    2017-10-17

    Srs2 is a super-family 1 helicase that promotes genome stability by dismantling toxic DNA recombination intermediates. However, the mechanisms by which Srs2 remodels or resolves recombination intermediates remain poorly understood. Here, single-molecule imaging is used to visualize Srs2 in real time as it acts on single-stranded DNA (ssDNA) bound by protein factors that function in recombination. We demonstrate that Srs2 is highly processive and translocates rapidly (∼170 nt per second) in the 3'→5' direction along ssDNA saturated with replication protein A (RPA). We show that RPA is evicted from DNA during the passage of Srs2. Remarkably, Srs2 also readily removes the recombination mediator Rad52 from RPA-ssDNA and, in doing so, promotes rapid redistribution of both Rad52 and RPA. These findings have important mechanistic implications for understanding how Srs2 and related nucleic acid motor proteins resolve potentially pathogenic nucleoprotein intermediates. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  9. DP97, a DEAD box DNA/RNA helicase, is a target gene-selective co-regulator of the constitutive androstane receptor

    International Nuclear Information System (INIS)

    Kanno, Yuichiro; Serikawa, Takafumi; Inajima, Jun; Inouye, Yoshio

    2012-01-01

    Highlights: ► DP97 interacts with nuclear receptor CAR. ► DP97 enhances CAR-mediated transcriptional activation. ► DP97 synergistically enhances transactivity of CAR by the co-expression of SRC-1 or PGC1α. ► DP97 is a gene-selective co-activator for hCAR. -- Abstract: The constitutive androstane receptor (CAR) plays a key role in the expression of xenobiotic/steroid and drug metabolizing enzymes and their transporters. In this study, we demonstrated that DP97, a member of the DEAD box DNA/RNA helicase protein family, is a novel CAR-interacting protein. Using HepG2 cells expressing human CAR in the presence of tetracycline, we showed that knockdown of DP97 with small interfering RNAs suppressed tetracycline-inducible mRNA expression of CYP2B6 and UGT1A1 but not CYP3A4. Thus, DP97 was found to be a gene (or promoter)-selective co-activator for hCAR. DP97-mediated CAR transactivation was synergistically enhanced by the co-expression of SRC-1 or PGC1α, therefore it might act as mediator between hCAR and appropriate co-activators.

  10. Posttranscriptional regulation of the karyogamy gene by Kem1p/Xrn1p exoribonuclease and Rok1p RNA helicase of Saccharomyces cerevisiae

    International Nuclear Information System (INIS)

    Kim, Jaehee; Jeon, Soonmee; Yang, Yun-Seok; Kim, Jinmi

    2004-01-01

    The major biochemical activities ascribed to Kem1p/Xrn1p of Saccharomyces cerevisiae are 5'-3' exoribonuclease functioning in RNA turnover and a microtubule-binding protein. Mutational analysis has shown that Kem1p/Xrn1p participates in microtubule-related functions such as nuclear fusion (karyogamy) during mating, chromosome transmission, and spindle pole body duplication. Here, evidence is presented that Kem1p plays a specific role in nuclear fusion by affecting, at the posttranscriptional level, the pheromone induction of the karyogamy-specific transcription factor Kar4p and the expression of Rok1p, a putative RNA helicase. We found that Rok1p itself also affects the pheromone induction of Kar4p and thereby participates in nuclear fusion. Analysis of the active-site mutations, xrn1-D206A or D208A, shows that nuclear fusion as well as the Rok1p synthesis do not require the exoribonuclease activity of Kem1p. Our data provide an important insight into the gene-specific regulatory function mediated by the general RNA-modulating enzymes

  11. A recessive founder mutation in regulator of telomere elongation helicase 1, RTEL1, underlies severe immunodeficiency and features of Hoyeraal Hreidarsson syndrome.

    Science.gov (United States)

    Ballew, Bari J; Joseph, Vijai; De, Saurav; Sarek, Grzegorz; Vannier, Jean-Baptiste; Stracker, Travis; Schrader, Kasmintan A; Small, Trudy N; O'Reilly, Richard; Manschreck, Chris; Harlan Fleischut, Megan M; Zhang, Liying; Sullivan, John; Stratton, Kelly; Yeager, Meredith; Jacobs, Kevin; Giri, Neelam; Alter, Blanche P; Boland, Joseph; Burdett, Laurie; Offit, Kenneth; Boulton, Simon J; Savage, Sharon A; Petrini, John H J

    2013-08-01

    Dyskeratosis congenita (DC) is a heterogeneous inherited bone marrow failure and cancer predisposition syndrome in which germline mutations in telomere biology genes account for approximately one-half of known families. Hoyeraal Hreidarsson syndrome (HH) is a clinically severe variant of DC in which patients also have cerebellar hypoplasia and may present with severe immunodeficiency and enteropathy. We discovered a germline autosomal recessive mutation in RTEL1, a helicase with critical telomeric functions, in two unrelated families of Ashkenazi Jewish (AJ) ancestry. The affected individuals in these families are homozygous for the same mutation, R1264H, which affects three isoforms of RTEL1. Each parent was a heterozygous carrier of one mutant allele. Patient-derived cell lines revealed evidence of telomere dysfunction, including significantly decreased telomere length, telomere length heterogeneity, and the presence of extra-chromosomal circular telomeric DNA. In addition, RTEL1 mutant cells exhibited enhanced sensitivity to the interstrand cross-linking agent mitomycin C. The molecular data and the patterns of inheritance are consistent with a hypomorphic mutation in RTEL1 as the underlying basis of the clinical and cellular phenotypes. This study further implicates RTEL1 in the etiology of DC/HH and immunodeficiency, and identifies the first known homozygous autosomal recessive disease-associated mutation in RTEL1.

  12. A recessive founder mutation in regulator of telomere elongation helicase 1, RTEL1, underlies severe immunodeficiency and features of Hoyeraal Hreidarsson syndrome.

    Directory of Open Access Journals (Sweden)

    Bari J Ballew

    2013-08-01

    Full Text Available Dyskeratosis congenita (DC is a heterogeneous inherited bone marrow failure and cancer predisposition syndrome in which germline mutations in telomere biology genes account for approximately one-half of known families. Hoyeraal Hreidarsson syndrome (HH is a clinically severe variant of DC in which patients also have cerebellar hypoplasia and may present with severe immunodeficiency and enteropathy. We discovered a germline autosomal recessive mutation in RTEL1, a helicase with critical telomeric functions, in two unrelated families of Ashkenazi Jewish (AJ ancestry. The affected individuals in these families are homozygous for the same mutation, R1264H, which affects three isoforms of RTEL1. Each parent was a heterozygous carrier of one mutant allele. Patient-derived cell lines revealed evidence of telomere dysfunction, including significantly decreased telomere length, telomere length heterogeneity, and the presence of extra-chromosomal circular telomeric DNA. In addition, RTEL1 mutant cells exhibited enhanced sensitivity to the interstrand cross-linking agent mitomycin C. The molecular data and the patterns of inheritance are consistent with a hypomorphic mutation in RTEL1 as the underlying basis of the clinical and cellular phenotypes. This study further implicates RTEL1 in the etiology of DC/HH and immunodeficiency, and identifies the first known homozygous autosomal recessive disease-associated mutation in RTEL1.

  13. DP97, a DEAD box DNA/RNA helicase, is a target gene-selective co-regulator of the constitutive androstane receptor

    Energy Technology Data Exchange (ETDEWEB)

    Kanno, Yuichiro, E-mail: ykanno@phar.toho-u.ac.jp [Faculty of Pharmaceutical Sciences, Toho University, Chiba (Japan); Serikawa, Takafumi; Inajima, Jun; Inouye, Yoshio [Faculty of Pharmaceutical Sciences, Toho University, Chiba (Japan)

    2012-09-14

    Highlights: Black-Right-Pointing-Pointer DP97 interacts with nuclear receptor CAR. Black-Right-Pointing-Pointer DP97 enhances CAR-mediated transcriptional activation. Black-Right-Pointing-Pointer DP97 synergistically enhances transactivity of CAR by the co-expression of SRC-1 or PGC1{alpha}. Black-Right-Pointing-Pointer DP97 is a gene-selective co-activator for hCAR. -- Abstract: The constitutive androstane receptor (CAR) plays a key role in the expression of xenobiotic/steroid and drug metabolizing enzymes and their transporters. In this study, we demonstrated that DP97, a member of the DEAD box DNA/RNA helicase protein family, is a novel CAR-interacting protein. Using HepG2 cells expressing human CAR in the presence of tetracycline, we showed that knockdown of DP97 with small interfering RNAs suppressed tetracycline-inducible mRNA expression of CYP2B6 and UGT1A1 but not CYP3A4. Thus, DP97 was found to be a gene (or promoter)-selective co-activator for hCAR. DP97-mediated CAR transactivation was synergistically enhanced by the co-expression of SRC-1 or PGC1{alpha}, therefore it might act as mediator between hCAR and appropriate co-activators.

  14. ARCPHdb: A comprehensive protein database for SF1 and SF2 helicase from archaea.

    Science.gov (United States)

    Moukhtar, Mirna; Chaar, Wafi; Abdel-Razzak, Ziad; Khalil, Mohamad; Taha, Samir; Chamieh, Hala

    2017-01-01

    Superfamily 1 and Superfamily 2 helicases, two of the largest helicase protein families, play vital roles in many biological processes including replication, transcription and translation. Study of helicase proteins in the model microorganisms of archaea have largely contributed to the understanding of their function, architecture and assembly. Based on a large phylogenomics approach, we have identified and classified all SF1 and SF2 protein families in ninety five sequenced archaea genomes. Here we developed an online webserver linked to a specialized protein database named ARCPHdb to provide access for SF1 and SF2 helicase families from archaea. ARCPHdb was implemented using MySQL relational database. Web interfaces were developed using Netbeans. Data were stored according to UniProt accession numbers, NCBI Ref Seq ID, PDB IDs and Entrez Databases. A user-friendly interactive web interface has been developed to browse, search and download archaeal helicase protein sequences, their available 3D structure models, and related documentation available in the literature provided by ARCPHdb. The database provides direct links to matching external databases. The ARCPHdb is the first online database to compile all protein information on SF1 and SF2 helicase from archaea in one platform. This database provides essential resource information for all researchers interested in the field. Copyright © 2016 Elsevier Ltd. All rights reserved.

  15. Combinatorial regulation of meiotic holliday junction resolution in C. elegans by HIM-6 (BLM) helicase, SLX-4, and the SLX-1, MUS-81 and XPF-1 nucleases.

    Science.gov (United States)

    Agostinho, Ana; Meier, Bettina; Sonneville, Remi; Jagut, Marlène; Woglar, Alexander; Blow, Julian; Jantsch, Verena; Gartner, Anton

    2013-01-01

    Holliday junctions (HJs) are cruciform DNA structures that are created during recombination events. It is a matter of considerable importance to determine the resolvase(s) that promote resolution of these structures. We previously reported that C. elegans GEN-1 is a symmetrically cleaving HJ resolving enzyme required for recombinational repair, but we could not find an overt role in meiotic recombination. Here we identify C. elegans proteins involved in resolving meiotic HJs. We found no evidence for a redundant meiotic function of GEN-1. In contrast, we discovered two redundant HJ resolution pathways likely coordinated by the SLX-4 scaffold protein and also involving the HIM-6/BLM helicase. SLX-4 associates with the SLX-1, MUS-81 and XPF-1 nucleases and has been implicated in meiotic recombination in C. elegans. We found that C. elegans [mus-81; xpf-1], [slx-1; xpf-1], [mus-81; him-6] and [slx-1; him-6] double mutants showed a similar reduction in survival rates as slx-4. Analysis of meiotic diakinesis chromosomes revealed a distinct phenotype in these double mutants. Instead of wild-type bivalent chromosomes, pairs of "univalents" linked by chromatin bridges occur. These linkages depend on the conserved meiosis-specific transesterase SPO-11 and can be restored by ionizing radiation, suggesting that they represent unresolved meiotic HJs. This suggests the existence of two major resolvase activities, one provided by XPF-1 and HIM-6, the other by SLX-1 and MUS-81. In all double mutants crossover (CO) recombination is reduced but not abolished, indicative of further redundancy in meiotic HJ resolution. Real time imaging revealed extensive chromatin bridges during the first meiotic division that appear to be eventually resolved in meiosis II, suggesting back-up resolution activities acting at or after anaphase I. We also show that in HJ resolution mutants, the restructuring of chromosome arms distal and proximal to the CO still occurs, suggesting that CO initiation

  16. RNA helicase HEL-1 promotes longevity by specifically activating DAF-16/FOXO transcription factor signaling in Caenorhabditis elegans

    Science.gov (United States)

    Seo, Mihwa; Seo, Keunhee; Hwang, Wooseon; Koo, Hee Jung; Hahm, Jeong-Hoon; Yang, Jae-Seong; Han, Seong Kyu; Hwang, Daehee; Kim, Sanguk; Jang, Sung Key; Lee, Yoontae; Nam, Hong Gil; Lee, Seung-Jae V.

    2015-01-01

    The homeostatic maintenance of the genomic DNA is crucial for regulating aging processes. However, the role of RNA homeostasis in aging processes remains unknown. RNA helicases are a large family of enzymes that regulate the biogenesis and homeostasis of RNA. However, the functional significance of RNA helicases in aging has not been explored. Here, we report that a large fraction of RNA helicases regulate the lifespan of Caenorhabditis elegans. In particular, we show that a DEAD-box RNA helicase, helicase 1 (HEL-1), promotes longevity by specifically activating the DAF-16/forkhead box O (FOXO) transcription factor signaling pathway. We find that HEL-1 is required for the longevity conferred by reduced insulin/insulin-like growth factor 1 (IGF-1) signaling (IIS) and is sufficient for extending lifespan. We further show that the expression of HEL-1 in the intestine and neurons contributes to longevity. HEL-1 enhances the induction of a large fraction of DAF-16 target genes. Thus, the RNA helicase HEL-1 appears to promote longevity in response to decreased IIS as a transcription coregulator of DAF-16. Because HEL-1 and IIS are evolutionarily well conserved, a similar mechanism for longevity regulation via an RNA helicase-dependent regulation of FOXO signaling may operate in mammals, including humans. PMID:26195740

  17. The Cellular DNA Helicase ChlR1 Regulates Chromatin and Nuclear Matrix Attachment of the Human Papillomavirus 16 E2 Protein and High-Copy-Number Viral Genome Establishment.

    Science.gov (United States)

    Harris, Leanne; McFarlane-Majeed, Laura; Campos-León, Karen; Roberts, Sally; Parish, Joanna L

    2017-01-01

    In papillomavirus infections, the viral genome is established as a double-stranded DNA episome. To segregate the episomes into daughter cells during mitosis, they are tethered to cellular chromatin by the viral E2 protein. We previously demonstrated that the E2 proteins of diverse papillomavirus types, including bovine papillomavirus (BPV) and human papillomavirus 16 (HPV16), associate with the cellular DNA helicase ChlR1. This virus-host interaction is important for the tethering of BPV E2 to mitotic chromatin and the stable maintenance of BPV episomes. The role of the association between E2 and ChlR1 in the HPV16 life cycle is unresolved. Here we show that an HPV16 E2 Y131A mutant (E2 Y131A ) had significantly reduced binding to ChlR1 but retained transcriptional activation and viral origin-dependent replication functions. Subcellular fractionation of keratinocytes expressing E2 Y131A showed a marked change in the localization of the protein. Compared to that of wild-type E2 (E2 WT ), the chromatin-bound pool of E2 Y131A was decreased, concomitant with an increase in nuclear matrix-associated protein. Cell cycle synchronization indicated that the shift in subcellular localization of E2 Y131A occurred in mid-S phase. A similar alteration between the subcellular pools of the E2 WT protein occurred upon ChlR1 silencing. Notably, in an HPV16 life cycle model in primary human keratinocytes, mutant E2 Y131A genomes were established as episomes, but at a markedly lower copy number than that of wild-type HPV16 genomes, and they were not maintained upon cell passage. Our studies indicate that ChlR1 is an important regulator of the chromatin association of E2 and of the establishment and maintenance of HPV16 episomes. Infections with high-risk human papillomaviruses (HPVs) are a major cause of anogenital and oropharyngeal cancers. During infection, the circular DNA genome of HPV persists within the nucleus, independently of the host cell chromatin. Persistence of infection

  18. Authentic interdomain communication in an RNA helicase reconstituted by expressed protein ligation of two helicase domains.

    Science.gov (United States)

    Karow, Anne R; Theissen, Bettina; Klostermeier, Dagmar

    2007-01-01

    RNA helicases mediate structural rearrangements of RNA or RNA-protein complexes at the expense of ATP hydrolysis. Members of the DEAD box helicase family consist of two flexibly connected helicase domains. They share nine conserved sequence motifs that are involved in nucleotide binding and hydrolysis, RNA binding, and helicase activity. Most of these motifs line the cleft between the two helicase domains, and extensive communication between them is required for RNA unwinding. The two helicase domains of the Bacillus subtilis RNA helicase YxiN were produced separately as intein fusions, and a functional RNA helicase was generated by expressed protein ligation. The ligated helicase binds adenine nucleotides with very similar affinities to the wild-type protein. Importantly, its intrinsically low ATPase activity is stimulated by RNA, and the Michaelis-Menten parameters are similar to those of the wild-type. Finally, ligated YxiN unwinds a minimal RNA substrate to an extent comparable to that of the wild-type helicase, confirming authentic interdomain communication.

  19. Archaeal orthologs of Cdc45 and GINS form a stable complex that stimulates the helicase activity of MCM.

    Science.gov (United States)

    Xu, Yuli; Gristwood, Tamzin; Hodgson, Ben; Trinidad, Jonathan C; Albers, Sonja-Verena; Bell, Stephen D

    2016-11-22

    The regulated recruitment of Cdc45 and GINS is key to activating the eukaryotic MCM(2-7) replicative helicase. We demonstrate that the homohexameric archaeal MCM helicase associates with orthologs of GINS and Cdc45 in vivo and in vitro. Association of these factors with MCM robustly stimulates the MCM helicase activity. In contrast to the situation in eukaryotes, archaeal Cdc45 and GINS form an extremely stable complex before binding MCM. Further, the archaeal GINS•Cdc45 complex contains two copies of Cdc45. Our analyses give insight into the function and evolution of the conserved core of the archaeal/eukaryotic replisome.

  20. Helicase-like transcription factor (Hltf regulates G2/M transition, Wt1/Gata4/Hif-1a cardiac transcription networks, and collagen biogenesis.

    Directory of Open Access Journals (Sweden)

    Rebecca A Helmer

    Full Text Available HLTF/Hltf regulates transcription, remodels chromatin, and coordinates DNA damage repair. Hltf is expressed in mouse brain and heart during embryonic and postnatal development. Silencing Hltf is semilethal. Seventy-four percent of congenic C57BL/6J Hltf knockout mice died, 75% within 12-24 hours of birth. Previous studies in neonatal (6-8 hour postpartum brain revealed silencing Hltf disrupted cell cycle progression, and attenuated DNA damage repair. An RNA-Seq snapshot of neonatal heart transcriptome showed 1,536 of 20,000 total transcripts were altered (p < 0.05 - 10 up- and 1,526 downregulated. Pathway enrichment analysis with MetaCore™ showed Hltf's regulation of the G2/M transition (p=9.726E(-15 of the cell cycle in heart is nearly identical to its role in brain. In addition, Brca1 and 12 members of the Brca1 associated genome surveillance complex are also downregulated. Activation of caspase 3 coincides with transcriptional repression of Bcl-2. Hltf loss caused downregulation of Wt1/Gata4/Hif-1a signaling cascades as well as Myh7b/miR499 transcription. Hltf-specific binding to promoters and/or regulatory regions of these genes was authenticated by ChIP-PCR. Hif-1a targets for prolyl (P4ha1, P4ha2 and lysyl (Plod2 collagen hydroxylation, PPIase enzymes (Ppid, Ppif, Ppil3 for collagen trimerization, and lysyl oxidase (Loxl2 for collagen-elastin crosslinking were downregulated. However, transcription of genes for collagens, fibronectin, Mmps and their inhibitors (Timps was unaffected. The collective downregulation of genes whose protein products control collagen biogenesis caused disorganization of the interstitial and perivascular myocardial collagen fibrillar network as viewed with picrosirius red-staining, and authenticated with spectral imaging. Wavy collagen bundles in control hearts contrasted with collagen fibers that were thin, short and disorganized in Hltf null hearts. Collagen bundles in Hltf null hearts were tangled and

  1. Internet pharmacy: issues of access, quality, costs, and regulation.

    Science.gov (United States)

    Crawford, Stephanie Y

    2003-02-01

    Internet pharmacy has been the focus of heightened interest over the past 3 years since the first major Web site was introduced in the United States. This paper addresses issues pertaining to Internet pharmacies that sell prescriptions and other products to consumers at the retail level. The Internet pharmacy industry has shifted rapidly in the short time span. This paper begins with a summary of historical considerations and the shifting organization of Internet pharmacy. The advantages and disadvantages of online pharmacy practice are listed. Issues of access, quality, and cost are described. The challenges in regulation at the state and federal levels are presented. Advice to consumers is offered regarding the use of Internet pharmacy sites for purchasing prescription drug products.

  2. EM structure of a helicase-loader complex depicting a 6:2 binding sub-stoichiometry from Geobacillus kaustophilus HTA426

    Energy Technology Data Exchange (ETDEWEB)

    Lin, Yen-Chen [Institute of Molecular Biology, Academia Sinica, Taipei 115, Taiwan (China); Naveen, Vankadari [Institute of Molecular Biology, Academia Sinica, Taipei 115, Taiwan (China); Molecular Cell Biology, Taiwan International Graduate Program, Institute of Molecular Biology, Academia Sinica, and Graduate Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan (China); Hsiao, Chwan-Deng, E-mail: hsiao@gate.sinica.edu.tw [Institute of Molecular Biology, Academia Sinica, Taipei 115, Taiwan (China); Molecular Cell Biology, Taiwan International Graduate Program, Institute of Molecular Biology, Academia Sinica, and Graduate Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan (China)

    2016-04-22

    During DNA replication, bacterial helicase is recruited as a complex in association with loader proteins to unwind the parental duplex. Previous structural studies have reported saturated 6:6 helicase-loader complexes with different conformations. However, structural information on the sub-stoichiometric conformations of these previously-documented helicase-loader complexes remains elusive. Here, with the aid of single particle electron-microscopy (EM) image reconstruction, we present the Geobacillus kaustophilus HTA426 helicase-loader (DnaC-DnaI) complex with a 6:2 binding stoichiometry in the presence of ATPγS. In the 19 Å resolution EM map, the undistorted and unopened helicase ring holds a robust loader density above the C-terminal RecA-like domain. Meanwhile, the path of the central DNA binding channel appears to be obstructed by the reconstructed loader density, implying its potential role as a checkpoint conformation to prevent the loading of immature complex onto DNA. Our data also reveals that the bound nucleotides and the consequently induced conformational changes in the helicase hexamer are essential for active association with loader proteins. These observations provide fundamental insights into the formation of the helicase-loader complex in bacteria that regulates the DNA replication process. - Highlights: • Helicase-loader complex structure with 6:2 sub-stoichiometry is resolved by EM. • Helicase hexamer in 6:2 sub-stoichiometry is constricted and un-opened. • 6:2 binding ratio of helicase-loader complex could act as a DNA loading checkpoint. • Nucleotides stabilize helicase-loader complex at low protein concentrations.

  3. EM structure of a helicase-loader complex depicting a 6:2 binding sub-stoichiometry from Geobacillus kaustophilus HTA426

    International Nuclear Information System (INIS)

    Lin, Yen-Chen; Naveen, Vankadari; Hsiao, Chwan-Deng

    2016-01-01

    During DNA replication, bacterial helicase is recruited as a complex in association with loader proteins to unwind the parental duplex. Previous structural studies have reported saturated 6:6 helicase-loader complexes with different conformations. However, structural information on the sub-stoichiometric conformations of these previously-documented helicase-loader complexes remains elusive. Here, with the aid of single particle electron-microscopy (EM) image reconstruction, we present the Geobacillus kaustophilus HTA426 helicase-loader (DnaC-DnaI) complex with a 6:2 binding stoichiometry in the presence of ATPγS. In the 19 Å resolution EM map, the undistorted and unopened helicase ring holds a robust loader density above the C-terminal RecA-like domain. Meanwhile, the path of the central DNA binding channel appears to be obstructed by the reconstructed loader density, implying its potential role as a checkpoint conformation to prevent the loading of immature complex onto DNA. Our data also reveals that the bound nucleotides and the consequently induced conformational changes in the helicase hexamer are essential for active association with loader proteins. These observations provide fundamental insights into the formation of the helicase-loader complex in bacteria that regulates the DNA replication process. - Highlights: • Helicase-loader complex structure with 6:2 sub-stoichiometry is resolved by EM. • Helicase hexamer in 6:2 sub-stoichiometry is constricted and un-opened. • 6:2 binding ratio of helicase-loader complex could act as a DNA loading checkpoint. • Nucleotides stabilize helicase-loader complex at low protein concentrations.

  4. Access Regulation for Naturally Monopolistic Port Terminals: Lessons from Regulated Network Industries

    OpenAIRE

    Defilippi, Enzo

    2010-01-01

    textabstractThe problem of access arises in industries where inputs from monopolistic and competitive markets are complementarily needed to provide a service. In these circumstances, the firm controlling the monopolistic segment has incentives to deter competition in the competitive segments (markets) to recover profits foregone by regulation (Paredes, 1997). In the port industry, for example, a number of services need to be jointly provided to complete the logistics chain: pilotage, towage, ...

  5. Purification and crystallization of Kokobera virus helicase

    International Nuclear Information System (INIS)

    De Colibus, Luigi; Speroni, Silvia; Coutard, Bruno; Forrester, Naomi L.; Gould, Ernest; Canard, Bruno; Mattevi, Andrea

    2007-01-01

    Kokobera virus is a mosquito-borne flavivirus belonging, like West Nile virus, to the Japanese encephalitis virus serocomplex. Crystals of the Kokobera virus helicase domain were obtained by the hanging-drop vapour-diffusion method and exhibit a diffraction limit of 2.3 Å. Kokobera virus is a mosquito-borne flavivirus belonging, like West Nile virus, to the Japanese encephalitis virus serocomplex. The flavivirus genus is characterized by a positive-sense single-stranded RNA genome. The unique open reading frame of the viral RNA is transcribed and translated as a single polyprotein which is post-translationally cleaved to yield three structural and seven nonstructural proteins, one of which is the NS3 gene that encodes a C-terminal helicase domain consisting of 431 amino acids. Helicase inhibitors are potential antiviral drugs as the helicase is essential to viral replication. Crystals of the Kokobera virus helicase domain were obtained by the hanging-drop vapour-diffusion method. The crystals belong to space group P3 1 21 (or P3 2 21), with unit-cell parameters a = 88.6, c = 138.6 Å, and exhibit a diffraction limit of 2.3 Å

  6. Helicase-dependent amplification of nucleic acids.

    Science.gov (United States)

    Cao, Yun; Kim, Hyun-Jin; Li, Ying; Kong, Huimin; Lemieux, Bertrand

    2013-10-11

    Helicase-dependent amplification (HDA) is a novel method for the isothermal in vitro amplification of nucleic acids. The HDA reaction selectively amplifies a target sequence by extension of two oligonucleotide primers. Unlike the polymerase chain reaction (PCR), HDA uses a helicase enzyme to separate the deoxyribonucleic acid (DNA) strands, rather than heat denaturation. This allows DNA amplification without the need for thermal cycling. The helicase used in HDA is a helicase super family II protein obtained from a thermophilic organism, Thermoanaerobacter tengcongensis (TteUvrD). This thermostable helicase is capable of unwinding blunt-end nucleic acid substrates at elevated temperatures (60° to 65°C). The HDA reaction can also be coupled with reverse transcription for ribonucleic acid (RNA) amplification. The products of this reaction can be detected during the reaction using fluorescent probes when incubations are conducted in a fluorimeter. Alternatively, products can be detected after amplification using a disposable amplicon containment device that contains an embedded lateral flow strip. Copyright © 2013 John Wiley & Sons, Inc.

  7. Purification and crystallization of Kokobera virus helicase

    Energy Technology Data Exchange (ETDEWEB)

    De Colibus, Luigi; Speroni, Silvia [Department of Genetics and Microbiology, University of Pavia, Via Ferrata 1, 27100 Pavia (Italy); Coutard, Bruno [Architecture et Fonction des Macromolécules Biologiques, UMR 6098 CNRS et Université Aix-Marseille I et II, ESIL, Campus de Luminy, 13288 Marseille CEDEX 09 (France); Forrester, Naomi L.; Gould, Ernest [Centre for Ecology and Hydrology (formerly Institute of Virology), Mansfield Road, Oxford OX1 3SR (United Kingdom); Canard, Bruno [Architecture et Fonction des Macromolécules Biologiques, UMR 6098 CNRS et Université Aix-Marseille I et II, ESIL, Campus de Luminy, 13288 Marseille CEDEX 09 (France); Mattevi, Andrea, E-mail: mattevi@ipvgen.unipv.it [Department of Genetics and Microbiology, University of Pavia, Via Ferrata 1, 27100 Pavia (Italy)

    2007-03-01

    Kokobera virus is a mosquito-borne flavivirus belonging, like West Nile virus, to the Japanese encephalitis virus serocomplex. Crystals of the Kokobera virus helicase domain were obtained by the hanging-drop vapour-diffusion method and exhibit a diffraction limit of 2.3 Å. Kokobera virus is a mosquito-borne flavivirus belonging, like West Nile virus, to the Japanese encephalitis virus serocomplex. The flavivirus genus is characterized by a positive-sense single-stranded RNA genome. The unique open reading frame of the viral RNA is transcribed and translated as a single polyprotein which is post-translationally cleaved to yield three structural and seven nonstructural proteins, one of which is the NS3 gene that encodes a C-terminal helicase domain consisting of 431 amino acids. Helicase inhibitors are potential antiviral drugs as the helicase is essential to viral replication. Crystals of the Kokobera virus helicase domain were obtained by the hanging-drop vapour-diffusion method. The crystals belong to space group P3{sub 1}21 (or P3{sub 2}21), with unit-cell parameters a = 88.6, c = 138.6 Å, and exhibit a diffraction limit of 2.3 Å.

  8. Interplay of cis- and trans-regulatory mechanisms in the spliceosomal RNA helicase Brr2.

    Science.gov (United States)

    Absmeier, Eva; Becke, Christian; Wollenhaupt, Jan; Santos, Karine F; Wahl, Markus C

    2017-01-02

    RNA helicase Brr2 is implicated in multiple phases of pre-mRNA splicing and thus requires tight regulation. Brr2 can be auto-inhibited via a large N-terminal region folding back onto its helicase core and auto-activated by a catalytically inactive C-terminal helicase cassette. Furthermore, it can be regulated in trans by the Jab1 domain of the Prp8 protein, which can inhibit Brr2 by intermittently inserting a C-terminal tail in the enzyme's RNA-binding tunnel or activate the helicase after removal of this tail. Presently it is unclear, whether these regulatory mechanisms functionally interact and to which extent they are evolutionarily conserved. Here, we report crystal structures of Saccharomyces cerevisiae and Chaetomium thermophilum Brr2-Jab1 complexes, demonstrating that Jab1-based inhibition of Brr2 presumably takes effect in all eukaryotes but is implemented via organism-specific molecular contacts. Moreover, the structures show that Brr2 auto-inhibition can act in concert with Jab1-mediated inhibition, and suggest that the N-terminal region influences how the Jab1 C-terminal tail interacts at the RNA-binding tunnel. Systematic RNA binding and unwinding studies revealed that the N-terminal region and the Jab1 C-terminal tail specifically interfere with accommodation of double-stranded and single-stranded regions of an RNA substrate, respectively, mutually reinforcing each other. Additionally, such analyses show that regulation based on the N-terminal region requires the presence of the inactive C-terminal helicase cassette. Together, our results outline an intricate system of regulatory mechanisms, which control Brr2 activities during snRNP assembly and splicing.

  9. The Helicase Activity of Hyperthermophilic Archaeal MCM is Enhanced at High Temperatures by Lysine Methylation.

    Science.gov (United States)

    Xia, Yisui; Niu, Yanling; Cui, Jiamin; Fu, Yang; Chen, Xiaojiang S; Lou, Huiqiang; Cao, Qinhong

    2015-01-01

    Lysine methylation and methyltransferases are widespread in the third domain of life, archaea. Nevertheless, the effects of methylation on archaeal proteins wait to be defined. Here, we report that recombinant sisMCM, an archaeal homolog of Mcm2-7 eukaryotic replicative helicase, is methylated by aKMT4 in vitro. Mono-methylation of these lysine residues occurs coincidently in the endogenous sisMCM protein purified from the hyperthermophilic Sulfolobus islandicus cells as indicated by mass spectra. The helicase activity of mini-chromosome maintenance (MCM) is stimulated by methylation, particularly at temperatures over 70°C. The methylated MCM shows optimal DNA unwinding activity after heat-treatment between 76 and 82°C, which correlates well with the typical growth temperatures of hyperthermophilic Sulfolobus. After methylation, the half life of MCM helicase is dramatically extended at 80°C. The methylated sites are located on the accessible protein surface, which might modulate the intra- and inter- molecular interactions through changing the hydrophobicity and surface charge. Furthermore, the methylation-mimic mutants of MCM show heat resistance helicase activity comparable to the methylated MCM. These data provide the biochemical evidence that posttranslational modifications such as methylation may enhance kinetic stability of proteins under the elevated growth temperatures of hyperthermophilic archaea.

  10. Comparative structural analysis of human DEAD-box RNA helicases.

    Directory of Open Access Journals (Sweden)

    Patrick Schütz

    2010-09-01

    Full Text Available DEAD-box RNA helicases play various, often critical, roles in all processes where RNAs are involved. Members of this family of proteins are linked to human disease, including cancer and viral infections. DEAD-box proteins contain two conserved domains that both contribute to RNA and ATP binding. Despite recent advances the molecular details of how these enzymes convert chemical energy into RNA remodeling is unknown. We present crystal structures of the isolated DEAD-domains of human DDX2A/eIF4A1, DDX2B/eIF4A2, DDX5, DDX10/DBP4, DDX18/myc-regulated DEAD-box protein, DDX20, DDX47, DDX52/ROK1, and DDX53/CAGE, and of the helicase domains of DDX25 and DDX41. Together with prior knowledge this enables a family-wide comparative structural analysis. We propose a general mechanism for opening of the RNA binding site. This analysis also provides insights into the diversity of DExD/H- proteins, with implications for understanding the functions of individual family members.

  11. Comparative structural analysis of human DEAD-box RNA helicases.

    Science.gov (United States)

    Schütz, Patrick; Karlberg, Tobias; van den Berg, Susanne; Collins, Ruairi; Lehtiö, Lari; Högbom, Martin; Holmberg-Schiavone, Lovisa; Tempel, Wolfram; Park, Hee-Won; Hammarström, Martin; Moche, Martin; Thorsell, Ann-Gerd; Schüler, Herwig

    2010-09-30

    DEAD-box RNA helicases play various, often critical, roles in all processes where RNAs are involved. Members of this family of proteins are linked to human disease, including cancer and viral infections. DEAD-box proteins contain two conserved domains that both contribute to RNA and ATP binding. Despite recent advances the molecular details of how these enzymes convert chemical energy into RNA remodeling is unknown. We present crystal structures of the isolated DEAD-domains of human DDX2A/eIF4A1, DDX2B/eIF4A2, DDX5, DDX10/DBP4, DDX18/myc-regulated DEAD-box protein, DDX20, DDX47, DDX52/ROK1, and DDX53/CAGE, and of the helicase domains of DDX25 and DDX41. Together with prior knowledge this enables a family-wide comparative structural analysis. We propose a general mechanism for opening of the RNA binding site. This analysis also provides insights into the diversity of DExD/H- proteins, with implications for understanding the functions of individual family members.

  12. Expanding Access to Insurance by the Poor : Policy, Regulation and ...

    International Development Research Centre (IDRC) Digital Library (Canada)

    This project aims to facilitate poor people's access to insurance products and ... Potential candidates include China, Colombia, India, Indonesia, Mali and South ... IDRC congratulates first cohort of Women in Climate Change Science Fellows.

  13. ELF4 Regulates GIGANTEA Chromatin Access through Subnuclear Sequestration

    Directory of Open Access Journals (Sweden)

    Yumi Kim

    2013-03-01

    Full Text Available Many organisms, including plants, use the circadian clock to measure the duration of day and night. Daily rhythms in the plant circadian system are generated by multiple interlocked transcriptional/translational loops and also by spatial regulations such as nuclear translocation. GIGANTEA (GI, one of the key clock components in Arabidopsis, makes distinctive nuclear bodies like other nuclear-localized circadian regulators. However, little is known about the dynamics or roles of GI subnuclear localization. Here, we characterize GI subnuclear compartmentalization and identify unexpected dynamic changes under diurnal conditions. We further identify EARLY FLOWERING 4 (ELF4 as a regulator of GI nuclear distribution through a physical interaction. ELF4 sequesters GI from the nucleoplasm, where GI binds the promoter of CONSTANS (CO, to discrete nuclear bodies. We suggest that the subnuclear compartmentalization of GI by ELF4 contributes to the regulation of photoperiodic flowering.

  14. Telecommunications (Interception and Access) and Its Regulation in Arab Countries

    OpenAIRE

    Kiswani, Nazzal

    2010-01-01

    Telecommunication has been a necessity in all countries this century. Communication has always been an essential part of our lives, education, family relations, business, government and other organizational activities. As telecommunication technology has advanced, so has the need for the interception of telecommunications and access by law enforcement authorities. In addition, lawful interception and the way it is performed have played an important role in the effectiveness of the monitoring ...

  15. RTEL1: functions of a disease-associated helicase.

    Science.gov (United States)

    Vannier, Jean-Baptiste; Sarek, Grzegorz; Boulton, Simon J

    2014-07-01

    DNA secondary structures that arise during DNA replication, repair, and recombination (3R) must be processed correctly to prevent genetic instability. Regulator of telomere length 1 (RTEL1) is an essential DNA helicase that disassembles a variety of DNA secondary structures to facilitate 3R processes and to maintain telomere integrity. The past few years have witnessed the emergence of RTEL1 variants that confer increased susceptibility to high-grade glioma, astrocytomas, and glioblastomas. Mutations in RTEL1 have also been implicated in Hoyeraal-Hreidarsson syndrome, a severe form of the bone-marrow failure and cancer predisposition disorder, dyskeratosis congenita. We review these recent findings and highlight its crucial link between DNA secondary-structure metabolism and human disease. Copyright © 2014 Elsevier Ltd. All rights reserved.

  16. Spatial Regulation, Politics of Access and Informal economic Policy ...

    African Journals Online (AJOL)

    ... to draw attention to the political contestation over space. The city was 'invaded' and 'conquered' by traders who were formerly excluded (Nesvag, 2000, 2001; Tsoeu, 2003). At the same time, attention is drawn to the current attempts by city managers to control and regulate this contested space in an environment of flux.

  17. 78 FR 30233 - Defense Federal Acquisition Regulation Supplement; Government Support Contractor Access to...

    Science.gov (United States)

    2013-05-22

    ... Number 0750-AG38 Defense Federal Acquisition Regulation Supplement; Government Support Contractor Access... Government support contractors to have access to proprietary technical data belonging to prime contractors and other third parties, provided that the technical data owner may require the support contractor to...

  18. Molecular architecture of the recombinant human MCM2-7 helicase in complex with nucleotides and DNA

    DEFF Research Database (Denmark)

    Boskovic, Jasminka; Bragado-Nilsson, Elisabeth; Saligram Prabhakar, Bhargav

    2016-01-01

    DNA replication is a key biological process that involves different protein complexes whose assembly is rigorously regulated in a successive order. One of these complexes is a replicative hexameric helicase, the MCM complex, which is essential for the initiation and elongation phases of replicati...

  19. RNA helicase DDX3 is a regulatory subunit of casein kinase 1 in Wnt-beta-catenin signaling

    NARCIS (Netherlands)

    Cruciat, C.M.; Dolde, C.; de Groot, R.E.; Ohkawara, B.; Reinhard, C.; Korswagen, H.C.; Niehrs, C.

    2013-01-01

    Casein kinase 1 (CK1) members play key roles in numerous biological processes. They are considered "rogue" kinases, because their enzymatic activity appears unregulated. Contrary to this notion, we have identified the DEAD-box RNA helicase DDX3 as a regulator of the Wnt-beta-catenin network, where

  20. Access regulation in the next generation access network environment: A comparative study of Hong Kong and Singapore from the transaction cost economics perspectives

    OpenAIRE

    Ho, Au Man

    2012-01-01

    Hong Kong and Singapore have adopted two different models in the regulation of the next generation access (NGA) networks. In Hong Kong, the government has decided that access regulation will not be applied to fibre-based access networks and its strategy will be to rely on facilities-based competition to promote investment in the NGA networks. Singapore, on the other hand, has promoted access/services-based competition over a next generation broadband infrastructure subsidised by public fundin...

  1. Access Denied: Should Youth Access to the Internet Be Regulated? A Resource Guide for Teachers.

    Science.gov (United States)

    Parrini, Michelle

    Much of the current public discussion in the United States about the Internet and speech rights focuses on the array of materials (particularly sexually explicit materials) that are available on the Internet, the effect of exposure to them on youth development, and whether the material should be regulated. In exploring questions about youth access…

  2. Crystal structure of Middle East respiratory syndrome coronavirus helicase.

    Directory of Open Access Journals (Sweden)

    Wei Hao

    2017-06-01

    Full Text Available Middle East respiratory syndrome coronavirus (MERS-CoV remains a threat to public health worldwide; however, effective vaccine or drug against CoVs remains unavailable. CoV helicase is one of the three evolutionary most conserved proteins in nidoviruses, thus making it an important target for drug development. We report here the first structure of full-length coronavirus helicase, MERS-CoV nsp13. MERS-CoV helicase has multiple domains, including an N-terminal Cys/His rich domain (CH with three zinc atoms, a beta-barrel domain and a C-terminal SF1 helicase core with two RecA-like subdomains. Our structural analyses show that while the domain organization of nsp13 is conserved throughout nidoviruses, the individual domains of nsp13 are closely related to the equivalent eukaryotic domains of Upf1 helicases. The most distinctive feature differentiating CoV helicases from eukaryotic Upf1 helicases is the interaction between CH domain and helicase core.

  3. Structural basis of Zika virus helicase in recognizing its substrates

    Directory of Open Access Journals (Sweden)

    Hongliang Tian

    2016-07-01

    Full Text Available Abstract The recent explosive outbreak of Zika virus (ZIKV infection has been reported in South and Central America and the Caribbean. Neonatal microcephaly associated with ZIKV infection has already caused a public health emergency of international concern. No specific vaccines or drugs are currently available to treat ZIKV infection. The ZIKV helicase, which plays a pivotal role in viral RNA replication, is an attractive target for therapy. We determined the crystal structures of ZIKV helicase-ATP-Mn2+ and ZIKV helicase-RNA. This is the first structure of any flavivirus helicase bound to ATP. Comparisons with related flavivirus helicases have shown that although the critical P-loop in the active site has variable conformations among different species, it adopts an identical mode to recognize ATP/Mn2+. The structure of ZIKV helicase-RNA has revealed that upon RNA binding, rotations of the motor domains can cause significant conformational changes. Strikingly, although ZIKV and dengue virus (DENV apo-helicases share conserved residues for RNA binding, their different manners of motor domain rotations result in distinct individual modes for RNA recognition. It suggests that flavivirus helicases could have evolved a conserved engine to convert chemical energy from nucleoside triphosphate to mechanical energy for RNA unwinding, but different motor domain rotations result in variable RNA recognition modes to adapt to individual viral replication.

  4. Nucleolin inhibits G4 oligonucleotide unwinding by Werner helicase.

    Directory of Open Access Journals (Sweden)

    Fred E Indig

    Full Text Available The Werner protein (WRNp, a member of the RecQ helicase family, is strongly associated with the nucleolus, as is nucleolin (NCL, an important nucleolar constituent protein. Both WRNp and NCL respond to the effects of DNA damaging agents. Therefore, we have investigated if these nuclear proteins interact and if this interaction has a possible functional significance in DNA damage repair.Here we report that WRNp interacts with the RNA-binding protein, NCL, based on immunoprecipitation, immunofluorescent co-localization in live and fixed cells, and direct binding of purified WRNp to nucleolin. We also map the binding region to the C-terminal domains of both proteins. Furthermore, treatment of U2OS cells with 15 µM of the Topoisomerase I inhibitor, camptothecin, causes the dissociation of the nucleolin-Werner complex in the nucleolus, followed by partial re-association in the nucleoplasm. Other DNA damaging agents, such as hydroxyurea, Mitomycin C, and aphidicolin do not have these effects. Nucleolin or its C-terminal fragment affected the helicase, but not the exonuclease activity of WRNp, by inhibiting WRN unwinding of G4 tetraplex DNA structures, as seen in activity assays and electrophoretic mobility shift assays (EMSA.These data suggest that nucleolin may regulate G4 DNA unwinding by WRNp, possibly in response to certain DNA damaging agents. We postulate that the NCL-WRNp complex may contain an inactive form of WRNp, which is released from the nucleolus upon DNA damage. Then, when required, WRNp is released from inhibition and can participate in the DNA repair processes.

  5. Escherichia coli and Neisseria gonorrhoeae UvrD helicase unwinds G4 DNA structures.

    Science.gov (United States)

    Shukla, Kaustubh; Thakur, Roshan Singh; Ganguli, Debayan; Rao, Desirazu Narasimha; Nagaraju, Ganesh

    2017-10-18

    G-quadruplex (G4) secondary structures have been implicated in various biological processes, including gene expression, DNA replication and telomere maintenance. However, unresolved G4 structures impede replication progression which can lead to the generation of DNA double-strand breaks and genome instability. Helicases have been shown to resolve G4 structures to facilitate faithful duplication of the genome. Escherichia coli UvrD (EcUvrD) helicase plays a crucial role in nucleotide excision repair, mismatch repair and in the regulation of homologous recombination. Here, we demonstrate a novel role of E. coli and Neisseria gonorrhoeae UvrD in resolving G4 tetraplexes. EcUvrD and N gonorrhoeae UvrD were proficient in unwinding previously characterized tetramolecular G4 structures. Notably, EcUvrD was equally efficient in resolving tetramolecular and bimolecular G4 DNA that were derived from the potential G4-forming sequences from the genome of E. coli Interestingly, in addition to resolving intermolecular G4 structures, EcUvrD was robust in unwinding intramolecular G4 structures. These data for the first time provide evidence for the role of UvrD in the resolution of G4 structures, which has implications for the in vivo role of UvrD helicase in G4 DNA resolution and genome maintenance. © 2017 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

  6. The helicase domain of Polθ counteracts RPA to promote alt-NHEJ.

    Science.gov (United States)

    Mateos-Gomez, Pedro A; Kent, Tatiana; Deng, Sarah K; McDevitt, Shane; Kashkina, Ekaterina; Hoang, Trung M; Pomerantz, Richard T; Sfeir, Agnel

    2017-12-01

    Mammalian polymerase theta (Polθ) is a multifunctional enzyme that promotes error-prone DNA repair by alternative nonhomologous end joining (alt-NHEJ). Here we present structure-function analyses that reveal that, in addition to the polymerase domain, Polθ-helicase activity plays a central role during double-strand break (DSB) repair. Our results show that the helicase domain promotes chromosomal translocations by alt-NHEJ in mouse embryonic stem cells and also suppresses CRISPR-Cas9- mediated gene targeting by homologous recombination (HR). In vitro assays demonstrate that Polθ-helicase activity facilitates the removal of RPA from resected DSBs to allow their annealing and subsequent joining by alt-NHEJ. Consistent with an antagonistic role for RPA during alt-NHEJ, inhibition of RPA1 enhances end joining and suppresses recombination. Taken together, our results reveal that the balance between HR and alt-NHEJ is controlled by opposing activities of Polθ and RPA, providing further insight into the regulation of repair-pathway choice in mammalian cells.

  7. The Arabidopsis thaliana homolog of the helicase RTEL1 plays multiple roles in preserving genome stability.

    Science.gov (United States)

    Recker, Julia; Knoll, Alexander; Puchta, Holger

    2014-12-01

    In humans, mutations in the DNA helicase Regulator of Telomere Elongation Helicase1 (RTEL1) lead to Hoyeraal-Hreidarsson syndrome, a severe, multisystem disorder. Here, we demonstrate that the RTEL1 homolog in Arabidopsis thaliana plays multiple roles in preserving genome stability. RTEL1 suppresses homologous recombination in a pathway parallel to that of the DNA translocase FANCM. Cytological analyses of root meristems indicate that RTEL1 is involved in processing DNA replication intermediates independently from FANCM and the nuclease MUS81. Moreover, RTEL1 is involved in interstrand and intrastrand DNA cross-link repair independently from FANCM and (in intrastrand cross-link repair) parallel to MUS81. RTEL1 contributes to telomere homeostasis; the concurrent loss of RTEL1 and the telomerase TERT leads to rapid, severe telomere shortening, which occurs much more rapidly than it does in the single-mutant line tert, resulting in developmental arrest after four generations. The double mutant rtel1-1 recq4A-4 exhibits massive growth defects, indicating that this RecQ family helicase, which is also involved in the suppression of homologous recombination and the repair of DNA lesions, can partially replace RTEL1 in the processing of DNA intermediates. The requirement for RTEL1 in multiple pathways to preserve genome stability in plants can be explained by its putative role in the destabilization of DNA loop structures, such as D-loops and T-loops. © 2014 American Society of Plant Biologists. All rights reserved.

  8. 76 FR 45403 - Bank Secrecy Act Regulations-Definitions and Other Regulations Relating to Prepaid Access

    Science.gov (United States)

    2011-07-29

    ... prepaid access products and services posing lower risks of money laundering and terrorist financing from... exceptions to: (1) Establish written anti-money laundering (AML) programs that are reasonably designed to prevent the MSB from being used to facilitate money laundering and the financing of terrorist activities...

  9. Access Regulation for Naturally Monopolistic Port Terminals: Lessons from Regulated Network Industries

    NARCIS (Netherlands)

    E.F. Defilippi (Enzo)

    2010-01-01

    textabstractThe problem of access arises in industries where inputs from monopolistic and competitive markets are complementarily needed to provide a service. In these circumstances, the firm controlling the monopolistic segment has incentives to deter competition in the competitive segments

  10. Access to Orphan Drugs: A Comprehensive Review of Legislations, Regulations and Policies in 35 Countries

    Science.gov (United States)

    Gammie, Todd

    2015-01-01

    Objective To review existing regulations and policies utilised by countries to enable patient access to orphan drugs. Methods A review of the literature (1998 to 2014) was performed to identify relevant, peer-reviewed articles. Using content analysis, we synthesised regulations and policies for access to orphan drugs by type and by country. Results Fifty seven articles and 35 countries were included in this review. Six broad categories of regulation and policy instruments were identified: national orphan drug policies, orphan drug designation, marketing authorization, incentives, marketing exclusivity, and pricing and reimbursement. The availability of orphan drugs depends on individual country’s legislation and regulations including national orphan drug policies, orphan drug designation, marketing authorization, marketing exclusivity and incentives such as tax credits to ensure research, development and marketing. The majority of countries (27/35) had in place orphan drug legislation. Access to orphan drugs depends on individual country’s pricing and reimbursement policies, which varied widely between countries. High prices and insufficient evidence often limit orphan drugs from meeting the traditional health technology assessment criteria, especially cost-effectiveness, which may influence access. Conclusions Overall many countries have implemented a combination of legislations, regulations and policies for orphan drugs in the last two decades. While these may enable the availability and access to orphan drugs, there are critical differences between countries in terms of range and types of legislations, regulations and policies implemented. Importantly, China and India, two of the largest countries by population size, both lack national legislation for orphan medicines and rare diseases, which could have substantial negative impacts on their patient populations with rare diseases. PMID:26451948

  11. Access to Orphan Drugs: A Comprehensive Review of Legislations, Regulations and Policies in 35 Countries.

    Science.gov (United States)

    Gammie, Todd; Lu, Christine Y; Babar, Zaheer Ud-Din

    2015-01-01

    To review existing regulations and policies utilised by countries to enable patient access to orphan drugs. A review of the literature (1998 to 2014) was performed to identify relevant, peer-reviewed articles. Using content analysis, we synthesised regulations and policies for access to orphan drugs by type and by country. Fifty seven articles and 35 countries were included in this review. Six broad categories of regulation and policy instruments were identified: national orphan drug policies, orphan drug designation, marketing authorization, incentives, marketing exclusivity, and pricing and reimbursement. The availability of orphan drugs depends on individual country's legislation and regulations including national orphan drug policies, orphan drug designation, marketing authorization, marketing exclusivity and incentives such as tax credits to ensure research, development and marketing. The majority of countries (27/35) had in place orphan drug legislation. Access to orphan drugs depends on individual country's pricing and reimbursement policies, which varied widely between countries. High prices and insufficient evidence often limit orphan drugs from meeting the traditional health technology assessment criteria, especially cost-effectiveness, which may influence access. Overall many countries have implemented a combination of legislations, regulations and policies for orphan drugs in the last two decades. While these may enable the availability and access to orphan drugs, there are critical differences between countries in terms of range and types of legislations, regulations and policies implemented. Importantly, China and India, two of the largest countries by population size, both lack national legislation for orphan medicines and rare diseases, which could have substantial negative impacts on their patient populations with rare diseases.

  12. Access to Orphan Drugs: A Comprehensive Review of Legislations, Regulations and Policies in 35 Countries.

    Directory of Open Access Journals (Sweden)

    Todd Gammie

    Full Text Available To review existing regulations and policies utilised by countries to enable patient access to orphan drugs.A review of the literature (1998 to 2014 was performed to identify relevant, peer-reviewed articles. Using content analysis, we synthesised regulations and policies for access to orphan drugs by type and by country.Fifty seven articles and 35 countries were included in this review. Six broad categories of regulation and policy instruments were identified: national orphan drug policies, orphan drug designation, marketing authorization, incentives, marketing exclusivity, and pricing and reimbursement. The availability of orphan drugs depends on individual country's legislation and regulations including national orphan drug policies, orphan drug designation, marketing authorization, marketing exclusivity and incentives such as tax credits to ensure research, development and marketing. The majority of countries (27/35 had in place orphan drug legislation. Access to orphan drugs depends on individual country's pricing and reimbursement policies, which varied widely between countries. High prices and insufficient evidence often limit orphan drugs from meeting the traditional health technology assessment criteria, especially cost-effectiveness, which may influence access.Overall many countries have implemented a combination of legislations, regulations and policies for orphan drugs in the last two decades. While these may enable the availability and access to orphan drugs, there are critical differences between countries in terms of range and types of legislations, regulations and policies implemented. Importantly, China and India, two of the largest countries by population size, both lack national legislation for orphan medicines and rare diseases, which could have substantial negative impacts on their patient populations with rare diseases.

  13. Ufd1-Npl4 Recruit Cdc48 for Disassembly of Ubiquitylated CMG Helicase at the End of Chromosome Replication

    Directory of Open Access Journals (Sweden)

    Marija Maric

    2017-03-01

    Full Text Available Disassembly of the Cdc45-MCM-GINS (CMG DNA helicase is the key regulated step during DNA replication termination in eukaryotes, involving ubiquitylation of the Mcm7 helicase subunit, leading to a disassembly process that requires the Cdc48 “segregase”. Here, we employ a screen to identify partners of budding yeast Cdc48 that are important for disassembly of ubiquitylated CMG helicase at the end of chromosome replication. We demonstrate that the ubiquitin-binding Ufd1-Npl4 complex recruits Cdc48 to ubiquitylated CMG. Ubiquitylation of CMG in yeast cell extracts is dependent upon lysine 29 of Mcm7, which is the only detectable site of ubiquitylation both in vitro and in vivo (though in vivo other sites can be modified when K29 is mutated. Mutation of K29 abrogates in vitro recruitment of Ufd1-Npl4-Cdc48 to the CMG helicase, supporting a model whereby Ufd1-Npl4 recruits Cdc48 to ubiquitylated CMG at the end of chromosome replication, thereby driving the disassembly reaction.

  14. Preliminary crystallographic characterization of an RNA helicase from Kunjin virus

    International Nuclear Information System (INIS)

    Mastrangelo, Eloise; Bollati, Michela; Milani, Mario; Brisbarre, Nadège; Lamballerie, Xavier de; Coutard, Bruno; Canard, Bruno; Khromykh, Alexander; Bolognesi, Martino

    2006-01-01

    The C-terminal 440 amino acids of the NS3 protein from Kunjin virus (Flaviviridae) code for a helicase. The protein has been overexpressed and crystallized. Characterization of the isolated monoclinic crystal form and diffraction data (at 3.0 Å resolution) are presented, together with a preliminary molecular-replacement solution. Kunjin virus is a member of the Flavivirus genus and is an Australian variant of West Nile virus. The C-terminal domain of the Kunjin virus NS3 protein displays helicase activity. The protein is thought to separate daughter and template RNA strands, assisting the initiation of replication by unwinding RNA secondary structure in the 3′ nontranslated region. Expression, purification and preliminary crystallographic characterization of the NS3 helicase domain are reported. It is shown that Kunjin virus helicase may adopt a dimeric assembly in absence of nucleic acids, oligomerization being a means to provide the helicases with multiple nucleic acid-binding capability, facilitating translocation along the RNA strands. Kunjin virus NS3 helicase domain is an attractive model for studying the molecular mechanisms of flavivirus replication, while simultaneously providing a new basis for the rational development of anti-flaviviral compounds

  15. DNA-conjugated gold nanoparticles based colorimetric assay to assess helicase activity: a novel route to screen potential helicase inhibitors

    Science.gov (United States)

    Deka, Jashmini; Mojumdar, Aditya; Parisse, Pietro; Onesti, Silvia; Casalis, Loredana

    2017-03-01

    Helicase are essential enzymes which are widespread in all life-forms. Due to their central role in nucleic acid metabolism, they are emerging as important targets for anti-viral, antibacterial and anti-cancer drugs. The development of easy, cheap, fast and robust biochemical assays to measure helicase activity, overcoming the limitations of the current methods, is a pre-requisite for the discovery of helicase inhibitors through high-throughput screenings. We have developed a method which exploits the optical properties of DNA-conjugated gold nanoparticles (AuNP) and meets the required criteria. The method was tested with the catalytic domain of the human RecQ4 helicase and compared with a conventional FRET-based assay. The AuNP-based assay produced similar results but is simpler, more robust and cheaper than FRET. Therefore, our nanotechnology-based platform shows the potential to provide a useful alternative to the existing conventional methods for following helicase activity and to screen small-molecule libraries as potential helicase inhibitors.

  16. RNases and Helicases in Gram-Positive Bacteria.

    Science.gov (United States)

    Durand, Sylvain; Condon, Ciaran

    2018-04-01

    RNases are key enzymes involved in RNA maturation and degradation. Although they play a crucial role in all domains of life, bacteria, archaea, and eukaryotes have evolved with their own sets of RNases and proteins modulating their activities. In bacteria, these enzymes allow modulation of gene expression to adapt to rapidly changing environments. Today, >20 RNases have been identified in both Escherichia coli and Bacillus subtilis , the paradigms of the Gram-negative and Gram-positive bacteria, respectively. However, only a handful of these enzymes are common to these two organisms and some of them are essential to only one. Moreover, although sets of RNases can be very similar in closely related bacteria such as the Firmicutes Staphylococcus aureus and B. subtilis , the relative importance of individual enzymes in posttranscriptional regulation in these organisms varies. In this review, we detail the role of the main RNases involved in RNA maturation and degradation in Gram-positive bacteria, with an emphasis on the roles of RNase J1, RNase III, and RNase Y. We also discuss how other proteins such as helicases can modulate the RNA-degradation activities of these enzymes.

  17. Can NGOs regulate medicines markets? Social enterprise in wholesaling, and access to essential medicines.

    Science.gov (United States)

    Mackintosh, Maureen; Chaudhuri, Sudip; Mujinja, Phares Gm

    2011-02-28

    Citizens of high income countries rely on highly regulated medicines markets. However low income countries' impoverished populations generally struggle for access to essential medicines through out-of-pocket purchase on poorly regulated markets; results include ill health, drug resistance and further impoverishment. While the role of health facilities owned by non-governmental organisations (NGOs) in low income countries is well documented, national and international wholesaling of essential medicines by NGOs is largely unstudied. This article describes and assesses the activity of NGOs and social enterprise in essential medicines wholesaling. The article is based on a set of interviews conducted in 2006-8 with trading NGOs and social enterprises operating in Europe, India and Tanzania. The analysis applies socio-legal and economic perspectives on social enterprise and market regulation. Trading NGOs can resist the perverse incentives inherent in medicines wholesaling and improve access to essential medicines; they can also, in definable circumstances, exercise a broader regulatory influence over their markets by influencing the behaviour of competitors. We explore reasons for success and failure of social enterprise in essential medicines wholesaling, including commercial manufacturers' market response; social enterprise traders' own market strategies; and patterns of market advantage, market segmentation and subsidy generated by donors. We conclude that, in the absence of effective governmental activity and regulation, social enterprise wholesaling can improve access to good quality essential medicines. This role should be valued and where appropriate supported in international health policy design. NGO regulatory impact can complement but should not replace state action.

  18. The open access regulation in Brazil; A regulacao do livre acesso no Brasil

    Energy Technology Data Exchange (ETDEWEB)

    Andrade, Maisa Medeiros Pacheco de; Siqueira, Mariana de; Xavier, Yanko Marcius de Alencar [Universidade Federal do Rio Grande do Norte (UFRN), Natal, RN (Brazil)

    2008-07-01

    The present research, attempting for the economic relevance of the natural gas sector, for the lack of a law that disciplines it and, still, for the structural question of the natural gas ducts activity; it analyzes the monopoly of the natural gas ducts activity and the mechanism of competition chosen to brighten up it: the open access. The regulation of the open access and the viability that the insert of this practice has to Brazil nowadays are very important points to be discussed and, in that way, the present research also analyses them. (author)

  19. The DEAD-Box RNA Helicase DDX3 Interacts with m6A RNA Demethylase ALKBH5

    Directory of Open Access Journals (Sweden)

    Abdullah Shah

    2017-01-01

    Full Text Available DDX3 is a member of the family of DEAD-box RNA helicases. DDX3 is a multifaceted helicase and plays essential roles in key biological processes such as cell cycle, stress response, apoptosis, and RNA metabolism. In this study, we found that DDX3 interacted with ALKBH5, an m6A RNA demethylase. The ATP domain of DDX3 and DSBH domain of ALKBH5 were indispensable to their interaction with each other. Furthermore, DDX3 could modulate the demethylation of mRNAs. We also showed that DDX3 regulated the methylation status of microRNAs and there was an interaction between DDX3 and AGO2. The dynamics of m6A RNA modification is still a field demanding further investigation, and here, we add a link by showing that RNA demethylation can be regulated by proteins such as DDX3.

  20. Dissection of the functional domains of an archaeal holliday junction helicase

    DEFF Research Database (Denmark)

    Hong, Ye; Chu, Mingzhu; Li, Yansheng

    2012-01-01

    Helicases and nucleases form complexes that play very important roles in DNA repair pathways some of which interact with each other at Holliday junctions. In this study, we present in vitro and in vivo analysis of Hjm and its interaction with Hjc in Sulfolobus. In vitro studies employed Hjm from...... conformation change of the enzyme. Furthermore, StoHjm is able to prevent the formation of Hjc/HJ high complex, suggesting a regulation mechanism of Hjm to the activity of Hjc. We show that Hjm is essential for cell viability using recently developed genetic system and mutant propagation assay, suggesting...

  1. Prevention of suicide with regulations aimed at restricting access to highly hazardous pesticides

    DEFF Research Database (Denmark)

    Gunnell, David; Knipe, Duleeka; Chang, Shu Sen

    2017-01-01

    Background: Pesticide self-poisoning accounts for 14–20% of suicides worldwide. Regulation aimed at restricting access to pesticides or banning highly hazardous pesticides is one approach to reducing these deaths. We systematically reviewed the evidence of the effectiveness of pesticide regulation...... in reducing the incidence of pesticide suicides and overall suicides. Methods: We did a systematic review of the international evidence. We searched MEDLINE, PsycINFO, and Embase for studies published between Jan 1, 1960, and Dec 31, 2016, which investigated the effect of national or regional bans, and sales...... or import restrictions, on the availability of one or more pesticides and the incidence of suicide in different countries. We excluded other interventions aimed at limiting community access to pesticides. We extracted data from studies presenting pesticide suicide data and overall suicide data from before...

  2. Improving access to new diagnostics through harmonised regulation: priorities for action

    Directory of Open Access Journals (Sweden)

    Ruth McNerney

    2014-04-01

    Full Text Available A new generation of diagnostic tests is being developed for use at the point of care that could save lives and reduce the spread of infectious diseases through early detection and treatment. It is important that patients in developing countries have access to these products at affordable prices and without delay. Regulation of medical products is intended to ensure safety and quality whilst balancing the need for timely access to beneficial new products. Current regulatory oversight of diagnostic tests in developing countries is highly variable and weak regulation allows poor-quality tests to enter the market. However, inefficient orover zealous regulation results in unnecessary delays, increases costs and acts as a barrier to innovation and market entry. Setting international standards and streamlining the regulatory process could reduce these barriers. Four priority activities have been identified where convergence of standards and protocols or joint review of data would be advantageous: (1 adoption of a common registration file for pre-market approval; (2 convergence of quality standards for manufacturing site inspections; (3 use of common evaluation protocols, aswell as joint review of data, to reduce unnecessary duplication of lengthy and costly clinical performance studies; and (4 use of networks of laboratories for post-market surveillance in order to monitor ongoing quality of diagnostic devices. The adoption and implementation of such measures in developing countries could accelerate access to new diagnostic tests that are safe and affordable.

  3. Improving access to new diagnostics through harmonised regulation: priorities for action.

    Science.gov (United States)

    McNerney, Ruth; Sollis, Kimberly; Peeling, Rosanna W

    2014-01-01

    A new generation of diagnostic tests is being developed for use at the point of care that could save lives and reduce the spread of infectious diseases through early detection and treatment. It is important that patients in developing countries have access to these products at affordable prices and without delay. Regulation of medical products is intended to ensure safety and quality whilst balancing the need for timely access to beneficial new products. Current regulatory oversight of diagnostic tests in developing countries is highly variable and weak regulation allows poor-quality tests to enter the market. However, inefficient or overzealous regulation results in unnecessary delays, increases costs and acts as a barrier to innovation and market entry. Setting international standards and streamlining the regulatory process could reduce these barriers. Four priority activities have been identified where convergence of standards and protocols or joint review of data would be advantageous: (1) adoption of a common registration file for pre-market approval; (2) convergence of quality standards for manufacturing site inspections; (3) use of common evaluation protocols, as well as joint review of data, to reduce unnecessary duplication of lengthy and costly clinical performance studies; and (4) use of networks of laboratories for post-market surveillance in order to monitor ongoing quality of diagnostic devices. The adoption and implementation of such measures in developing countries could accelerate access to new diagnostic tests that are safe and affordable.

  4. Regional differences in electricity distribution costs and their consequences for yardstick regulation of access prices

    International Nuclear Information System (INIS)

    Filippini, M.; Wild, J.

    2001-01-01

    In this paper we estimate an average-cost function for a panel of 59 Swiss local and regional electricity distribution utilities as a basis for yardstick regulation of the distribution-network access prices. Shleifer (1985) proposed yardstick competition in terms of price to regulate local monopolies producing a homogeneous good. The regulated price for the individual firms depends on the average costs of identical firms. The yardstick competition concept can also be applied to firms that produce heterogeneous goods if these goods differ only in observable characteristics. To correct the yardstick for heterogeneity the regulator can use a multivariate estimation of an average-cost function. In the case of electricity distribution, the heterogeneity of output consists mainly of different characteristics of the distribution service areas. In this paper we follow Shleifer's suggestion to estimate a multivariate average-cost function that can be employed by the regulatory commission to benchmark network access prices at the distribution level. Several exogenous variables measuring the heterogeneity of the service areas were included in the cost model specification. We find that the regional differences of the service areas - e.g. area shares of forests, agricultural areas or unproductive land and population density - significantly influence electricity distribution costs

  5. Accessibility

    DEFF Research Database (Denmark)

    Brooks, Anthony Lewis

    2017-01-01

    This contribution is timely as it addresses accessibility in regards system hardware and software aligned with introduction of the Twenty-First Century Communications and Video Accessibility Act (CVAA) and adjoined game industry waiver that comes into force January 2017. This is an act created...... by the USA Federal Communications Commission (FCC) to increase the access of persons with disabilities to modern communications, and for other purposes. The act impacts advanced communications services and products including text messaging; e-mail; instant messaging; video communications; browsers; game...... platforms; and games software. However, the CVAA has no legal status in the EU. This text succinctly introduces and questions implications, impact, and wider adoption. By presenting the full CVAA and game industry waiver the text targets to motivate discussions and further publications on the subject...

  6. Can NGOs regulate medicines markets? Social enterprise in wholesaling, and access to essential medicines

    Directory of Open Access Journals (Sweden)

    Chaudhuri Sudip

    2011-02-01

    Full Text Available Abstract Background Citizens of high income countries rely on highly regulated medicines markets. However low income countries' impoverished populations generally struggle for access to essential medicines through out-of-pocket purchase on poorly regulated markets; results include ill health, drug resistance and further impoverishment. While the role of health facilities owned by non-governmental organisations (NGOs in low income countries is well documented, national and international wholesaling of essential medicines by NGOs is largely unstudied. This article describes and assesses the activity of NGOs and social enterprise in essential medicines wholesaling. Methods The article is based on a set of interviews conducted in 2006-8 with trading NGOs and social enterprises operating in Europe, India and Tanzania. The analysis applies socio-legal and economic perspectives on social enterprise and market regulation. Results Trading NGOs can resist the perverse incentives inherent in medicines wholesaling and improve access to essential medicines; they can also, in definable circumstances, exercise a broader regulatory influence over their markets by influencing the behaviour of competitors. We explore reasons for success and failure of social enterprise in essential medicines wholesaling, including commercial manufacturers' market response; social enterprise traders' own market strategies; and patterns of market advantage, market segmentation and subsidy generated by donors. Conclusions We conclude that, in the absence of effective governmental activity and regulation, social enterprise wholesaling can improve access to good quality essential medicines. This role should be valued and where appropriate supported in international health policy design. NGO regulatory impact can complement but should not replace state action.

  7. Can NGOs regulate medicines markets? Social enterprise in wholesaling, and access to essential medicines

    Science.gov (United States)

    2011-01-01

    Background Citizens of high income countries rely on highly regulated medicines markets. However low income countries' impoverished populations generally struggle for access to essential medicines through out-of-pocket purchase on poorly regulated markets; results include ill health, drug resistance and further impoverishment. While the role of health facilities owned by non-governmental organisations (NGOs) in low income countries is well documented, national and international wholesaling of essential medicines by NGOs is largely unstudied. This article describes and assesses the activity of NGOs and social enterprise in essential medicines wholesaling. Methods The article is based on a set of interviews conducted in 2006-8 with trading NGOs and social enterprises operating in Europe, India and Tanzania. The analysis applies socio-legal and economic perspectives on social enterprise and market regulation. Results Trading NGOs can resist the perverse incentives inherent in medicines wholesaling and improve access to essential medicines; they can also, in definable circumstances, exercise a broader regulatory influence over their markets by influencing the behaviour of competitors. We explore reasons for success and failure of social enterprise in essential medicines wholesaling, including commercial manufacturers' market response; social enterprise traders' own market strategies; and patterns of market advantage, market segmentation and subsidy generated by donors. Conclusions We conclude that, in the absence of effective governmental activity and regulation, social enterprise wholesaling can improve access to good quality essential medicines. This role should be valued and where appropriate supported in international health policy design. NGO regulatory impact can complement but should not replace state action. PMID:21356076

  8. RecQ helicases and cellular responses to DNA damage

    International Nuclear Information System (INIS)

    Wu, Leonard; Hickson, Ian D.

    2002-01-01

    The faithful replication of the genome is essential for the survival of all organisms. It is not surprising therefore that numerous mechanisms have evolved to ensure that duplication of the genome occurs with only minimal risk of mutation induction. One mechanism of genome destabilization is replication fork demise, which can occur when a translocating fork meets a lesion or adduct in the template. Indeed, the collapse of replication forks has been suggested to occur in every replicative cell cycle making this a potentially significant problem for all proliferating cells. The RecQ helicases, which are essential for the maintenance of genome stability, are thought to function during DNA replication. In particular, RecQ helicase mutants display replication defects and have phenotypes consistent with an inability to efficiently reinitiate replication following replication fork demise. Here, we review some current models for how replication fork repair might be effected, and discuss potential roles for RecQ helicases in this process

  9. The nuclear import of RNA helicase A is mediated by importin-α3

    International Nuclear Information System (INIS)

    Aratani, Satoko; Oishi, Takayuki; Fujita, Hidetoshi; Nakazawa, Minako; Fujii, Ryouji; Imamoto, Naoko; Yoneda, Yoshihiro; Fukamizu, Akiyoshi; Nakajima, Toshihiro

    2006-01-01

    RNA helicase A (RHA), an ATPase/helicase, regulates the gene expression at various steps including transcriptional activation and RNA processing. RHA is known to shuttle between the nucleus and cytoplasm. We identified the nuclear localization signal (NLS) of RHA and analyzed the nuclear import mechanisms. The NLS of RHA (RHA-NLS) consisting of 19 amino acid residues is highly conserved through species and does not have the consensus classical NLS. In vitro nuclear import assays revealed that the nuclear import of RHA was Ran-dependent and mediated with the classical importin-α/β-dependent pathway. The binding assay indicated that the basic residues in RHA-NLS were used for interaction with importin-α. Furthermore, the nuclear import of RHA-NLS was supported by importin-α1 and preferentially importin-α3. Our results indicate that the nuclear import of RHA is mediated by the importin-α3/importin-β-dependent pathway and suggest that the specificity for importin may regulate the functions of cargo proteins

  10. Bloom syndrome helicase in meiosis: Pro-crossover functions of an anti-crossover protein.

    Science.gov (United States)

    Hatkevich, Talia; Sekelsky, Jeff

    2017-09-01

    The functions of the Bloom syndrome helicase (BLM) and its orthologs are well characterized in mitotic DNA damage repair, but their roles within the context of meiotic recombination are less clear. In meiotic recombination, multiple repair pathways are used to repair meiotic DSBs, and current studies suggest that BLM may regulate the use of these pathways. Based on literature from Saccharomyces cerevisiae, Arabidopsis thaliana, Mus musculus, Drosophila melanogaster, and Caenorhabditis elegans, we present a unified model for a critical meiotic role of BLM and its orthologs. In this model, BLM and its orthologs utilize helicase activity to regulate the use of various pathways in meiotic recombination by continuously disassembling recombination intermediates. This unwinding activity provides the meiotic program with a steady pool of early recombination substrates, increasing the probability for a DSB to be processed by the appropriate pathway. As a result of BLM activity, crossovers are properly placed throughout the genome, promoting proper chromosomal disjunction at the end of meiosis. This unified model can be used to further refine the complex role of BLM and its orthologs in meiotic recombination. © 2017 WILEY Periodicals, Inc.

  11. G-quadruplexes Significantly Stimulate Pif1 Helicase-catalyzed Duplex DNA Unwinding*

    Science.gov (United States)

    Duan, Xiao-Lei; Liu, Na-Nv; Yang, Yan-Tao; Li, Hai-Hong; Li, Ming; Dou, Shuo-Xing; Xi, Xu-Guang

    2015-01-01

    The evolutionarily conserved G-quadruplexes (G4s) are faithfully inherited and serve a variety of cellular functions such as telomere maintenance, gene regulation, DNA replication initiation, and epigenetic regulation. Different from the Watson-Crick base-pairing found in duplex DNA, G4s are formed via Hoogsteen base pairing and are very stable and compact DNA structures. Failure of untangling them in the cell impedes DNA-based transactions and leads to genome instability. Cells have evolved highly specific helicases to resolve G4 structures. We used a recombinant nuclear form of Saccharomyces cerevisiae Pif1 to characterize Pif1-mediated DNA unwinding with a substrate mimicking an ongoing lagging strand synthesis stalled by G4s, which resembles a replication origin and a G4-structured flap in Okazaki fragment maturation. We find that the presence of G4 may greatly stimulate the Pif1 helicase to unwind duplex DNA. Further studies reveal that this stimulation results from G4-enhanced Pif1 dimerization, which is required for duplex DNA unwinding. This finding provides new insights into the properties and functions of G4s. We discuss the observed activation phenomenon in relation to the possible regulatory role of G4s in the rapid rescue of the stalled lagging strand synthesis by helping the replicator recognize and activate the replication origin as well as by quickly removing the G4-structured flap during Okazaki fragment maturation. PMID:25627683

  12. Mycobacterium tuberculosis DinG is a structure-specific helicase that unwinds G4 DNA: implications for targeting G4 DNA as a novel therapeutic approach.

    Science.gov (United States)

    Thakur, Roshan Singh; Desingu, Ambika; Basavaraju, Shivakumar; Subramanya, Shreelakshmi; Rao, Desirazu N; Nagaraju, Ganesh

    2014-09-05

    The significance of G-quadruplexes and the helicases that resolve G4 structures in prokaryotes is poorly understood. The Mycobacterium tuberculosis genome is GC-rich and contains >10,000 sequences that have the potential to form G4 structures. In Escherichia coli, RecQ helicase unwinds G4 structures. However, RecQ is absent in M. tuberculosis, and the helicase that participates in G4 resolution in M. tuberculosis is obscure. Here, we show that M. tuberculosis DinG (MtDinG) exhibits high affinity for ssDNA and ssDNA translocation with a 5' → 3' polarity. Interestingly, MtDinG unwinds overhangs, flap structures, and forked duplexes but fails to unwind linear duplex DNA. Our data with DNase I footprinting provide mechanistic insights and suggest that MtDinG is a 5' → 3' polarity helicase. Notably, in contrast to E. coli DinG, MtDinG catalyzes unwinding of replication fork and Holliday junction structures. Strikingly, we find that MtDinG resolves intermolecular G4 structures. These data suggest that MtDinG is a multifunctional structure-specific helicase that unwinds model structures of DNA replication, repair, and recombination as well as G4 structures. We finally demonstrate that promoter sequences of M. tuberculosis PE_PGRS2, mce1R, and moeB1 genes contain G4 structures, implying that G4 structures may regulate gene expression in M. tuberculosis. We discuss these data and implicate targeting G4 structures and DinG helicase in M. tuberculosis could be a novel therapeutic strategy for culminating the infection with this pathogen. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  13. The Human Genome Project: Information access, management, and regulation. Final report

    Energy Technology Data Exchange (ETDEWEB)

    McInerney, J.D.; Micikas, L.B.

    1996-08-31

    The Human Genome Project is a large, internationally coordinated effort in biological research directed at creating a detailed map of human DNA. This report describes the access of information, management, and regulation of the project. The project led to the development of an instructional module titled The Human Genome Project: Biology, Computers, and Privacy, designed for use in high school biology classes. The module consists of print materials and both Macintosh and Windows versions of related computer software-Appendix A contains a copy of the print materials and discs containing the two versions of the software.

  14. Cognitive radio policy and regulation techno-economic studies to facilitate dynamic spectrum access

    CERN Document Server

    Holland, Oliver

    2014-01-01

    This book offers a timely reflection on how the proliferation of advanced wireless communications technologies, particularly cognitive radio (CR) can be enabled by thoroughly-considered policy and appropriate regulation. It looks at the prospects of CR from the divergent standpoints of technological development and economic market reality. The book provides a broad survey of various techno-economic and policy aspects of CR development, and provides the reader with an understanding of the complexities involved as well as a toolbox of possible solutions to enable the evolutionary leap towards successful implementation of disruptive CR technology or indeed any other novel wireless technologies. Cognitive Radio Policy and Regulation showcases the original ideas and concepts introduced into the field of CR and dynamic spectrum access policy over nearly four years of work within COST Action IC0905 TERRA, a think-tank with participants from more than 20 countries. The book’s subject matter includes: • deploymen...

  15. MCM Paradox: Abundance of Eukaryotic Replicative Helicases and Genomic Integrity.

    Science.gov (United States)

    Das, Mitali; Singh, Sunita; Pradhan, Satyajit; Narayan, Gopeshwar

    2014-01-01

    As a crucial component of DNA replication licensing system, minichromosome maintenance (MCM) 2-7 complex acts as the eukaryotic DNA replicative helicase. The six related MCM proteins form a heterohexamer and bind with ORC, CDC6, and Cdt1 to form the prereplication complex. Although the MCMs are well known as replicative helicases, their overabundance and distribution patterns on chromatin present a paradox called the "MCM paradox." Several approaches had been taken to solve the MCM paradox and describe the purpose of excess MCMs distributed beyond the replication origins. Alternative functions of these MCMs rather than a helicase had also been proposed. This review focuses on several models and concepts generated to solve the MCM paradox coinciding with their helicase function and provides insight into the concept that excess MCMs are meant for licensing dormant origins as a backup during replication stress. Finally, we extend our view towards the effect of alteration of MCM level. Though an excess MCM constituent is needed for normal cells to withstand stress, there must be a delineation of the threshold level in normal and malignant cells. This review also outlooks the future prospects to better understand the MCM biology.

  16. Emerging Importance of Helicases in Plant Stress Tolerance: Characterization of Oryza sativa Repair Helicase XPB2 Promoter and Its Functional Validation in Tobacco under Multiple Stresses.

    Science.gov (United States)

    Raikwar, Shailendra; Srivastava, Vineet K; Gill, Sarvajeet S; Tuteja, Renu; Tuteja, Narendra

    2015-01-01

    Genetic material always remains at the risk of spontaneous or induced damage which challenges the normal functioning of DNA molecule, thus, DNA repair is vital to protect the organisms against genetic damage. Helicases, the unique molecular motors, are emerged as prospective molecules to engineer stress tolerance in plants and are involved in nucleic acid metabolism including DNA repair. The repair helicase, XPB is an evolutionary conserved protein present in different organisms, including plants. Availability of few efficient promoters for gene expression in plants provoked us to study the promoter of XPB for better understanding of gene regulation under stress conditions. Here, we report the in silico analysis of novel stress inducible promoter of Oryza sativa XPB2 (OsXPB2). The in vivo validation of functionality/activity of OsXPB2 promoter under abiotic and hormonal stress conditions was performed by Agrobacterium-mediated transient assay in tobacco leaves using OsXPB2::GUS chimeric construct. The present research revealed that OsXPB2 promoter contains cis-elements accounting for various abiotic stresses (salt, dehydration, or cold) and hormone (Auxin, ABA, or MeJA) induced GUS expression/activity in the promoter-reporter assay. The promoter region of OsXPB2 contains CACG, GTAACG, CACGTG, CGTCA CCGCCGCGCT cis acting-elements which are reported to be salt, dehydration, cold, MeJA, or ABA responsive, respectively. Functional analysis was done by Agrobacterium-mediated transient assay using agroinfiltration in tobacco leaves, followed by GUS staining and fluorescence quantitative analyses. The results revealed high induction of GUS activity under multiple abiotic stresses as compared to mock treated control. The present findings suggest that OsXPB2 promoter is a multi-stress inducible promoter and has potential applications in sustainable crop production under abiotic stresses by regulating desirable pattern of gene expression.

  17. The Arabidopsis thaliana Homolog of the Helicase RTEL1 Plays Multiple Roles in Preserving Genome Stability[C][W

    Science.gov (United States)

    Recker, Julia; Knoll, Alexander; Puchta, Holger

    2014-01-01

    In humans, mutations in the DNA helicase Regulator of Telomere Elongation Helicase1 (RTEL1) lead to Hoyeraal-Hreidarsson syndrome, a severe, multisystem disorder. Here, we demonstrate that the RTEL1 homolog in Arabidopsis thaliana plays multiple roles in preserving genome stability. RTEL1 suppresses homologous recombination in a pathway parallel to that of the DNA translocase FANCM. Cytological analyses of root meristems indicate that RTEL1 is involved in processing DNA replication intermediates independently from FANCM and the nuclease MUS81. Moreover, RTEL1 is involved in interstrand and intrastrand DNA cross-link repair independently from FANCM and (in intrastrand cross-link repair) parallel to MUS81. RTEL1 contributes to telomere homeostasis; the concurrent loss of RTEL1 and the telomerase TERT leads to rapid, severe telomere shortening, which occurs much more rapidly than it does in the single-mutant line tert, resulting in developmental arrest after four generations. The double mutant rtel1-1 recq4A-4 exhibits massive growth defects, indicating that this RecQ family helicase, which is also involved in the suppression of homologous recombination and the repair of DNA lesions, can partially replace RTEL1 in the processing of DNA intermediates. The requirement for RTEL1 in multiple pathways to preserve genome stability in plants can be explained by its putative role in the destabilization of DNA loop structures, such as D-loops and T-loops. PMID:25516598

  18. Three-dimensional structure of N-terminal domain of DnaB helicase and helicase-primase interactions in Helicobacter pylori.

    Directory of Open Access Journals (Sweden)

    Tara Kashav

    2009-10-01

    Full Text Available Replication initiation is a crucial step in genome duplication and homohexameric DnaB helicase plays a central role in the replication initiation process by unwinding the duplex DNA and interacting with several other proteins during the process of replication. N-terminal domain of DnaB is critical for helicase activity and for DnaG primase interactions. We present here the crystal structure of the N-terminal domain (NTD of H. pylori DnaB (HpDnaB helicase at 2.2 A resolution and compare the structural differences among helicases and correlate with the functional differences. The structural details of NTD suggest that the linker region between NTD and C-terminal helicase domain plays a vital role in accurate assembly of NTD dimers. The sequence analysis of the linker regions from several helicases reveals that they should form four helix bundles. We also report the characterization of H. pylori DnaG primase and study the helicase-primase interactions, where HpDnaG primase stimulates DNA unwinding activity of HpDnaB suggesting presence of helicase-primase cohort at the replication fork. The protein-protein interaction study of C-terminal domain of primase and different deletion constructs of helicase suggests that linker is essential for proper conformation of NTD to interact strongly with HpDnaG. The surface charge distribution on the primase binding surface of NTDs of various helicases suggests that DnaB-DnaG interaction and stability of the complex is most probably charge dependent. Structure of the linker and helicase-primase interactions indicate that HpDnaB differs greatly from E.coli DnaB despite both belong to gram negative bacteria.

  19. Velocity and processivity of helicase unwinding of double-stranded nucleic acids

    International Nuclear Information System (INIS)

    Betterton, M D; Juelicher, F

    2005-01-01

    Helicases are molecular motors which unwind double-stranded nucleic acids (dsNA) in cells. Many helicases move with directional bias on single-stranded (ss) nucleic acids, and couple their directional translocation to strand separation. A model of the coupling between translocation and unwinding uses an interaction potential to represent passive and active helicase mechanisms. A passive helicase must wait for thermal fluctuations to open dsNA base pairs before it can advance and inhibit NA closing. An active helicase directly destabilizes dsNA base pairs, accelerating the opening rate. Here we extend this model to include helicase unbinding from the nucleic-acid strand. The helicase processivity depends on the form of the interaction potential. A passive helicase has a mean attachment time which does not change between ss translocation and ds unwinding, while an active helicase in general shows a decrease in attachment time during unwinding relative to ss translocation. In addition, we describe how helicase unwinding velocity and processivity vary if the base-pair binding free energy is changed

  20. Requirement for the E1 Helicase C-Terminal Domain in Papillomavirus DNA Replication In Vivo.

    Science.gov (United States)

    Bergvall, Monika; Gagnon, David; Titolo, Steve; Lehoux, Michaël; D'Abramo, Claudia M; Melendy, Thomas; Archambault, Jacques

    2016-01-06

    The papillomavirus (PV) E1 helicase contains a conserved C-terminal domain (CTD), located next to its ATP-binding site, whose function in vivo is still poorly understood. The CTD is comprised of an alpha helix followed by an acidic region (AR) and a C-terminal extension termed the C-tail. Recent biochemical studies on bovine papillomavirus 1 (BPV1) E1 showed that the AR and C-tail regulate the oligomerization of the protein into a double hexamer at the origin. In this study, we assessed the importance of the CTD of human papillomavirus 11 (HPV11) E1 in vivo, using a cell-based DNA replication assay. Our results indicate that combined deletion of the AR and C-tail drastically reduces DNA replication, by 85%, and that further truncation into the alpha-helical region compromises the structural integrity of the E1 helicase domain and its interaction with E2. Surprisingly, removal of the C-tail alone or mutation of highly conserved residues within the domain still allows significant levels of DNA replication (55%). This is in contrast to the absolute requirement for the C-tail reported for BPV1 E1 in vitro and confirmed here in vivo. Characterization of chimeric proteins in which the AR and C-tail from HPV11 E1 were replaced by those of BPV1 indicated that while the function of the AR is transferable, that of the C-tail is not. Collectively, these findings define the contribution of the three CTD subdomains to the DNA replication activity of E1 in vivo and suggest that the function of the C-tail has evolved in a PV type-specific manner. While much is known about hexameric DNA helicases from superfamily 3, the papillomavirus E1 helicase contains a unique C-terminal domain (CTD) adjacent to its ATP-binding site. We show here that this CTD is important for the DNA replication activity of HPV11 E1 in vivo and that it can be divided into three functional subdomains that roughly correspond to the three conserved regions of the CTD: an alpha helix, needed for the structural

  1. Requirement for the E1 Helicase C-Terminal Domain in Papillomavirus DNA Replication In Vivo

    Science.gov (United States)

    Bergvall, Monika; Gagnon, David; Titolo, Steve; Lehoux, Michaël; D'Abramo, Claudia M.

    2016-01-01

    ABSTRACT The papillomavirus (PV) E1 helicase contains a conserved C-terminal domain (CTD), located next to its ATP-binding site, whose function in vivo is still poorly understood. The CTD is comprised of an alpha helix followed by an acidic region (AR) and a C-terminal extension termed the C-tail. Recent biochemical studies on bovine papillomavirus 1 (BPV1) E1 showed that the AR and C-tail regulate the oligomerization of the protein into a double hexamer at the origin. In this study, we assessed the importance of the CTD of human papillomavirus 11 (HPV11) E1 in vivo, using a cell-based DNA replication assay. Our results indicate that combined deletion of the AR and C-tail drastically reduces DNA replication, by 85%, and that further truncation into the alpha-helical region compromises the structural integrity of the E1 helicase domain and its interaction with E2. Surprisingly, removal of the C-tail alone or mutation of highly conserved residues within the domain still allows significant levels of DNA replication (55%). This is in contrast to the absolute requirement for the C-tail reported for BPV1 E1 in vitro and confirmed here in vivo. Characterization of chimeric proteins in which the AR and C-tail from HPV11 E1 were replaced by those of BPV1 indicated that while the function of the AR is transferable, that of the C-tail is not. Collectively, these findings define the contribution of the three CTD subdomains to the DNA replication activity of E1 in vivo and suggest that the function of the C-tail has evolved in a PV type-specific manner. IMPORTANCE While much is known about hexameric DNA helicases from superfamily 3, the papillomavirus E1 helicase contains a unique C-terminal domain (CTD) adjacent to its ATP-binding site. We show here that this CTD is important for the DNA replication activity of HPV11 E1 in vivo and that it can be divided into three functional subdomains that roughly correspond to the three conserved regions of the CTD: an alpha helix, needed

  2. Editorial. Special issue on strategies and self-regulation in self-access learning

    Directory of Open Access Journals (Sweden)

    Heath Rose

    2012-12-01

    Full Text Available Learner strategy and self-regulation theory have been in a state of flux in recent years, and this is an exciting time to share new ideas, conceptualizations and models of research in order to move the field forward. Therefore, the editor was eager to pursue a special issue where emerging voices in these fields could be heard, and these new ideas could be shared. The representation of strategic learning in recent conferences is indicative of a growing trend in the field to move towards a self-access and learner autonomy perspective. There is great potential to share knowledge between these fields. This special issue brings these fields, which have already been gravitating together, closer in a more concrete and published format.

  3. The SH2 domain of Abl kinases regulates kinase autophosphorylation by controlling activation loop accessibility

    Science.gov (United States)

    Lamontanara, Allan Joaquim; Georgeon, Sandrine; Tria, Giancarlo; Svergun, Dmitri I.; Hantschel, Oliver

    2014-11-01

    The activity of protein kinases is regulated by multiple molecular mechanisms, and their disruption is a common driver of oncogenesis. A central and almost universal control element of protein kinase activity is the activation loop that utilizes both conformation and phosphorylation status to determine substrate access. In this study, we use recombinant Abl tyrosine kinases and conformation-specific kinase inhibitors to quantitatively analyse structural changes that occur after Abl activation. Allosteric SH2-kinase domain interactions were previously shown to be essential for the leukemogenesis caused by the Bcr-Abl oncoprotein. We find that these allosteric interactions switch the Abl activation loop from a closed to a fully open conformation. This enables the trans-autophosphorylation of the activation loop and requires prior phosphorylation of the SH2-kinase linker. Disruption of the SH2-kinase interaction abolishes activation loop phosphorylation. Our analysis provides a molecular mechanism for the SH2 domain-dependent activation of Abl that may also regulate other tyrosine kinases.

  4. A Brownian motor mechanism of translocation and strand separation by hepatitis C virus helicase.

    Science.gov (United States)

    Levin, Mikhail K; Gurjar, Madhura; Patel, Smita S

    2005-05-01

    Helicases translocate along their nucleic acid substrates using the energy of ATP hydrolysis and by changing conformations of their nucleic acid-binding sites. Our goal is to characterize the conformational changes of hepatitis C virus (HCV) helicase at different stages of ATPase cycle and to determine how they lead to translocation. We have reported that ATP binding reduces HCV helicase affinity for nucleic acid. Now we identify the stage of the ATPase cycle responsible for translocation and unwinding. We show that a rapid directional movement occurs upon helicase binding to DNA in the absence of ATP, resulting in opening of several base pairs. We propose that HCV helicase translocates as a Brownian motor with a simple two-stroke cycle. The directional movement step is fueled by single-stranded DNA binding energy while ATP binding allows for a brief period of random movement that prepares the helicase for the next cycle.

  5. GINS complex protein Sld5 recruits SIK1 to activate MCM helicase during DNA replication.

    Science.gov (United States)

    Joshi, Kiranmai; Shah, Varun Jayeshkumar; Maddika, Subbareddy

    2016-12-01

    In eukaryotes, proper loading and activation of MCM helicase at chromosomal origins plays a central role in DNA replication. Activation of MCM helicase requires its association with CDC45-GINS complex, but the mechanism of how this complex activates MCM helicase is poorly understood. Here we identified SIK1 (salt-inducible kinase 1), an AMPK related protein kinase, as a molecular link that connects GINS complex with MCM helicase activity. We demonstrated that Sld5 a component of GINS complex interacts with SIK1 and recruits it to the sites of DNA replication at the onset of S phase. Depletion of SIK1 leads to defective DNA replication. Further, we showed that SIK1 phosphorylates MCM2 at five conserved residues at its N-terminus, which is essential for the activation of MCM helicase. Collectively, our results suggest SIK1 as a novel integral component of CMG replicative helicase during eukaryotic DNA replication. Copyright © 2016 Elsevier Inc. All rights reserved.

  6. Inhibition of RNA Helicases of ssRNA+ Virus Belonging to Flaviviridae, Coronaviridae and Picornaviridae Families

    Directory of Open Access Journals (Sweden)

    Irene Briguglio

    2011-01-01

    Full Text Available Many viral pathogens encode the motor proteins named RNA helicases which display various functions in genome replication. General strategies to design specific and selective drugs targeting helicase for the treatment of viral infections could act via one or more of the following mechanisms: inhibition of the NTPase activity, by interferences with ATP binding and therefore by limiting the energy required for the unwinding and translocation, or by allosteric mechanism and therefore by stabilizing the conformation of the enzyme in low helicase activity state; inhibition of nucleic acids binding to the helicase; inhibition of coupling of ATP hydrolysis to unwinding; inhibition of unwinding by sterically blocking helicase translocation. Recently, by in vitro screening studies, it has been reported that several benzotriazole, imidazole, imidazodiazepine, phenothiazine, quinoline, anthracycline, triphenylmethane, tropolone, pyrrole, acridone, small peptide, and Bananin derivatives are endowed with helicase inhibition of pathogen viruses belonging to Flaviviridae, Coronaviridae, and Picornaviridae families.

  7. The patient's safety and access to experimental drugs after the termination of clinical trials: regulations and trends.

    Science.gov (United States)

    da Silva, Ricardo Eccard; Amato, Angélica Amorim; Sousa, Thiago do Rego; de Carvalho, Marta Rodrigues; Novaes, Maria Rita Carvalho Garbi

    2018-05-12

    Participants' rights and safety must be guaranteed not only while a clinical trial is being conducted but also when a clinical trial finishes. The criteria for post-trial access to experimental drugs, however, are unclear in various countries. The objectives of this study were (i) to ascertain if there were regulations or guidelines related to patients' access to drugs after the end of clinical trials in the countries selected in the study and (ii) to analyze trends in post-trial access in countries classified by their level of economic development. This study is a retrospective review. The data are from the records of clinical trials from 2014 registered in the World Health Organization's International Clinical Trials Registry Platform (ICTRP) database. Among the countries selected, provision of drugs post-trial is mandatory only in Argentina, Brazil, Chile, Finland, and Peru. The plans for post-trial access tend to be more present in low- and middle-income and upper middle-income countries, in comparison with high-income countries. Studies involving vulnerable populations are 2.53 times more likely to have plans for post-trial access than studies which do not. The guaranteeing of post-trial access remains mandatory in few countries. Considering that individuals seen as vulnerable have been included in clinical trials without plans for post-trial access, stakeholders must discuss the need to develop regulations mandating the guaranteeing of post-trial access in specified situations.

  8. Saccharomyces cerevisiae Hrq1 requires a long 3′-tailed DNA substrate for helicase activity

    International Nuclear Information System (INIS)

    Kwon, Sung-Hun; Choi, Do-Hee; Lee, Rina; Bae, Sung-Ho

    2012-01-01

    Highlights: ► Hrq1 has intrinsic 3′–5′ helicase and DNA strand annealing activities. ► Hrq1 requires a long 3′-tail for efficient DNA unwinding. ► Helicase activity of Hrq1 is stimulated by a fork structure. ► Hrq1 is a moderately processive helicase. -- Abstract: RecQ helicases are well conserved proteins from bacteria to human and function in various DNA metabolism for maintenance of genome stability. Five RecQ helicases are found in humans, whereas only one RecQ helicase has been described in lower eukaryotes. However, recent studies predicted the presence of a second RecQ helicase, Hrq1, in fungal genomes and verified it as a functional gene in fission yeast. Here we show that 3′–5′ helicase activity is intrinsically associated with Hrq1 of Saccharomyces cerevisiae. We also determined several biochemical properties of Hrq1 helicase distinguishable from those of other RecQ helicase members. Hrq1 is able to unwind relatively long duplex DNA up to 120-bp and is significantly stimulated by a preexisting fork structure. Further, the most striking feature of Hrq1 is its absolute requirement for a long 3′-tail (⩾70-nt) for efficient unwinding of duplex DNA. We also found that Hrq1 has potent DNA strand annealing activity. Our results indicate that Hrq1 has vigorous helicase activity that deserves further characterization to expand our understanding of RecQ helicases.

  9. Mechanism of Archaeal MCM Helicase Recruitment to DNA Replication Origins

    Science.gov (United States)

    Samson, Rachel Y.; Abeyrathne, Priyanka D.; Bell, Stephen D.

    2015-01-01

    Summary Cellular DNA replication origins direct the recruitment of replicative helicases via the action of initiator proteins belonging to the AAA+ superfamily of ATPases. Archaea have a simplified subset of the eukaryotic DNA replication machinery proteins and possess initiators that appear ancestral to both eukaryotic Orc1 and Cdc6. We have reconstituted origin-dependent recruitment of the homohexameric archaeal MCM in vitro with purified recombinant proteins. Using this system, we reveal that archaeal Orc1-1 fulfills both Orc1 and Cdc6 functions by binding to a replication origin and directly recruiting MCM helicase. We identify the interaction interface between these proteins and reveal how ATP binding by Orc1-1 modulates recruitment of MCM. Additionally, we provide evidence that an open-ring form of the archaeal MCM homohexamer is loaded at origins. PMID:26725007

  10. microRNAs targeting DEAD-box helicases are involved in salinity stress response in rice (Oryza sativa L.

    Directory of Open Access Journals (Sweden)

    Macovei Anca

    2012-10-01

    Full Text Available Abstract Background Rice (Oryza sativa L., one of the most important food crop in the world, is considered to be a salt-sensitive crop. Excess levels of salt adversely affect all the major metabolic activities, including cell wall damage, cytoplasmic lysis and genomic stability. In order to cope with salt stress, plants have evolved high degrees of developmental plasticity, including adaptation via cascades of molecular networks and changes in gene expression profiles. Posttranscriptional regulation, through the activity of microRNAs, also plays an important role in the plant response to salinity conditions. MicroRNAs are small endogenous RNAs that modulate gene expression and are involved in the most essential physiological processes, including plant development and adaptation to environmental changes. Results In the present study, we investigated the expression profiles of osa-MIR414, osa-MIR408 and osa-MIR164e along with their targeted genes, under salinity stress conditions in wild type and transgenic rice plants ectopically expressing the PDH45 (Pea DNA Helicase gene. The present miRNAs were predicted to target the OsABP (ATP-Binding Protein, OsDSHCT (DOB1/SK12/helY-like DEAD-box Helicase and OsDBH (DEAD-Box Helicase genes, included in the DEAD-box helicase family. An in silico characterization of the proteins was performed and the miRNAs predicted targets were validated by RLM-5′RACE. The qRT-PCR analysis showed that the OsABP, OsDBH and OsDSHCT genes were up-regulated in response to 100 and 200 mM NaCl treatments. The present study also highlighted an increased accumulation of the gene transcripts in wild type plants, with the exception of the OsABP mRNA which showed the highest level (15.1-fold change compared to control in the transgenic plants treated with 200 mM NaCl. Salinity treatments also affected the expression of osa-MIR414, osa-MIR164e and osa-MIR408, found to be significantly down-regulated, although the changes in mi

  11. The MCM Helicase Motor of the Eukaryotic Replisome.

    Science.gov (United States)

    Abid Ali, Ferdos; Costa, Alessandro

    2016-05-08

    The MCM motor of the CMG helicase powers ahead of the eukaryotic replication machinery to unwind DNA, in a process that requires ATP hydrolysis. The reconstitution of DNA replication in vitro has established the succession of events that lead to replication origin activation by the MCM and recent studies have started to elucidate the structural basis of duplex DNA unwinding. Despite the exciting progress, how the MCM translocates on DNA remains a matter of debate. Copyright © 2016. Published by Elsevier Ltd.

  12. Saccharomyces cerevisiae Hrq1 requires a long 3'-tailed DNA substrate for helicase activity.

    Science.gov (United States)

    Kwon, Sung-Hun; Choi, Do-Hee; Lee, Rina; Bae, Sung-Ho

    2012-10-26

    RecQ helicases are well conserved proteins from bacteria to human and function in various DNA metabolism for maintenance of genome stability. Five RecQ helicases are found in humans, whereas only one RecQ helicase has been described in lower eukaryotes. However, recent studies predicted the presence of a second RecQ helicase, Hrq1, in fungal genomes and verified it as a functional gene in fission yeast. Here we show that 3'-5' helicase activity is intrinsically associated with Hrq1 of Saccharomyces cerevisiae. We also determined several biochemical properties of Hrq1 helicase distinguishable from those of other RecQ helicase members. Hrq1 is able to unwind relatively long duplex DNA up to 120-bp and is significantly stimulated by a preexisting fork structure. Further, the most striking feature of Hrq1 is its absolute requirement for a long 3'-tail (⩾70-nt) for efficient unwinding of duplex DNA. We also found that Hrq1 has potent DNA strand annealing activity. Our results indicate that Hrq1 has vigorous helicase activity that deserves further characterization to expand our understanding of RecQ helicases. Copyright © 2012 Elsevier Inc. All rights reserved.

  13. In TFIIH, XPD helicase is exclusively devoted to DNA repair.

    Directory of Open Access Journals (Sweden)

    Jochen Kuper

    2014-09-01

    Full Text Available The eukaryotic XPD helicase is an essential subunit of TFIIH involved in both transcription and nucleotide excision repair (NER. Mutations in human XPD are associated with several inherited diseases such as xeroderma pigmentosum, Cockayne syndrome, and trichothiodystrophy. We performed a comparative analysis of XPD from Homo sapiens and Chaetomium thermophilum (a closely related thermostable fungal orthologue to decipher the different molecular prerequisites necessary for either transcription or DNA repair. In vitro and in vivo assays demonstrate that mutations in the 4Fe4S cluster domain of XPD abrogate the NER function of TFIIH and do not affect its transcriptional activity. We show that the p44-dependent activation of XPD is promoted by the stimulation of its ATPase activity. Furthermore, we clearly demonstrate that XPD requires DNA binding, ATPase, and helicase activity to function in NER. In contrast, these enzymatic properties are dispensable for transcription initiation. XPD helicase is thus exclusively devoted to NER and merely acts as a structural scaffold to maintain TFIIH integrity during transcription.

  14. RTEL1 is a replisome-associated helicase that promotes telomere and genome-wide replication.

    Science.gov (United States)

    Vannier, Jean-Baptiste; Sandhu, Sumit; Petalcorin, Mark I R; Wu, Xiaoli; Nabi, Zinnatun; Ding, Hao; Boulton, Simon J

    2013-10-11

    Regulator of telomere length 1 (RTEL1) is an essential DNA helicase that disassembles telomere loops (T loops) and suppresses telomere fragility to maintain the integrity of chromosome ends. We established that RTEL1 also associates with the replisome through binding to proliferating cell nuclear antigen (PCNA). Mouse cells disrupted for the RTEL1-PCNA interaction (PIP mutant) exhibited accelerated senescence, replication fork instability, reduced replication fork extension rates, and increased origin usage. Although T-loop disassembly at telomeres was unaffected in the mutant cells, telomere replication was compromised, leading to fragile sites at telomeres. RTEL1-PIP mutant mice were viable, but loss of the RTEL1-PCNA interaction accelerated the onset of tumorigenesis in p53-deficient mice. We propose that RTEL1 plays a critical role in both telomere and genome-wide replication, which is crucial for genetic stability and tumor avoidance.

  15. The RNA helicase Rm62 cooperates with SU(VAR3-9 to re-silence active transcription in Drosophila melanogaster.

    Directory of Open Access Journals (Sweden)

    Joern Boeke

    Full Text Available Gene expression is highly dynamic and many genes show a wide range in expression over several orders of magnitude. This regulation is often mediated by sequence specific transcription factors. In addition, the tight packaging of DNA into chromatin can provide an additional layer of control resulting in a dynamic range of gene expression covering several orders of magnitude. During transcriptional activation, chromatin barriers have to be eliminated to allow an efficient progression of the RNA polymerase. This repressive chromatin structure has to be re-established quickly after it has been activated in order to tightly regulate gene activity. We show that the DExD/H box containing RNA helicase Rm62 is targeted to a site of rapid induction of transcription where it is responsible for an increased degree of methylation at H3K9 at the heat shock locus after removal of the heat shock stimulus. The RNA helicase interacts with the well-characterized histone methyltransferase SU(VAR3-9 via its N-terminus, which provides a potential mechanism for the targeting of H3K9 methylation to highly regulated genes. The recruitment of SU(VAR3-9 through interaction with a RNA helicase to a site of active transcription might be a general mechanism that allows an efficient silencing of highly regulated genes thereby enabling a cell to fine tune its gene activity over a wide range.

  16. What Remains on Your Mind After You Are Done?: Flexible Regulation of Knowledge Accessibility

    Science.gov (United States)

    Hedberg, Per H.; Higgins, E. Tory

    2011-01-01

    The accessibility of stored knowledge has been found to decline over time after activation without further stimulation. A special case is goal pursuit; goal-related knowledge remains accessible until goal completion, and then its accessibility declines rapidly. We hypothesized that after goal completion the decline in accessibility of goal-related knowledge would be especially rapid for strong promotion-focused individuals because their motivation to eagerly advance beyond the status quo would make accessibility of this knowledge an irrelevant detriment. We hypothesized an opposite effect for strongly prevention-predominant individuals because their motivation to vigilantly maintain a satisfactory state would make accessibility of this knowledge continually relevant. The results of two studies supported both these predicted moderators of accessibility change. Indeed, we found that for strongly prevention-predominant participants, knowledge accessibility actually increased over time after goal completion. We discuss how even basic cognitive mechanisms, like changes in accessibility, can be affected by general motivational concerns. PMID:21765541

  17. Emerging importance of helicases in plant stress tolerance: characterization of Oryza sativa repair helicase XPB2 promoter and its functional validation in tobacco under multiple stresses

    Directory of Open Access Journals (Sweden)

    Shailendra eRaikwar

    2015-12-01

    Full Text Available Genetic material always remains at the risk of spontaneous or induced damage which challenges the normal functioning of DNA molecule, thus, DNA repair is vital to protect the organisms against genetic damage. DNA hHelicases, the unique molecular motors, are emerged as potentialprospective molecules to engineer stress tolerance in plants and are involved in a variety of DNA nucleic acid metabolismc processes including DNA repair. The DNA repair helicase, OsXPB2 is an evolutionary conserved protein present in different organisms, including plants. Availability of few efficient promoters for gene expression in plants provoked us to study the promoter of XPB for better understanding of gene regulation under stress The analysis of promoter sequence from plant genome is important in understanding the gene regulation. Hereconditions. Here, we report the in silico analysis of novel stress inducible promoter of rice Oryza sativa OsXPB2 (OsXPB2. gene is reported. The in vivo validation of functionality/activity of novel stress inducible promoter of rice OsXPB2 gene promoter under abiotic and hormonal stress conditions was performed by Agrobacterium-mediated transient assay in tobacco leaves using OsXPB2::GUS chimeric construct. Our resultsThe present research revealed that OsXPB2 promoter contains cis-elements accounting for various abiotic stresses (salt, dehydration or cold and hormone (Auxin, ABA or MeJA induced GUS expression/activity in the promoter-reporter assay. The promoter region of OsXPB2 contains CACG, GTAACG, CACGTG, CGTCA CCGCCGCGCT cis acting-elements which are reported to be salt, dehydration, cold, MeJA or ABA responsive, respectively. Functional analysis was done by Agrobacterium-transient assays using agroinfiltration in tobacco leaves, followed by GUS staining and fluorescence quantitative analyses. The results revealed high induction of GUS activity under multiple abiotic stresses as compared to mock treated control. The present

  18. Suicide Rates and State Laws Regulating Access and Exposure to Handguns.

    Science.gov (United States)

    Anestis, Michael D; Anestis, Joye C

    2015-10-01

    Using previous research, we examined the impact of 4 handgun laws (waiting periods, universal background checks, gun locks, and open carrying regulations) on suicide rates. We used publicly available databases to collect information on statewide laws, suicide rates, and demographic characteristics for 2013. Each law was associated with significantly lower firearm suicide rates and the proportion of suicides resulting from firearms. In addition, each law, except for that which required a waiting period, was associated with a lower overall suicide rate. Follow-up analyses showed a significant indirect effect on overall suicide rates through the proportion of suicides by firearms, indicating that the reduced overall suicide rate was attributable to fewer suicide attempts, fewer handguns in the home, suicide attempts using less lethal means, or a combination of these factors. States that implemented any of these laws saw a decreased suicide rate in subsequent years, whereas the only state that repealed 1 of these laws saw an increased suicide rate. Our results were supportive of a potentially vital role in suicide prevention for state legislation that limits access and exposure to handguns.

  19. Sun-mediated mechanical LINC between nucleus and cytoskeleton regulates βcatenin nuclear access.

    Science.gov (United States)

    Uzer, Gunes; Bas, Guniz; Sen, Buer; Xie, Zhihui; Birks, Scott; Olcum, Melis; McGrath, Cody; Styner, Maya; Rubin, Janet

    2018-06-06

    βcatenin acts as a primary intracellular signal transducer for mechanical and Wnt signaling pathways to control cell function and fate. Regulation of βcatenin in the cytoplasm has been well studied but βcatenin nuclear trafficking and function remains unclear. In a previous study we showed that, in mesenchymal stem cells (MSC), mechanical blockade of adipogenesis relied on inhibition of βcatenin destruction complex element GSK3β (glycogen synthase kinase 3β) to increase nuclear βcatenin as well as the function of Linker of Cytoskeleton and Nucleoskeleton (LINC) complexes, suggesting that these two mechanisms may be linked. Here we show that shortly after inactivation of GSK3β due to either low intensity vibration (LIV), substrate strain or pharmacologic inhibition, βcatenin associates with the nucleoskeleton, defined as the insoluble nuclear fraction that provides structure to the integrated nuclear envelope, nuclear lamina and chromatin. Co-depleting LINC elements Sun-1 and Sun-2 interfered with both nucleoskeletal association and nuclear entry of βcatenin, resulting in decreased nuclear βcatenin levels. Our findings reveal that the insoluble structural nucleoskeleton actively participates in βcatenin dynamics. As the cytoskeleton transmits applied mechanical force to the nuclear surface to influence the nucleoskeleton and its LINC mediated interaction, our results suggest a pathway by which LINC mediated connectivity may play a role in signaling pathways that depend on nuclear access of βcatenin. Copyright © 2018 Elsevier Ltd. All rights reserved.

  20. Genome-wide identification of SF1 and SF2 helicases from archaea.

    Science.gov (United States)

    Chamieh, Hala; Ibrahim, Hiba; Kozah, Juliana

    2016-01-15

    Archaea microorganisms have long been used as model organisms for the study of protein molecular machines. Archaeal proteins are particularly appealing to study since archaea, even though prokaryotic, possess eukaryotic-like cellular processes. Super Family I (SF1) and Super Family II (SF2) helicase families have been studied in many model organisms, little is known about their presence and distribution in archaea. We performed an exhaustive search of homologs of SF1 and SF2 helicase proteins in 95 complete archaeal genomes. In the present study, we identified the complete sets of SF1 and SF2 helicases in archaea. Comparative analysis between archaea, human and the bacteria E. coli SF1 and SF2 helicases, resulted in the identification of seven helicase families conserved among representatives of the domains of life. This analysis suggests that these helicase families are highly conserved throughout evolution. We highlight the conserved motifs of each family and characteristic domains of the detected families. Distribution of SF1/SF2 families show that Ski2-like, Lhr, Sfth and Rad3-like helicases are ubiquitous among archaeal genomes while the other families are specific to certain archaeal groups. We also report the presence of a novel SF2 helicase specific to archaea domain named Archaea Specific Helicase (ASH). Phylogenetic analysis indicated that ASH has evolved in Euryarchaeota and is evolutionary related to the Ski2-like family with specific characteristic domains. Our study provides the first exhaustive analysis of SF1 and SF2 helicases from archaea. It expands the variety of SF1 and SF2 archaeal helicases known to exist to date and provides a starting point for new biochemical and genetic studies needed to validate their biological functions. Copyright © 2015 Elsevier B.V. All rights reserved.

  1. Research on the Legal Regulation of Market Access for Agricultural Products in China

    Institute of Scientific and Technical Information of China (English)

    2011-01-01

    On the basis of defining the concept of market access for agricultural products,this paper analyzes the necessity of establishing market access system of agricultural products,proposes the conception of establishing market access system of agricultural products in China;sets up the frame of market access system of agricultural products;analyzes the rationality of the frame of market access system of agricultural products;poses the consideration of economic law regarding setup of market access system of agricultural products.This paper also puts forward the legislative suggestions for establishing market access system of agricultural products as follows:establish the frame of market access system of agricultural products taking quality access as core;establish and perfect the compensation and relief system of guaranteeing benefit of manager and producers;establish the market access system of agricultural products with hierarchical structure;sort out existing laws and form the sound the legal frame of market access system of agricultural products.

  2. Identification of Hydroxyanthraquinones as Novel Inhibitors of Hepatitis C Virus NS3 Helicase

    Science.gov (United States)

    Furuta, Atsushi; Tsubuki, Masayoshi; Endoh, Miduki; Miyamoto, Tatsuki; Tanaka, Junichi; Abdus Salam, Kazi; Akimitsu, Nobuyoshi; Tani, Hidenori; Yamashita, Atsuya; Moriishi, Kohji; Nakakoshi, Masamichi; Sekiguchi, Yuji; Tsuneda, Satoshi; Noda, Naohiro

    2015-01-01

    Hepatitis C virus (HCV) is an important etiological agent of severe liver diseases, including cirrhosis and hepatocellular carcinoma. The HCV genome encodes nonstructural protein 3 (NS3) helicase, which is a potential anti-HCV drug target because its enzymatic activity is essential for viral replication. Some anthracyclines are known to be NS3 helicase inhibitors and have a hydroxyanthraquinone moiety in their structures; mitoxantrone, a hydroxyanthraquinone analogue, is also known to inhibit NS3 helicase. Therefore, we hypothesized that the hydroxyanthraquinone moiety alone could also inhibit NS3 helicase. Here, we performed a structure–activity relationship study on a series of hydroxyanthraquinones by using a fluorescence-based helicase assay. Hydroxyanthraquinones inhibited NS3 helicase with IC50 values in the micromolar range. The inhibitory activity varied depending on the number and position of the phenolic hydroxyl groups, and among different hydroxyanthraquinones examined, 1,4,5,8-tetrahydroxyanthraquinone strongly inhibited NS3 helicase with an IC50 value of 6 µM. Furthermore, hypericin and sennidin A, which both have two hydroxyanthraquinone-like moieties, were found to exert even stronger inhibition with IC50 values of 3 and 0.8 µM, respectively. These results indicate that the hydroxyanthraquinone moiety can inhibit NS3 helicase and suggest that several key chemical structures are important for the inhibition. PMID:26262613

  3. Identification of Hydroxyanthraquinones as Novel Inhibitors of Hepatitis C Virus NS3 Helicase

    Directory of Open Access Journals (Sweden)

    Atsushi Furuta

    2015-08-01

    Full Text Available Hepatitis C virus (HCV is an important etiological agent of severe liver diseases, including cirrhosis and hepatocellular carcinoma. The HCV genome encodes nonstructural protein 3 (NS3 helicase, which is a potential anti-HCV drug target because its enzymatic activity is essential for viral replication. Some anthracyclines are known to be NS3 helicase inhibitors and have a hydroxyanthraquinone moiety in their structures; mitoxantrone, a hydroxyanthraquinone analogue, is also known to inhibit NS3 helicase. Therefore, we hypothesized that the hydroxyanthraquinone moiety alone could also inhibit NS3 helicase. Here, we performed a structure–activity relationship study on a series of hydroxyanthraquinones by using a fluorescence-based helicase assay. Hydroxyanthraquinones inhibited NS3 helicase with IC50 values in the micromolar range. The inhibitory activity varied depending on the number and position of the phenolic hydroxyl groups, and among different hydroxyanthraquinones examined, 1,4,5,8-tetrahydroxyanthraquinone strongly inhibited NS3 helicase with an IC50 value of 6 µM. Furthermore, hypericin and sennidin A, which both have two hydroxyanthraquinone-like moieties, were found to exert even stronger inhibition with IC50 values of 3 and 0.8 µM, respectively. These results indicate that the hydroxyanthraquinone moiety can inhibit NS3 helicase and suggest that several key chemical structures are important for the inhibition.

  4. A role for the fission yeast Rqh1 helicase in chromosome segregation

    DEFF Research Database (Denmark)

    Win, Thein Z; Mankouri, Hocine W; Hickson, Ian D

    2005-01-01

    Schizosaccharomyces pombe Rqh1 protein is a member of the RecQ DNA helicase family. Members of this protein family are mutated in several human genome instability syndromes, including Bloom, Werner and Rothmund-Thomson syndromes. RecQ helicases participate in recombination repair of stalled...

  5. Proposed regulations could limit access to affordable health coverage for workers' children and family members.

    Science.gov (United States)

    Jacobs, Ken; Graham-Squire, Dave; Roby, Dylan H; Kominski, Gerald F; Kinane, Christina M; Needleman, Jack; Watson, Greg; Gans, Daphna

    2011-12-01

    Key Findings. The Patient Protection and Affordable Care Act (ACA) is designed to offer premium subsidies to help eligible individuals and their families purchase insurance coverage when affordable job-based coverage is not available. However, the law is unclear on how this affordability protection is applied in those instances where self-only coverage offered by an employer is affordable but family coverage is not. Regulations recently proposed by the Department of the Treasury would make family members ineligible for subsidized coverage in the exchange if an employee is offered affordable self-only coverage by an employer, even if family coverage is unaffordable. This could have significant financial consequences for low- and moderate-income families that fall in this gap. Using an alternative interpretation of the law could allow the entire family to enter the exchange when family coverage is unaffordable, which would broaden access to coverage. However, this option has been cited as cost prohibitive. In this brief we consider a middle ground alternative that would base eligibility for the individual worker on the cost of self-only coverage, but would use the additional cost to the employee for family coverage as the basis for determining affordability and eligibility for subsidies for the remaining family members. We find that: Under the middle ground alternative scenario an additional 144,000 Californians would qualify for and use premium subsidies in the California Health Benefit Exchange, half of whom are children. Less than 1 percent of those with employer-based coverage would move to subsidized coverage in the California Health Benefit Exchange as a result of having unaffordable coverage on the job.

  6. Emerging Importance of Helicases in Plant Stress Tolerance: Characterization of Oryza sativa Repair Helicase XPB2 Promoter and Its Functional Validation in Tobacco under Multiple Stresses

    OpenAIRE

    Raikwar, Shailendra; Srivastava, Vineet K.; Gill, Sarvajeet S.; Tuteja, Renu; Tuteja, Narendra

    2015-01-01

    Genetic material always remains at the risk of spontaneous or induced damage which challenges the normal functioning of DNA molecule, thus, DNA repair is vital to protect the organisms against genetic damage. Helicases, the unique molecular motors, are emerged as prospective molecules to engineer stress tolerance in plants and are involved in nucleic acid metabolism including DNA repair. The repair helicase, XPB is an evolutionary conserved protein present in different organisms, including pl...

  7. Emerging importance of helicases in plant stress tolerance: characterization of Oryza sativa repair helicase XPB2 promoter and its functional validation in tobacco under multiple stresses

    OpenAIRE

    Shailendra eRaikwar; Vineet Kumar Shrivastava; Sarvajeet Singh Gill; Renu eTuteja; Narendra eTuteja; Narendra eTuteja

    2015-01-01

    Genetic material always remains at the risk of spontaneous or induced damage which challenges the normal functioning of DNA molecule, thus, DNA repair is vital to protect the organisms against genetic damage. DNA hHelicases, the unique molecular motors, are emerged as potentialprospective molecules to engineer stress tolerance in plants and are involved in a variety of DNA nucleic acid metabolismc processes including DNA repair. The DNA repair helicase, OsXPB2 is an evolutionary conserved pr...

  8. Mycobacterium smegmatis HelY Is an RNA-Activated ATPase/dATPase and 3'-to-5' Helicase That Unwinds 3'-Tailed RNA Duplexes and RNA:DNA Hybrids.

    Science.gov (United States)

    Uson, Maria Loressa; Ordonez, Heather; Shuman, Stewart

    2015-10-01

    Mycobacteria have a large and distinctive ensemble of DNA helicases that function in DNA replication, repair, and recombination. Little is known about the roster of RNA helicases in mycobacteria or their roles in RNA transactions. The 912-amino-acid Mycobacterium smegmatis HelY (MSMEG_3885) protein is a bacterial homolog of the Mtr4 and Ski2 helicases that regulate RNA 3' processing and turnover by the eukaryal exosome. Here we characterize HelY as an RNA-stimulated ATPase/dATPase and an ATP/dATP-dependent 3'-to-5' helicase. HelY requires a 3' single-strand RNA tail (a loading RNA strand) to displace the complementary strand of a tailed RNA:RNA or RNA:DNA duplex. The findings that HelY ATPase is unresponsive to a DNA polynucleotide cofactor and that HelY is unable to unwind a 3'-tailed duplex in which the loading strand is DNA distinguish HelY from other mycobacterial nucleoside triphosphatases/helicases characterized previously. The biochemical properties of HelY, which resemble those of Mtr4/Ski2, hint at a role for HelY in mycobacterial RNA catabolism. RNA helicases play crucial roles in transcription, RNA processing, and translation by virtue of their ability to alter RNA secondary structure or remodel RNA-protein interactions. In eukarya, the RNA helicases Mtr4 and Ski2 regulate RNA 3' resection by the exosome. Mycobacterium smegmatis HelY, a bacterial homolog of Mtr4/Ski2, is characterized here as a unidirectional helicase, powered by RNA-dependent ATP/dATP hydrolysis, that tracks 3' to 5' along a loading RNA strand to displace the complementary strand of a tailed RNA:RNA or RNA:DNA duplex. The biochemical properties of HelY suggest a role in bacterial RNA transactions. HelY homologs are present in pathogenic mycobacteria (e.g., M. tuberculosis and M. leprae) and are widely prevalent in Actinobacteria and Cyanobacteria but occur sporadically elsewhere in the bacterial domain. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  9. Uncoupling of Protease trans-Cleavage and Helicase Activities in Pestivirus NS3.

    Science.gov (United States)

    Zheng, Fengwei; Lu, Guoliang; Li, Ling; Gong, Peng; Pan, Zishu

    2017-11-01

    The nonstructural protein NS3 from the Flaviviridae family is a multifunctional protein that contains an N-terminal protease and a C-terminal helicase, playing essential roles in viral polyprotein processing and genome replication. Here we report a full-length crystal structure of the classical swine fever virus (CSFV) NS3 in complex with its NS4A protease cofactor segment (PCS) at a 2.35-Å resolution. The structure reveals a previously unidentified ∼2,200-Å 2 intramolecular protease-helicase interface comprising three clusters of interactions, representing a "closed" global conformation related to the NS3-NS4A cis -cleavage event. Although this conformation is incompatible with protease trans -cleavage, it appears to be functionally important and beneficial to the helicase activity, as the mutations designed to perturb this conformation impaired both the helicase activities in vitro and virus production in vivo Our work reveals important features of protease-helicase coordination in pestivirus NS3 and provides a key basis for how different conformational states may explicitly contribute to certain functions of this natural protease-helicase fusion protein. IMPORTANCE Many RNA viruses encode helicases to aid their RNA genome replication and transcription by unwinding structured RNA. Being naturally fused to a protease participating in viral polyprotein processing, the NS3 helicases encoded by the Flaviviridae family viruses are unique. Therefore, how these two enzyme modules coordinate in a single polypeptide is of particular interest. Here we report a previously unidentified conformation of pestivirus NS3 in complex with its NS4A protease cofactor segment (PCS). This conformational state is related to the protease cis -cleavage event and is optimal for the function of helicase. This work provides an important basis to understand how different enzymatic activities of NS3 may be achieved by the coordination between the protease and helicase through different

  10. RecQL5 promotes genome stabilization through two parallel mechanisms--interacting with RNA polymerase II and acting as a helicase.

    Science.gov (United States)

    Islam, M Nurul; Fox, David; Guo, Rong; Enomoto, Takemi; Wang, Weidong

    2010-05-01

    The RecQL5 helicase is essential for maintaining genome stability and reducing cancer risk. To elucidate its mechanism of action, we purified a RecQL5-associated complex and identified its major component as RNA polymerase II (Pol II). Bioinformatics and structural modeling-guided mutagenesis revealed two conserved regions in RecQL5 as KIX and SRI domains, already known in transcriptional regulators for Pol II. The RecQL5-KIX domain binds both initiation (Pol IIa) and elongation (Pol IIo) forms of the polymerase, whereas the RecQL5-SRI domain interacts only with the elongation form. Fully functional RecQL5 requires both helicase activity and associations with the initiation polymerase, because mutants lacking either activity are partially defective in the suppression of sister chromatid exchange and resistance to camptothecin-induced DNA damage, and mutants lacking both activities are completely defective. We propose that RecQL5 promotes genome stabilization through two parallel mechanisms: by participation in homologous recombination-dependent DNA repair as a RecQ helicase and by regulating the initiation of Pol II to reduce transcription-associated replication impairment and recombination.

  11. Regulation of natural gas: the issue of open access; Regulacao em materia de gas natural: a questao do livre acesso

    Energy Technology Data Exchange (ETDEWEB)

    Sandes, Ingrid; Siqueira, Mariana [Universidade Federal do Rio Grande do Norte (UFRN), Natal, RN (Brazil). Faculdade de Direito

    2004-07-01

    The present work discusses the question of the open access to the natural gas ducts, trying to show the most relevant aspects of it. Analyzing the regulation existent in Brazil and the main important characteristics of the natural gas activities, it will be tried to suggest new directions to be taken in the future. The open access it is a very important way to introduce new agents on the sector and, in that way, the discussions that try to show new aspects of it, are very relevant (author)

  12. The Drosophila Helicase MLE Targets Hairpin Structures in Genomic Transcripts.

    Directory of Open Access Journals (Sweden)

    Simona Cugusi

    2016-01-01

    Full Text Available RNA hairpins are a common type of secondary structures that play a role in every aspect of RNA biochemistry including RNA editing, mRNA stability, localization and translation of transcripts, and in the activation of the RNA interference (RNAi and microRNA (miRNA pathways. Participation in these functions often requires restructuring the RNA molecules by the association of single-strand (ss RNA-binding proteins or by the action of helicases. The Drosophila MLE helicase has long been identified as a member of the MSL complex responsible for dosage compensation. The complex includes one of two long non-coding RNAs and MLE was shown to remodel the roX RNA hairpin structures in order to initiate assembly of the complex. Here we report that this function of MLE may apply to the hairpins present in the primary RNA transcripts that generate the small molecules responsible for RNA interference. Using stocks from the Transgenic RNAi Project and the Vienna Drosophila Research Center, we show that MLE specifically targets hairpin RNAs at their site of transcription. The association of MLE at these sites is independent of sequence and chromosome location. We use two functional assays to test the biological relevance of this association and determine that MLE participates in the RNAi pathway.

  13. Molecular Dynamics of the ZIKA Virus NS3 Helicase

    Science.gov (United States)

    Raubenolt, Bryan; Rick, Steven; The Rick Group Team

    The recent outbreaks of the ZIKA virus (ZIKV) and its connection to microcephaly in newborns has raised its awareness as a global threat and many scientific research efforts are currently underway in attempt to create a vaccine. Molecular Dynamics is a powerful method of investigating the physical behavior of protein complexes. ZIKV is comprised of 3 structural and 7 nonstructural proteins. The NS3 helicase protein appears to play a significant role in the replication complex and its inhibition could be a crucial source of antiviral drug design. This research primarily focuses on studying the structural dynamics, over the course of few hundred nanoseconds, of NS3 helicase in the free state, as well as in complex form with human ssRNA, ATP, and an analogue of GTP. RMSD and RMSF plots of each simulation will provide details on the forces involved in the overall stability of the active and inactive states. Furthermore, free energy calculations on a per residue level will reveal the most interactive residues between states and ultimately the primary driving force behind these interactions. Together these analyses will provide highly relevant information on the binding surface chemistry and thus serve as the basis for potential drug design.

  14. Molecular determinants of nucleolar translocation of RNA helicase A

    International Nuclear Information System (INIS)

    Liu Zhe; Kenworthy, Rachael; Green, Christopher; Tang, Hengli

    2007-01-01

    RNA helicase A (RHA) is a member of the DEAH-box family of DNA/RNA helicases involved in multiple cellular processes and the life cycles of many viruses. The subcellular localization of RHA is dynamic despite its steady-state concentration in the nucleoplasm. We have previously shown that it shuttles rapidly between the nucleus and the cytoplasm by virtue of a bidirectional nuclear transport domain (NTD) located in its carboxyl terminus. Here, we investigate the molecular determinants for its translocation within the nucleus and, more specifically, its redistribution from the nucleoplasm to nucleolus or the perinucleolar region. We found that low temperature treatment, transcription inhibition or replication of hepatitis C virus caused the intranuclear redistribution of the protein, suggesting that RHA shuttles between the nucleolus and nucleoplasm and becomes trapped in the nucleolus or the perinucleolar region upon blockade of transport to the nucleoplasm. Both the NTD and ATPase activity were essential for RHA's transport to the nucleolus or perinucleolar region. One of the double-stranded RNA binding domains (dsRBD II) was also required for this nucleolar translocation (NoT) phenotype. RNA interference studies revealed that RHA is essential for survival of cultured hepatoma cells and the ATPase activity appears to be important for this critical role

  15. Inherited mutations in the helicase RTEL1 cause telomere dysfunction and Hoyeraal-Hreidarsson syndrome.

    Science.gov (United States)

    Deng, Zhong; Glousker, Galina; Molczan, Aliah; Fox, Alan J; Lamm, Noa; Dheekollu, Jayaraju; Weizman, Orr-El; Schertzer, Michael; Wang, Zhuo; Vladimirova, Olga; Schug, Jonathan; Aker, Memet; Londoño-Vallejo, Arturo; Kaestner, Klaus H; Lieberman, Paul M; Tzfati, Yehuda

    2013-09-03

    Telomeres repress the DNA damage response at the natural chromosome ends to prevent cell-cycle arrest and maintain genome stability. Telomeres are elongated by telomerase in a tightly regulated manner to ensure a sufficient number of cell divisions throughout life, yet prevent unlimited cell division and cancer development. Hoyeraal-Hreidarsson syndrome (HHS) is characterized by accelerated telomere shortening and a broad range of pathologies, including bone marrow failure, immunodeficiency, and developmental defects. HHS-causing mutations have previously been found in telomerase and the shelterin component telomeric repeat binding factor 1 (TRF1)-interacting nuclear factor 2 (TIN2). We identified by whole-genome exome sequencing compound heterozygous mutations in four siblings affected with HHS, in the gene encoding the regulator of telomere elongation helicase 1 (RTEL1). Rtel1 was identified in mouse by its genetic association with telomere length. However, its mechanism of action and whether it regulates telomere length in human remained unknown. Lymphoblastoid cell lines obtained from a patient and from the healthy parents carrying heterozygous RTEL1 mutations displayed telomere shortening, fragility and fusion, and growth defects in culture. Ectopic expression of WT RTEL1 suppressed the telomere shortening and growth defect, confirming the causal role of the RTEL1 mutations in HHS and demonstrating the essential function of human RTEL1 in telomere protection and elongation. Finally, we show that human RTEL1 interacts with the shelterin protein TRF1, providing a potential recruitment mechanism of RTEL1 to telomeres.

  16. Inherited mutations in the helicase RTEL1 cause telomere dysfunction and Hoyeraal–Hreidarsson syndrome

    Science.gov (United States)

    Deng, Zhong; Glousker, Galina; Molczan, Aliah; Fox, Alan J.; Lamm, Noa; Dheekollu, Jayaraju; Weizman, Orr-El; Schertzer, Michael; Wang, Zhuo; Vladimirova, Olga; Schug, Jonathan; Aker, Memet; Londoño-Vallejo, Arturo; Kaestner, Klaus H.; Lieberman, Paul M.; Tzfati, Yehuda

    2013-01-01

    Telomeres repress the DNA damage response at the natural chromosome ends to prevent cell-cycle arrest and maintain genome stability. Telomeres are elongated by telomerase in a tightly regulated manner to ensure a sufficient number of cell divisions throughout life, yet prevent unlimited cell division and cancer development. Hoyeraal–Hreidarsson syndrome (HHS) is characterized by accelerated telomere shortening and a broad range of pathologies, including bone marrow failure, immunodeficiency, and developmental defects. HHS-causing mutations have previously been found in telomerase and the shelterin component telomeric repeat binding factor 1 (TRF1)-interacting nuclear factor 2 (TIN2). We identified by whole-genome exome sequencing compound heterozygous mutations in four siblings affected with HHS, in the gene encoding the regulator of telomere elongation helicase 1 (RTEL1). Rtel1 was identified in mouse by its genetic association with telomere length. However, its mechanism of action and whether it regulates telomere length in human remained unknown. Lymphoblastoid cell lines obtained from a patient and from the healthy parents carrying heterozygous RTEL1 mutations displayed telomere shortening, fragility and fusion, and growth defects in culture. Ectopic expression of WT RTEL1 suppressed the telomere shortening and growth defect, confirming the causal role of the RTEL1 mutations in HHS and demonstrating the essential function of human RTEL1 in telomere protection and elongation. Finally, we show that human RTEL1 interacts with the shelterin protein TRF1, providing a potential recruitment mechanism of RTEL1 to telomeres. PMID:23959892

  17. Genome-Wide Analysis of the RNA Helicase Gene Family in Gossypium raimondii

    Directory of Open Access Journals (Sweden)

    Jie Chen

    2014-03-01

    Full Text Available The RNA helicases, which help to unwind stable RNA duplexes, and have important roles in RNA metabolism, belong to a class of motor proteins that play important roles in plant development and responses to stress. Although this family of genes has been the subject of systematic investigation in Arabidopsis, rice, and tomato, it has not yet been characterized in cotton. In this study, we identified 161 putative RNA helicase genes in the genome of the diploid cotton species Gossypium raimondii. We classified these genes into three subfamilies, based on the presence of either a DEAD-box (51 genes, DEAH-box (52 genes, or DExD/H-box (58 genes in their coding regions. Chromosome location analysis showed that the genes that encode RNA helicases are distributed across all 13 chromosomes of G. raimondii. Syntenic analysis revealed that 62 of the 161 G. raimondii helicase genes (38.5% are within the identified syntenic blocks. Sixty-six (40.99% helicase genes from G. raimondii have one or several putative orthologs in tomato. Additionally, GrDEADs have more conserved gene structures and more simple domains than GrDEAHs and GrDExD/Hs. Transcriptome sequencing data demonstrated that many of these helicases, especially GrDEADs, are highly expressed at the fiber initiation stage and in mature leaves. To our knowledge, this is the first report of a genome-wide analysis of the RNA helicase gene family in cotton.

  18. PBDE: Structure-Activity Studies for the Inhibition of Hepatitis C Virus NS3 Helicase

    Directory of Open Access Journals (Sweden)

    Kazi Abdus Salam

    2014-04-01

    Full Text Available The helicase portion of the hepatitis C virus nonstructural protein 3 (NS3 is considered one of the most validated targets for developing direct acting antiviral agents. We isolated polybrominated diphenyl ether (PBDE 1 from a marine sponge as an NS3 helicase inhibitor. In this study, we evaluated the inhibitory effects of PBDE (1 on the essential activities of NS3 protein such as RNA helicase, ATPase, and RNA binding activities. The structure-activity relationship analysis of PBDE (1 against the HCV ATPase revealed that the biphenyl ring, bromine, and phenolic hydroxyl group on the benzene backbone might be a basic scaffold for the inhibitory potency.

  19. ATPase activity measurement of DNA replicative helicase from Bacillus stearothermophilus by malachite green method.

    Science.gov (United States)

    Yang, Mu; Wang, Ganggang

    2016-09-15

    The DnaB helicase from Bacillus stearothermophilus (DnaBBst) was a model protein for studying the bacterial DNA replication. In this work, a non-radioactive method for measuring ATPase activity of DnaBBst helicase was described. The working parameters and conditions were optimized. Furthermore, this method was applied to investigate effects of DnaG primase, ssDNA and helicase loader protein (DnaI) on ATPase activity of DnaBBst. Our results showed this method was sensitive and efficient. Moreover, it is suitable for the investigation of functional interaction between DnaB and related factors. Copyright © 2016 Elsevier Inc. All rights reserved.

  20. Insights into the Structure of Dimeric RNA Helicase CsdA and Indispensable Role of Its C-Terminal Regions.

    Science.gov (United States)

    Xu, Ling; Wang, Lijun; Peng, Junhui; Li, Fudong; Wu, Lijie; Zhang, Beibei; Lv, Mengqi; Zhang, Jiahai; Gong, Qingguo; Zhang, Rongguang; Zuo, Xiaobing; Zhang, Zhiyong; Wu, Jihui; Tang, Yajun; Shi, Yunyu

    2017-12-05

    CsdA has been proposed to be essential for the biogenesis of ribosome and gene regulation after cold shock. However, the structure of CsdA and the function of its long C-terminal regions are still unclear. Here, we solved all of the domain structures of CsdA and found two previously uncharacterized auxiliary domains: a dimerization domain (DD) and an RNA-binding domain (RBD). Small-angle X-ray scattering experiments helped to track the conformational flexibilities of the helicase core domains and C-terminal regions. Biochemical assays revealed that DD is indispensable for stabilizing the CsdA dimeric structure. We also demonstrate for the first time that CsdA functions as a stable dimer at low temperature. The C-terminal regions are critical for RNA binding and efficient enzymatic activities. CsdA_RBD could specifically bind to the regions with a preference for single-stranded G-rich RNA, which may help to bring the helicase core to unwind the adjacent duplex. Copyright © 2017 Elsevier Ltd. All rights reserved.

  1. The N-terminal domain of human DNA helicase Rtel1 contains a redox active iron-sulfur cluster.

    Science.gov (United States)

    Landry, Aaron P; Ding, Huangen

    2014-01-01

    Human telomere length regulator Rtel1 is a superfamily II DNA helicase and is essential for maintaining proper length of telomeres in chromosomes. Here we report that the N-terminal domain of human Rtel1 (RtelN) expressed in Escherichia coli cells produces a protein that contains a redox active iron-sulfur cluster with the redox midpoint potential of -248 ± 10 mV (pH 8.0). The iron-sulfur cluster in RtelN is sensitive to hydrogen peroxide and nitric oxide, indicating that reactive oxygen/nitrogen species may modulate the DNA helicase activity of Rtel1 via modification of its iron-sulfur cluster. Purified RtelN retains a weak binding affinity for the single-stranded (ss) and double-stranded (ds) DNA in vitro. However, modification of the iron-sulfur cluster by hydrogen peroxide or nitric oxide does not significantly affect the DNA binding activity of RtelN, suggesting that the iron-sulfur cluster is not directly involved in the DNA interaction in the N-terminal domain of Rtel1.

  2. The N-Terminal Domain of Human DNA Helicase Rtel1 Contains a Redox Active Iron-Sulfur Cluster

    Directory of Open Access Journals (Sweden)

    Aaron P. Landry

    2014-01-01

    Full Text Available Human telomere length regulator Rtel1 is a superfamily II DNA helicase and is essential for maintaining proper length of telomeres in chromosomes. Here we report that the N-terminal domain of human Rtel1 (RtelN expressed in Escherichia coli cells produces a protein that contains a redox active iron-sulfur cluster with the redox midpoint potential of −248 ± 10 mV (pH 8.0. The iron-sulfur cluster in RtelN is sensitive to hydrogen peroxide and nitric oxide, indicating that reactive oxygen/nitrogen species may modulate the DNA helicase activity of Rtel1 via modification of its iron-sulfur cluster. Purified RtelN retains a weak binding affinity for the single-stranded (ss and double-stranded (ds DNA in vitro. However, modification of the iron-sulfur cluster by hydrogen peroxide or nitric oxide does not significantly affect the DNA binding activity of RtelN, suggesting that the iron-sulfur cluster is not directly involved in the DNA interaction in the N-terminal domain of Rtel1.

  3. Affective regulation of stereotype activation: It’s the (accessible) thought that counts

    Science.gov (United States)

    Huntsinger, Jeffrey R.; Sinclair, Stacey; Dunn, Elizabeth; Clore, Gerald L.

    2010-01-01

    Extant research demonstrates that positive affect, compared to negative affect, increases stereotyping. In four experiments we explore whether the link between affect and stereotyping depends, critically, on the relative accessibility of stereotype-relevant thoughts and response tendencies. As well as manipulating mood, we measured or manipulated the accessibility of egalitarian response tendencies (Experiments 1-2) and counter-stereotypic thoughts (Experiments 3-4). In the absence of such response tendencies and thoughts, people in positive moods displayed greater stereotype activation —consistent with past research. By contrast, in the presence of accessible egalitarian response tendencies or counter-stereotypic thoughts, people in positive moods exhibited less stereotype activation than those in negative moods. PMID:20363909

  4. A mechanical mechanism for translocation of ring-shaped helicases on DNA and its demonstration in a macroscopic simulation system

    Science.gov (United States)

    Chou, Y. C.

    2018-04-01

    The asymmetry in the two-layered ring structure of helicases and the random thermal fluctuations of the helicase and DNA molecules are considered as the bases for the generation of the force required for translocation of the ring-shaped helicase on DNA. The helicase comprises a channel at its center with two unequal ends, through which strands of DNA can pass. The random collisions between the portion of the DNA strand in the central channel and the wall of the channel generate an impulsive force toward the small end. This impulsive force is the starting point for the helicase to translocate along the DNA with the small end in front. Such a physical mechanism may serve as a complementary for the chemomechanical mechanism of the translocation of helicase on DNA. When the helicase arrives at the junction of ssDNA and dsDNA (a fork), the collision between the helicase and the closest base pair may produce a sufficient impulsive force to break the weak hydrogen bond of the base pair. Thus, the helicase may advance and repeat the process of unwinding the dsDNA strand. This mechanism was tested in a macroscopic simulation system where the helicase was simulated using a truncated-cone structure and DNA was simulated with bead chains. Many features of translocation and unwinding such as translocation on ssDNA and dsDNA, unwinding of dsDNA, rewinding, strand switching, and Holliday junction resolution were reproduced.

  5. Identification of the molecular switch that regulates access of 5alpha-DHT to the androgen receptor.

    Science.gov (United States)

    Penning, Trevor M; Bauman, David R; Jin, Yi; Rizner, Tea Lanisik

    2007-02-01

    Pairs of hydroxysteroid dehydrogenases (HSDs) govern ligand access to steroid receptors in target tissues and act as molecular switches. By acting as reductases or oxidases, HSDs convert potent ligands into their cognate inactive metabolites or vice versa. This pre-receptor regulation of steroid hormone action may have profound effects on hormonal response. We have identified the HSDs responsible for regulating ligand access to the androgen receptor (AR) in human prostate. Type 3 3alpha-hydroxysteroid dehydrogenase (aldo-keto reductase 1C2) acts solely as a reductase to convert 5alpha-dihydrotestosterone (DHT), a potent ligand for the AR (K(d)=10(-11)M for the AR), to the inactive androgen 3alpha-androstanediol (K(d)=10(-6)M for the AR); while RoDH like 3alpha-HSD (a short-chain dehydrogenase/reductase (SDR)) acts solely as an oxidase to convert 3alpha-androstanediol back to 5alpha-DHT. Our studies suggest that aldo-keto reductase (AKRs) and SDRs function as reductases and oxidases, respectively, to control ligand access to nuclear receptors.

  6. Labor Law Reform: The Regulation of Free Speech and Equal Access in NLRB Representational Elections.

    Science.gov (United States)

    University of Pennsylvania Law Review, 1979

    1979-01-01

    After examining the existing legal standards governing free speech and equal access in representation elections, the author analyzes various reform possibilities and suggests a unified approach for legislative action. Available from the University of Pennsylvania Law School, 3400 Chestnut Street, Philadelphia, Pennsylvania 19104; $3.00 sc.…

  7. High-throughput screening assay of hepatitis C virus helicase inhibitors using fluorescence-quenching phenomenon

    International Nuclear Information System (INIS)

    Tani, Hidenori; Akimitsu, Nobuyoshi; Fujita, Osamu; Matsuda, Yasuyoshi; Miyata, Ryo; Tsuneda, Satoshi; Igarashi, Masayuki; Sekiguchi, Yuji; Noda, Naohiro

    2009-01-01

    We have developed a novel high-throughput screening assay of hepatitis C virus (HCV) nonstructural protein 3 (NS3) helicase inhibitors using the fluorescence-quenching phenomenon via photoinduced electron transfer between fluorescent dyes and guanine bases. We prepared double-stranded DNA (dsDNA) with a 5'-fluorescent-dye (BODIPY FL)-labeled strand hybridized with a complementary strand, the 3'-end of which has guanine bases. When dsDNA is unwound by helicase, the dye emits fluorescence owing to its release from the guanine bases. Our results demonstrate that this assay is suitable for quantitative assay of HCV NS3 helicase activity and useful for high-throughput screening for inhibitors. Furthermore, we applied this assay to the screening for NS3 helicase inhibitors from cell extracts of microorganisms, and found several cell extracts containing potential inhibitors.

  8. Crystal structures of the methyltransferase and helicase from the ZIKA 1947 MR766 Uganda strain

    Energy Technology Data Exchange (ETDEWEB)

    Bukrejewska, Malgorzata; Derewenda, Urszula; Radwanska, Malwina; Engel, Daniel A.; Derewenda, Zygmunt S.

    2017-08-15

    Two nonstructural proteins encoded byZika virusstrain MR766 RNA, a methyltransferase and a helicase, were crystallized and their structures were solved and refined at 2.10 and 2.01 Å resolution, respectively. The NS5 methyltransferase contains a boundS-adenosyl-L-methionine (SAM) co-substrate. The NS3 helicase is in the apo form. Comparison with published crystal structures of the helicase in the apo, nucleotide-bound and single-stranded RNA (ssRNA)-bound states suggests that binding of ssRNA to the helicase may occur through conformational selection rather than induced fit.

  9. Survey of International Rules and Practices Regarding Delineation of and Access to Regulated Areas for Radiation Protection - Final report

    International Nuclear Information System (INIS)

    Schieber, C.; Crouail, P.; Beltrami, L.-A.; Reaud, C.; Lehtinen, Maaret; Stritt, Nicolas; Thomas, Gareth

    2013-06-01

    European requirements for radiological protection, especially work on transposing the new EURATOM Directive on the basic radiological protection standards, are currently being revised. The Direction generale du travail (DGT - General Directorate of Labour) and the Autorite de Surete Nucleaire (ASN - Nuclear Safety Authority) therefore commissioned the Radiological Protection Standing Groups of experts (GPRAD and GPMED)1 to engage in a forward-looking debate on the delimitation of and access to regulated areas, within an ad hoc working group (called hereafter 'Classification of Area WG'). To fuel its debates, the 'Classification of Area WG' sought elements on international regulations and practices focusing on problem exposure situations in various areas of activity (nuclear, industrial, research, medical, transport and natural boosted). CEPN was entrusted with this study. This report presents a summary of rules applicable in seven countries in terms of delimitation of and access to regulated radiological protection areas. The countries are: Belgium, Spain, United States, Finland, United Kingdom, Sweden and Switzerland. Detailed sheets for each country can be found in the Annex. Based on these summaries, three countries have been selected to apply their rules and practices in force to a dozen or so particular cases put together by the 'Classification of Area WG' that are representative of exposure situations. The three countries are Finland, United Kingdom and Switzerland. The case studies applied to each country are presented in the second part of this report

  10. Ebselen Inhibits Hepatitis C Virus NS3 Helicase Binding to Nucleic Acid and Prevents Viral Replication

    OpenAIRE

    Mukherjee, Sourav; Weiner, Warren S.; Schroeder, Chad E.; Simpson, Denise S.; Hanson, Alicia M.; Sweeney, Noreena L.; Marvin, Rachel K.; Ndjomou, Jean; Kolli, Rajesh; Isailovic, Dragan; Schoenen, Frank J.; Frick, David N.

    2014-01-01

    The hepatitis C virus (HCV) nonstructural protein 3 (NS3) is both a protease, which cleaves viral and host proteins, and a helicase that separates nucleic acid strands, using ATP hydrolysis to fuel the reaction. Many antiviral drugs, and compounds in clinical trials, target the NS3 protease, but few helicase inhibitors that function as antivirals have been reported. This study focuses on the analysis of the mechanism by which ebselen (2-phenyl-1,2-benzisoselenazol-3-one), a compound previousl...

  11. Ebselen inhibits hepatitis C virus NS3 helicase binding to nucleic acid and prevents viral replication.

    Science.gov (United States)

    Mukherjee, Sourav; Weiner, Warren S; Schroeder, Chad E; Simpson, Denise S; Hanson, Alicia M; Sweeney, Noreena L; Marvin, Rachel K; Ndjomou, Jean; Kolli, Rajesh; Isailovic, Dragan; Schoenen, Frank J; Frick, David N

    2014-10-17

    The hepatitis C virus (HCV) nonstructural protein 3 (NS3) is both a protease, which cleaves viral and host proteins, and a helicase that separates nucleic acid strands, using ATP hydrolysis to fuel the reaction. Many antiviral drugs, and compounds in clinical trials, target the NS3 protease, but few helicase inhibitors that function as antivirals have been reported. This study focuses on the analysis of the mechanism by which ebselen (2-phenyl-1,2-benzisoselenazol-3-one), a compound previously shown to be a HCV antiviral agent, inhibits the NS3 helicase. Ebselen inhibited the abilities of NS3 to unwind nucleic acids, to bind nucleic acids, and to hydrolyze ATP, and about 1 μM ebselen was sufficient to inhibit each of these activities by 50%. However, ebselen had no effect on the activity of the NS3 protease, even at 100 times higher ebselen concentrations. At concentrations below 10 μM, the ability of ebselen to inhibit HCV helicase was reversible, but prolonged incubation of HCV helicase with higher ebselen concentrations led to irreversible inhibition and the formation of covalent adducts between ebselen and all 14 cysteines present in HCV helicase. Ebselen analogues with sulfur replacing the selenium were just as potent HCV helicase inhibitors as ebselen, but the length of the linker between the phenyl and benzisoselenazol rings was critical. Modifications of the phenyl ring also affected compound potency over 30-fold, and ebselen was a far more potent helicase inhibitor than other, structurally unrelated, thiol-modifying agents. Ebselen analogues were also more effective antiviral agents, and they were less toxic to hepatocytes than ebselen. Although the above structure-activity relationship studies suggest that ebselen targets a specific site on NS3, we were unable to confirm binding to either the NS3 ATP binding site or nucleic acid binding cleft by examining the effects of ebselen on NS3 proteins lacking key cysteines.

  12. Solving the Problems of Physical and Economic Accessibility of Foodstuff in the Region by Means of AIC State Regulation

    Directory of Open Access Journals (Sweden)

    Babich Tatyana Vladimirovna

    2015-09-01

    Full Text Available The article deals with the physical and economic accessibility to foodstuff in the region. The effects of economic sanctions are analyzed, the main types of risks of the domestic agricultural producers’ competitiveness are described, and the methods of their reduction are identified. The concept of food security in terms of physical and economic accessibility is considered. The analysis of the Volgograd region data on the development of agricultural production let conclude that there is the problem of ensuring the physical availability of foodstuff in the region. The state regulation of agricultural production is identified as an important factor of food security in the modern world. The authors identified the basic problems of agricultural production, including the problem of irrigation, economic accessibility of food products, depreciation of fixed assets, the use of obsolete and resource-intensive technologies of production, decline in qualification level of staff employed in the industry, underfunding of agricultural science, low competitiveness of agricultural and food policy. Moreover, in the current situation the further growth of food prices and reduced purchasing power of the population, as a result of inflation, would further reduce the economic affordability of food and decrease food security in the region and in the country, as a whole. As a result, аs part of the solution to the problem of providing physical and economic access to food, the authors offered and proved complex measures on improving state regulation of agro-industrial complex. These measures include conducting large-scale works on restoration of the complex reclamation of the region; implementation of technical and technological modernization of agriculture, food industry and agrobusiness production services; formation of the system of professional agricultural education; formation of modern social infrastructure in rural areas; development of the program of food

  13. Regulating internet access in UK public libraries: legal compliance and ethical dilemmas

    OpenAIRE

    Muir, Adrienne; Spacey, Rachel; Cooke, Louise; Creaser, Claire

    2016-01-01

    Purpose – This paper aims to consider selected results from the Arts and Humanities Research Council (AHRC) funded “Managing Access to the internet in Public Libraries” (MAIPLE) project, from 2012-2014. MAIPLE has explored the ways in which public library services manage use of the internet connections that they provide for the public. This included the how public library services balance their legal obligations and the needs of their communities in a public space and the ethical dilemmas tha...

  14. Close encounters for the first time: Helicase interactions with DNA damage.

    Science.gov (United States)

    Khan, Irfan; Sommers, Joshua A; Brosh, Robert M

    2015-09-01

    DNA helicases are molecular motors that harness the energy of nucleoside triphosphate hydrolysis to unwinding structured DNA molecules that must be resolved during cellular replication, DNA repair, recombination, and transcription. In vivo, DNA helicases are expected to encounter a wide spectrum of covalent DNA modifications to the sugar phosphate backbone or the nitrogenous bases; these modifications can be induced by endogenous biochemical processes or exposure to environmental agents. The frequency of lesion abundance can vary depending on the lesion type. Certain adducts such as oxidative base modifications can be quite numerous, and their effects can be helix-distorting or subtle perturbations to DNA structure. Helicase encounters with specific DNA lesions and more novel forms of DNA damage will be discussed. We will also review the battery of assays that have been used to characterize helicase-catalyzed unwinding of damaged DNA substrates. Characterization of the effects of specific DNA adducts on unwinding by various DNA repair and replication helicases has proven to be insightful for understanding mechanistic and biological aspects of helicase function in cellular DNA metabolism. Published by Elsevier B.V.

  15. XPD Helicase Structures and Activities: Insights into the Cancer and Aging Phenotypes from XPD Mutations

    Energy Technology Data Exchange (ETDEWEB)

    Tainer, John; Fan, Li; Fuss, Jill O.; Cheng, Quen J.; Arvai, Andrew S.; Hammel, Michal; Roberts, Victoria A.; Cooper, Priscilla K.; Tainer, John A.

    2008-06-02

    Mutations in XPD helicase, required for nucleotide excision repair (NER) as part of the transcription/repair complex TFIIH, cause three distinct phenotypes: cancer-prone xeroderma pigmentosum (XP), or aging disorders Cockayne syndrome (CS), and trichothiodystrophy (TTD). To clarify molecular differences underlying these diseases, we determined crystal structures of the XPD catalytic core from Sulfolobus acidocaldarius and measured mutant enzyme activities. Substrate-binding grooves separate adjacent Rad51/RecA-like helicase domains (HD1, HD2) and an arch formed by 4FeS and Arch domains. XP mutations map along the HD1 ATP-binding edge and HD2 DNA-binding channel and impair helicase activity essential for NER. XP/CS mutations both impair helicase activity and likely affect HD2 functional movement. TTD mutants lose or retain helicase activity but map to sites in all four domains expected to cause framework defects impacting TFIIH integrity. These results provide a foundation for understanding disease consequences of mutations in XPD and related 4Fe-4S helicases including FancJ.

  16. XPD Helicase Structures And Activities: Insights Into the Cancer And Aging Phenotypes From XPD Mutations

    Energy Technology Data Exchange (ETDEWEB)

    Fan, L.; Fuss, J.O.; Cheng, Q.J.; Arvai, A.S.; Hammel, M.; Roberts, V.A.; Cooper, P.K.; Tainer, J.A.

    2009-05-18

    Mutations in XPD helicase, required for nucleotide excision repair (NER) as part of the transcription/repair complex TFIIH, cause three distinct phenotypes: cancer-prone xeroderma pigmentosum (XP), or aging disorders Cockayne syndrome (CS), and trichothiodystrophy (TTD). To clarify molecular differences underlying these diseases, we determined crystal structures of the XPD catalytic core from Sulfolobus acidocaldarius and measured mutant enzyme activities. Substrate-binding grooves separate adjacent Rad51/RecA-like helicase domains (HD1, HD2) and an arch formed by 4FeS and Arch domains. XP mutations map along the HD1 ATP-binding edge and HD2 DNA-binding channel and impair helicase activity essential for NER. XP/CS mutations both impair helicase activity and likely affect HD2 functional movement. TTD mutants lose or retain helicase activity but map to sites in all four domains expected to cause framework defects impacting TFIIH integrity. These results provide a foundation for understanding disease consequences of mutations in XPD and related 4Fe-4S helicases including FancJ.

  17. Targeting Dengue Virus NS-3 Helicase by Ligand based Pharmacophore Modeling and Structure based Virtual Screening

    Science.gov (United States)

    Halim, Sobia A.; Khan, Shanza; Khan, Ajmal; Wadood, Abdul; Mabood, Fazal; Hussain, Javid; Al-Harrasi, Ahmed

    2017-10-01

    Dengue fever is an emerging public health concern, with several million viral infections occur annually, for which no effective therapy currently exist. Non-structural protein 3 (NS-3) Helicase encoded by the dengue virus (DENV) is considered as a potential drug target to design new and effective drugs against dengue. Helicase is involved in unwinding of dengue RNA. This study was conducted to design new NS-3 Helicase inhibitor by in silico ligand- and structure based approaches. Initially ligand-based pharmacophore model was generated that was used to screen a set of 1201474 compounds collected from ZINC Database. The compounds matched with the pharmacophore model were docked into the active site of NS-3 helicase. Based on docking scores and binding interactions, twenty five compounds are suggested to be potential inhibitors of NS3 Helicase. The pharmacokinetic properties of these hits were predicted. The selected hits revealed acceptable ADMET properties. This study identified potential inhibitors of NS-3 Helicase in silico, and can be helpful in the treatment of Dengue.

  18. Supporting Student Self-Regulation to Access the General Education Curriculum

    Science.gov (United States)

    Korinek, Lori; deFur, Sharon H.

    2016-01-01

    Educators express an almost universal desire for students to exhibit self-control--that is, manage, monitor, and assess their own social and academic behaviors. These skills comprise self-regulation, a complex set of functions derived from several fields of research, including social cognition (Zimmerman, 2000), self-determination (Wehmeyer &…

  19. Access and Retention in French Higher Education: Student Drop-Out as a Form of Regulation

    Science.gov (United States)

    Bodin, Romuald; Orange, Sophie

    2018-01-01

    Drawing on Bourdieu's theory and using Durkheim's concepts of "social fact" and "regulation", this article examines the place held by public universities within the French higher education (HE) system, breaking with the purely bureaucratic vision prevailing in France today. By setting aside some of the main received ideas about…

  20. DNA replication restart and cellular dynamics of Hef helicase/nuclease protein in Haloferax volcanii.

    Science.gov (United States)

    Lestini, Roxane; Delpech, Floriane; Myllykallio, Hannu

    2015-11-01

    Understanding how frequently spontaneous replication arrests occur and how archaea deal with these arrests are very interesting and challenging research topics. Here we will described how genetic and imaging studies have revealed the central role of the archaeal helicase/nuclease Hef belonging to the XPF/MUS81/FANCM family of endonucleases in repair of arrested replication forks. Special focus will be on description of a recently developed combination of genetic and imaging tools to study the dynamic localization of a functional Hef::GFP (Green Fluorescent Protein) fusion protein in the living cells of halophilic archaea Haloferax volcanii. As Archaea provide an excellent and unique model for understanding how DNA replication is regulated to allow replication of a circular DNA molecule either from single or multiple replication origins, we will also summarize recent studies that have revealed peculiar features regarding DNA replication, particularly in halophilic archaea. We strongly believe that fundamental knowledge of our on-going studies will shed light on the evolutionary history of the DNA replication machinery and will help to establish general rules concerning replication restart and the key role of recombination proteins not only in bacteria, yeast and higher eukaryotes but also in archaea. Copyright © 2015 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

  1. AAA-ATPase FIDGETIN-LIKE 1 and Helicase FANCM Antagonize Meiotic Crossovers by Distinct Mechanisms.

    Directory of Open Access Journals (Sweden)

    Chloe Girard

    2015-07-01

    Full Text Available Meiotic crossovers (COs generate genetic diversity and are critical for the correct completion of meiosis in most species. Their occurrence is tightly constrained but the mechanisms underlying this limitation remain poorly understood. Here we identified the conserved AAA-ATPase FIDGETIN-LIKE-1 (FIGL1 as a negative regulator of meiotic CO formation. We show that Arabidopsis FIGL1 limits CO formation genome-wide, that FIGL1 controls dynamics of the two conserved recombinases DMC1 and RAD51 and that FIGL1 hinders the interaction between homologous chromosomes, suggesting that FIGL1 counteracts DMC1/RAD51-mediated inter-homologue strand invasion to limit CO formation. Further, depleting both FIGL1 and the previously identified anti-CO helicase FANCM synergistically increases crossover frequency. Additionally, we showed that the effect of mutating FANCM on recombination is much lower in F1 hybrids contrasting from the phenotype of inbred lines, while figl1 mutation equally increases crossovers in both contexts. This shows that the modes of action of FIGL1 and FANCM are differently affected by genomic contexts. We propose that FIGL1 and FANCM represent two successive barriers to CO formation, one limiting strand invasion, the other disassembling D-loops to promote SDSA, which when both lifted, leads to a large increase of crossovers, without impairing meiotic progression.

  2. Do no harm: the role of community pharmacists in regulating public access to prescription drugs in Saudi Arabia.

    Science.gov (United States)

    Bahnassi, Anas

    2016-04-01

    Pharmacists have a crucial role to ensure regulated public access to prescription drugs. The study aimed to investigate the views of community pharmacists practising in Saudi Arabia on their role in the unauthorised supply of prescription drugs, consider the possible contributory factors and report pharmacists' suggested strategies to regulate supply. One hundred community pharmacists were invited to participate in an interview-based survey, including questions on demographic characteristics, and the unauthorised supply of prescription drugs. Descriptive statistics were conducted, and associations between categorical responses tested; a P value of ≤0.05 was considered significant. Responses to open questions were analysed thematically. In Saudi Arabia, there is widespread unregulated supply of prescription drugs; pharmacists are under pressure from patients to provide prescription drugs for a wide range of clinical conditions. There are safety and appropriateness concerns when drugs are provided based on patient demand rather than clinical need. Pharmacists do not maintain patient records with information on drugs supplied and associated actions. While most pharmacists supply prescription drugs without the necessary prescriber authorisation, they also this may jeopardise patients safety. While we have many concerns about this practice its present form, we believe pharmacists should have certain prescribing privileges within their areas of competence. A legal framework is needed to guarantee proper pharmacists' training, support, mentorship and access to the tools required to provide safe pharmacy practice. © 2015 Royal Pharmaceutical Society.

  3. Physical and functional interactions of Caenorhabditis elegans WRN-1 helicase with RPA-1.

    Science.gov (United States)

    Hyun, Moonjung; Park, Sojin; Kim, Eunsun; Kim, Do-Hyung; Lee, Se-Jin; Koo, Hyeon-Sook; Seo, Yeon-Soo; Ahn, Byungchan

    2012-02-21

    The Caenorhabditis elegans Werner syndrome protein, WRN-1, a member of the RecQ helicase family, has a 3'-5' DNA helicase activity. Worms with defective wrn-1 exhibit premature aging phenotypes and an increased level of genome instability. In response to DNA damage, WRN-1 participates in the initial stages of checkpoint activation in concert with C. elegans replication protein A (RPA-1). WRN-1 helicase is stimulated by RPA-1 on long DNA duplex substrates. However, the mechanism by which RPA-1 stimulates DNA unwinding and the function of the WRN-1-RPA-1 interaction are not clearly understood. We have found that WRN-1 physically interacts with two RPA-1 subunits, CeRPA73 and CeRPA32; however, full-length WRN-1 helicase activity is stimulated by only the CeRPA73 subunit, while the WRN-1(162-1056) fragment that harbors the helicase activity requires both the CeRPA73 and CeRPA32 subunits for the stimulation. We also found that the CeRPA73(1-464) fragment can stimulate WRN-1 helicase activity and that residues 335-464 of CeRPA73 are important for physical interaction with WRN-1. Because CeRPA73 and the CeRPA73(1-464) fragment are able to bind single-stranded DNA (ssDNA), the stimulation of WRN-1 helicase by RPA-1 is most likely due to the ssDNA binding activity of CeRPA73 and the direct interaction of WRN-1 and CeRPA73.

  4. Structure-Based Mutational Analysis of the Hepatitis C Virus NS3 Helicase

    Science.gov (United States)

    Tai, Chun-Ling; Pan, Wen-Ching; Liaw, Shwu-Huey; Yang, Ueng-Cheng; Hwang, Lih-Hwa; Chen, Ding-Shinn

    2001-01-01

    The carboxyl terminus of the hepatitis C virus (HCV) nonstructural protein 3 (NS3) possesses ATP-dependent RNA helicase activity. Based on the conserved sequence motifs and the crystal structures of the helicase domain, 17 mutants of the HCV NS3 helicase were generated. The ATP hydrolysis, RNA binding, and RNA unwinding activities of the mutant proteins were examined in vitro to determine the functional role of the mutated residues. The data revealed that Lys-210 in the Walker A motif and Asp-290, Glu-291, and His-293 in the Walker B motif were crucial to ATPase activity and that Thr-322 and Thr-324 in motif III and Arg-461 in motif VI significantly influenced ATPase activity. When the pairing between His-293 and Gln-460, referred to as gatekeepers, was replaced with the Asp-293/His-460 pair, which makes the NS3 helicase more like the DEAD helicase subgroup, ATPase activity was not restored. It thus indicated that the whole microenvironment surrounding the gatekeepers, rather than the residues per se, was important to the enzymatic activities. Arg-461 and Trp-501 are important residues for RNA binding, while Val-432 may only play a coadjutant role. The data demonstrated that RNA helicase activity was possibly abolished by the loss of ATPase activity or by reduced RNA binding activity. Nevertheless, a low threshold level of ATPase activity was found sufficient for helicase activity. Results in this study provide a valuable reference for efforts under way to develop anti-HCV therapeutic drugs targeting NS3. PMID:11483774

  5. Cdt1 stabilizes an open MCM ring for helicase loading.

    Science.gov (United States)

    Frigola, Jordi; He, Jun; Kinkelin, Kerstin; Pye, Valerie E; Renault, Ludovic; Douglas, Max E; Remus, Dirk; Cherepanov, Peter; Costa, Alessandro; Diffley, John F X

    2017-06-23

    ORC, Cdc6 and Cdt1 act together to load hexameric MCM, the motor of the eukaryotic replicative helicase, into double hexamers at replication origins. Here we show that Cdt1 interacts with MCM subunits Mcm2, 4 and 6, which both destabilizes the Mcm2-5 interface and inhibits MCM ATPase activity. Using X-ray crystallography, we show that Cdt1 contains two winged-helix domains in the C-terminal half of the protein and a catalytically inactive dioxygenase-related N-terminal domain, which is important for MCM loading, but not for subsequent replication. We used these structures together with single-particle electron microscopy to generate three-dimensional models of MCM complexes. These show that Cdt1 stabilizes MCM in a left-handed spiral open at the Mcm2-5 gate. We propose that Cdt1 acts as a brace, holding MCM open for DNA entry and bound to ATP until ORC-Cdc6 triggers ATP hydrolysis by MCM, promoting both Cdt1 ejection and MCM ring closure.

  6. Regulating the Access to Genetic Resources in Brazil: Suggestions for a New Scenario

    Directory of Open Access Journals (Sweden)

    Nilo Luiz Saccaro-Junior

    2013-12-01

    Full Text Available The relationship between the use of genetic resources, social justice and biodiversity conservation has proved to be one of the main matters in current environmental issues. Brazil exercises a leadership role in international debates about genetic resources and is a pioneer in the creation of a specific internal regulation. This, however, has been strongly criticized by the stakeholders, as well as the lack of incentive for biodiversity research in this country. This paper discusses some of the main conflicting points and how a new scenario of control and promotion can overcome them, based on the regulatory experience since the creation of the current regulatory framework and national indicators available in research with Brazilian species. The suggestions are based on three pillars: a new relationship between government and bioprospectors, guided by encouraging legal activities; a diffuse distribution of benefits, with simple rules, being able to capture some profit from bioprospecting; and a synergy between the existence of traditional/indigenous communities and biodiversity maintenance.

  7. Cyclosporin A associated helicase-like protein facilitates the association of hepatitis C virus RNA polymerase with its cellular cyclophilin B.

    Directory of Open Access Journals (Sweden)

    Kengo Morohashi

    Full Text Available BACKGROUND: Cyclosporin A (CsA is well known as an immunosuppressive drug useful for allogeneic transplantation. It has been reported that CsA inhibits hepatitis C virus (HCV genome replication, which indicates that cellular targets of CsA regulate the viral replication. However, the regulation mechanisms of HCV replication governed by CsA target proteins have not been fully understood. PRINCIPAL FINDINGS: Here we show a chemical biology approach that elucidates a novel mechanism of HCV replication. We developed a phage display screening to investigate compound-peptide interaction and identified a novel cellular target molecule of CsA. This protein, named CsA associated helicase-like protein (CAHL, possessed RNA-dependent ATPase activity that was negated by treatment with CsA. The downregulation of CAHL in the cells resulted in a decrease of HCV genome replication. CAHL formed a complex with HCV-derived RNA polymerase NS5B and host-derived cyclophilin B (CyPB, known as a cellular cofactor for HCV replication, to regulate NS5B-CyPB interaction. CONCLUSIONS: We found a cellular factor, CAHL, as CsA associated helicase-like protein, which would form trimer complex with CyPB and NS5B of HCV. The strategy using a chemical compound and identifying its target molecule by our phage display analysis is useful to reveal a novel mechanism underlying cellular and viral physiology.

  8. Cyclosporin A associated helicase-like protein facilitates the association of hepatitis C virus RNA polymerase with its cellular cyclophilin B.

    Science.gov (United States)

    Morohashi, Kengo; Sahara, Hiroeki; Watashi, Koichi; Iwabata, Kazuki; Sunoki, Takashi; Kuramochi, Kouji; Takakusagi, Kaori; Miyashita, Hiroki; Sato, Noriyuki; Tanabe, Atsushi; Shimotohno, Kunitada; Kobayashi, Susumu; Sakaguchi, Kengo; Sugawara, Fumio

    2011-04-29

    Cyclosporin A (CsA) is well known as an immunosuppressive drug useful for allogeneic transplantation. It has been reported that CsA inhibits hepatitis C virus (HCV) genome replication, which indicates that cellular targets of CsA regulate the viral replication. However, the regulation mechanisms of HCV replication governed by CsA target proteins have not been fully understood. Here we show a chemical biology approach that elucidates a novel mechanism of HCV replication. We developed a phage display screening to investigate compound-peptide interaction and identified a novel cellular target molecule of CsA. This protein, named CsA associated helicase-like protein (CAHL), possessed RNA-dependent ATPase activity that was negated by treatment with CsA. The downregulation of CAHL in the cells resulted in a decrease of HCV genome replication. CAHL formed a complex with HCV-derived RNA polymerase NS5B and host-derived cyclophilin B (CyPB), known as a cellular cofactor for HCV replication, to regulate NS5B-CyPB interaction. We found a cellular factor, CAHL, as CsA associated helicase-like protein, which would form trimer complex with CyPB and NS5B of HCV. The strategy using a chemical compound and identifying its target molecule by our phage display analysis is useful to reveal a novel mechanism underlying cellular and viral physiology.

  9. The C-terminal domain of the Bloom syndrome DNA helicase is essential for genomic stability

    Directory of Open Access Journals (Sweden)

    Noonan James P

    2001-07-01

    Full Text Available Abstract Background Bloom syndrome is a rare cancer-prone disorder in which the cells of affected persons have a high frequency of somatic mutation and genomic instability. Bloom syndrome cells have a distinctive high frequency of sister chromatid exchange and quadriradial formation. BLM, the protein altered in BS, is a member of the RecQ DNA helicase family, whose members share an average of 40% identity in the helicase domain and have divergent N-terminal and C-terminal flanking regions of variable lengths. The BLM DNA helicase has been shown to localize to the ND10 (nuclear domain 10 or PML (promyelocytic leukemia nuclear bodies, where it associates with TOPIIIα, and to the nucleolus. Results This report demonstrates that the N-terminal domain of BLM is responsible for localization of the protein to the nuclear bodies, while the C-terminal domain directs the protein to the nucleolus. Deletions of the N-terminal domain of BLM have little effect on sister chromatid exchange frequency and chromosome stability as compared to helicase and C-terminal mutations which can increase SCE frequency and chromosome abnormalities. Conclusion The helicase activity and the C-terminal domain of BLM are critical for maintaining genomic stability as measured by the sister chromatid exchange assay. The localization of BLM into the nucleolus by the C-terminal domain appears to be more important to genomic stability than localization in the nuclear bodies.

  10. Unzippers, Resolvers and Sensors: A Structural and Functional Biochemistry Tale of RNA Helicases

    Directory of Open Access Journals (Sweden)

    Ana Lúcia Leitão

    2015-01-01

    Full Text Available The centrality of RNA within the biological world is an irrefutable fact that currently attracts increasing attention from the scientific community. The panoply of functional RNAs requires the existence of specific biological caretakers, RNA helicases, devoted to maintain the proper folding of those molecules, resolving unstable structures. However, evolution has taken advantage of the specific position and characteristics of RNA helicases to develop new functions for these proteins, which are at the interface of the basic processes for transference of information from DNA to proteins. RNA helicases are involved in many biologically relevant processes, not only as RNA chaperones, but also as signal transducers, scaffolds of molecular complexes, and regulatory elements. Structural biology studies during the last decade, founded in X-ray crystallography, have characterized in detail several RNA-helicases. This comprehensive review summarizes the structural knowledge accumulated in the last two decades within this family of proteins, with special emphasis on the structure-function relationships of the most widely-studied families of RNA helicases: the DEAD-box, RIG-I-like and viral NS3 classes.

  11. A case series of patients using medicinal marihuana for management of chronic pain under the Canadian Marihuana Medical Access Regulations.

    Science.gov (United States)

    Lynch, Mary E; Young, Judee; Clark, Alexander J

    2006-11-01

    The Canadian Marihuana Medical Access Regulations (MMAR) program allows Health Canada to grant access to marihuana for medical use to those who are suffering from grave and debilitating illnesses. This is a report on a case series of 30 patients followed at a tertiary care pain management center in Nova Scotia who have used medicinal marihuana for 1-5 years under the MMAR program. Patients completed a follow-up questionnaire containing demographic and dosing information, a series of 11-point numerical symptom relief rating scales, a side effect checklist, and a subjective measure of improvement in function. Doses of marihuana ranged from less than 1 to 5g per day via the smoked or oral route of administration. Ninety-three percent of patients reported moderate or greater pain relief. Side effects were reported by 76% of patients, the most common of which were increased appetite and a sense of well-being, weight gain, and slowed thoughts. Limitations of the study include self-selection bias, small size, and lack of a control group. The need for further study using controlled trials is discussed along with an overview of the MMAR program.

  12. Demonstration of helicase activity in the nonstructural protein, NSs, of the negative-sense RNA virus, groundnut bud necrosis virus.

    Science.gov (United States)

    Bhushan, Lokesh; Abraham, Ambily; Choudhury, Nirupam Roy; Rana, Vipin Singh; Mukherjee, Sunil Kumar; Savithri, Handanahal Subbarao

    2015-04-01

    The nonstructural protein NSs, encoded by the S RNA of groundnut bud necrosis virus (GBNV) (genus Tospovirus, family Bunyaviridae) has earlier been shown to possess nucleic-acid-stimulated NTPase and 5' α phosphatase activity. ATP hydrolysis is an essential function of a true helicase. Therefore, NSs was tested for DNA helicase activity. The results demonstrated that GBNV NSs possesses bidirectional DNA helicase activity. An alanine mutation in the Walker A motif (K189A rNSs) decreased DNA helicase activity substantially, whereas a mutation in the Walker B motif resulted in a marginal decrease in this activity. The parallel loss of the helicase and ATPase activity in the K189A mutant confirms that NSs acts as a non-canonical DNA helicase. Furthermore, both the wild-type and K189A NSs could function as RNA silencing suppressors, demonstrating that the suppressor activity of NSs is independent of its helicase or ATPase activity. This is the first report of a true helicase from a negative-sense RNA virus.

  13. Structural view of the helicase reveals that Zika virus uses a conserved mechanism for unwinding RNA.

    Science.gov (United States)

    Li, Lei; Wang, Jin; Jia, Zhihui; Shaw, Neil

    2018-04-01

    Recent studies suggest a link between infection by Zika virus (ZIKV) and the development of neurological complications. The lack of ZIKV-specific therapeutics has alarmed healthcare professionals worldwide. Here, crystal structures of apo and AMPPNP- and Mn 2+ -bound forms of the essential helicase of ZIKV refined to 1.78 and 1.3 Å resolution, respectively, are reported. The structures reveal a conserved trimodular topology of the helicase. ATP and Mn 2+ are tethered between two RecA-like domains by conserved hydrogen-bonding interactions. The binding of ligands induces the movement of backbone Cα and side-chain atoms. Numerous solvent molecules are observed in the vicinity of the AMPPNP, suggesting a role in catalysis. These high-resolution structures could be useful for the design of inhibitors targeting the helicase of ZIKV for the treatment of infections caused by ZIKV.

  14. Distinct functions of human RecQ helicases during DNA replication.

    Science.gov (United States)

    Urban, Vaclav; Dobrovolna, Jana; Janscak, Pavel

    2017-06-01

    DNA replication is the most vulnerable process of DNA metabolism in proliferating cells and therefore it is tightly controlled and coordinated with processes that maintain genomic stability. Human RecQ helicases are among the most important factors involved in the maintenance of replication fork integrity, especially under conditions of replication stress. RecQ helicases promote recovery of replication forks being stalled due to different replication roadblocks of either exogenous or endogenous source. They prevent generation of aberrant replication fork structures and replication fork collapse, and are involved in proper checkpoint signaling. The essential role of human RecQ helicases in the genome maintenance during DNA replication is underlined by association of defects in their function with cancer predisposition. Copyright © 2016 Elsevier B.V. All rights reserved.

  15. DNA binding and unwinding by Hel308 helicase requires dual functions of a winged helix domain.

    Science.gov (United States)

    Northall, Sarah J; Buckley, Ryan; Jones, Nathan; Penedo, J Carlos; Soultanas, Panos; Bolt, Edward L

    2017-09-01

    Hel308 helicases promote genome stability linked to DNA replication in archaea, and have homologues in metazoans. In the crystal structure of archaeal Hel308 bound to a tailed DNA duplex, core helicase domains encircle single-stranded DNA (ssDNA) in a "ratchet" for directional translocation. A winged helix domain (WHD) is also present, but its function is mysterious. We investigated the WHD in full-length Hel308, identifying that mutations in a solvent exposed α-helix resulted in reduced DNA binding and unwinding activities. When isolated from the rest of Hel308, the WHD protein alone bound to duplex DNA but not ssDNA, and DNA binding by WHD protein was abolished by the same mutations as were analyzed in full-length Hel308. Isolated WHD from a human Hel308 homologue (HelQ) also bound to duplex DNA. By disrupting the interface between the Hel308 WHD and a RecA-like domain, a topology typical of Ski2 helicases, we show that this is crucial for ATPase and helicase activities. The data suggest a model in which the WHD promotes activity of Hel308 directly, through binding to duplex DNA that is distinct from ssDNA binding by core helicase, and indirectly through interaction with the RecA-like domain. We propose how the WHD may contribute to ssDNA translocation, resulting in DNA helicase activity or in removal of other DNA bound proteins by "reeling" ssDNA. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. RNA helicase A is not required for RISC activity.

    Science.gov (United States)

    Liang, Xue-Hai; Crooke, Stanley T

    2013-10-01

    It has been shown that siRNAs can compete with each other or with endogenous miRNAs for RISC components. This competition may complicate the interpretations of phenotypes observed through siRNA-mediated knockdown of genes, especially those genes implicated in the RISC pathway. In this study, we re-examined the function of RNA helicase A (RHA), which has been previously proposed to function in RISC loading based on siRNA-mediated knockdown studies. Here we show that reduced RISC activity or loading of siRNAs was observed only in cells depleted of RHA using siRNA, but not using RNaseH-dependent antisense oligonucleotides (ASOs), suggesting that the impaired RISC function stems from the competition between pre-existing and newly transfected siRNAs, but not from reduction of the RHA protein. This view is further supported by the findings that cells depleted of a control protein, NCL1, using siRNA, but not ASO, exhibited similar defects on the loading and activity of a subsequently transfected siRNA. Transfection of RHA or NCL1 siRNAs, but not ASOs, reduced the levels of endogenous miRNAs, suggesting a competition mechanism. As a positive control, we showed that reduction of MOV10 by either siRNA or ASO decreased siRNA activity, confirming its role in RISC function. Together, our results indicate that RHA is not required for RISC activity or loading, and suggest that proper controls are required when using siRNAs to functionalize genes to avoid competition effects. © 2013. Published by Elsevier B.V. All rights reserved.

  17. Cooperation of DNA-PKcs and WRN helicase in the maintenance of telomeric D-loops

    DEFF Research Database (Denmark)

    Kusumoto-Matsuo, Rika; Opresko, Patricia L; Ramsden, Dale

    2010-01-01

    Werner syndrome is an inherited human progeriod syndrome caused by mutations in the gene encoding the Werner Syndrome protein, WRN. It has both 3'-5' DNA helicase and exonuclease activities, and is suggested to have roles in many aspects of DNA metabolism, including DNA repair and telomere...... D-loop model substrate. In addition, the length of telomeric G-tails decreases in DNA-PKcs knockdown cells, and this phenotype is reversed by overexpression of WRN helicase. These results suggest that WRN and DNA-PKcs may cooperatively prevent G-tail shortening in vivo....

  18. Biophysical Characterization of G-Quadruplex Recognition in the PITX1 mRNA by the Specificity Domain of the Helicase RHAU.

    Directory of Open Access Journals (Sweden)

    Emmanuel O Ariyo

    Full Text Available Nucleic acids rich in guanine are able to fold into unique structures known as G-quadruplexes. G-quadruplexes consist of four tracts of guanylates arranged in parallel or antiparallel strands that are aligned in stacked G-quartet planes. The structure is further stabilized by Hoogsteen hydrogen bonds and monovalent cations centered between the planes. RHAU (RNA helicase associated with AU-rich element is a member of the ATP-dependent DExH/D family of RNA helicases and can bind and resolve G-quadruplexes. RHAU contains a core helicase domain with an N-terminal extension that enables recognition and full binding affinity to RNA and DNA G-quadruplexes. PITX1, a member of the bicoid class of homeobox proteins, is a transcriptional activator active during development of vertebrates, chiefly in the anterior pituitary gland and several other organs. We have previously demonstrated that RHAU regulates PITX1 levels through interaction with G-quadruplexes at the 3'-end of the PITX1 mRNA. To understand the structural basis of G-quadruplex recognition by RHAU, we characterize a purified minimal PITX1 G-quadruplex using a variety of biophysical techniques including electrophoretic mobility shift assays, UV-VIS spectroscopy, circular dichroism, dynamic light scattering, small angle X-ray scattering and nuclear magnetic resonance spectroscopy. Our biophysical analysis provides evidence that the RNA G-quadruplex, but not its DNA counterpart, can adopt a parallel orientation, and that only the RNA can interact with N-terminal domain of RHAU via the tetrad face of the G-quadruplex. This work extends our insight into how the N-terminal region of RHAU recognizes parallel G-quadruplexes.

  19. Helicase and Polymerase Move Together Close to the Fork Junction and Copy DNA in One-Nucleotide Steps

    Directory of Open Access Journals (Sweden)

    Manjula Pandey

    2014-03-01

    Full Text Available By simultaneously measuring DNA synthesis and dNTP hydrolysis, we show that T7 DNA polymerase and T7 gp4 helicase move in sync during leading-strand synthesis, taking one-nucleotide steps and hydrolyzing one dNTP per base-pair unwound/copied. The cooperative catalysis enables the helicase and polymerase to move at a uniformly fast rate without guanine:cytosine (GC dependency or idling with futile NTP hydrolysis. We show that the helicase and polymerase are located close to the replication fork junction. This architecture enables the polymerase to use its strand-displacement synthesis to increase the unwinding rate, whereas the helicase aids this process by translocating along single-stranded DNA and trapping the unwound bases. Thus, in contrast to the helicase-only unwinding model, our results suggest a model in which the helicase and polymerase are moving in one-nucleotide steps, DNA synthesis drives fork unwinding, and a role of the helicase is to trap the unwound bases and prevent DNA reannealing.

  20. Prevention of suicide with regulations aimed at restricting access to highly hazardous pesticides: a systematic review of the international evidence.

    Science.gov (United States)

    Gunnell, David; Knipe, Duleeka; Chang, Shu-Sen; Pearson, Melissa; Konradsen, Flemming; Lee, Won Jin; Eddleston, Michael

    2017-10-01

    Pesticide self-poisoning accounts for 14-20% of suicides worldwide. Regulation aimed at restricting access to pesticides or banning highly hazardous pesticides is one approach to reducing these deaths. We systematically reviewed the evidence of the effectiveness of pesticide regulation in reducing the incidence of pesticide suicides and overall suicides. We did a systematic review of the international evidence. We searched MEDLINE, PsycINFO, and Embase for studies published between Jan 1, 1960, and Dec 31, 2016, which investigated the effect of national or regional bans, and sales or import restrictions, on the availability of one or more pesticides and the incidence of suicide in different countries. We excluded other interventions aimed at limiting community access to pesticides. We extracted data from studies presenting pesticide suicide data and overall suicide data from before and after national sales restrictions. Two reviewers independently assessed papers for inclusion, extracted data, and assessed risk of bias. We undertook a narrative synthesis of the data in each report, and where data were available for the years before and after a ban, we pooled data for the 3 years before and the 3 years after to obtain a crude estimate of the effect of the ban. This study is registered through PROSPERO, number CRD42017053329. We identified 27 studies undertaken in 16 countries-five low-income or middle-income countries (Bangladesh, Colombia, India, Jordan and Sri Lanka), and 11 high-income countries (Denmark, Finland, Germany, Greece, Hungary, Ireland, Japan, South Korea, Taiwan, UK, and USA). Assessments largely focused on national bans of specific pesticides (12 studies of bans in six countries-Jordan, Sri Lanka, Bangladesh, Greece [Crete], South Korea, and Taiwan) or sales restrictions (eight studies of restrictions in five countries- India, Denmark, Ireland, the UK and the USA). Only five studies used optimum analytical methods. National bans on commonly ingested

  1. A Small Molecule Inhibitor of the BLM Helicase Modulates Chromosome Stability in Human Cells

    DEFF Research Database (Denmark)

    Nguyen, Giang Huong; Dexheimer, Thomas S; Rosenthal, Andrew S

    2013-01-01

    The Bloom's syndrome protein, BLM, is a member of the conserved RecQ helicase family. Although cell lines lacking BLM exist, these exhibit progressive genomic instability that makes distinguishing primary from secondary effects of BLM loss problematic. In order to be able to acutely disable BLM f...

  2. BLM helicase measures DNA unwound before switching strands and hRPA promotes unwinding reinitiation

    Czech Academy of Sciences Publication Activity Database

    Yodh, J.G.; Stevens, B.C.; Kanagaraj, R.; Janščák, Pavel; Ha, T.

    2009-01-01

    Roč. 28, č. 4 (2009), s. 405-416 ISSN 0261-4189 Institutional research plan: CEZ:AV0Z50520514 Keywords : Bloom syndrome * FRET * helicase * hRPA * single molecule Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 8.993, year: 2009

  3. BLM helicase suppresses recombination at G-quadruplex motifs in transcribed genes

    NARCIS (Netherlands)

    van Wietmarschen, Niek; Merzouk, Sarra; Halsema, Nancy; Spierings, Diana C J; Guryev, Victor; Lansdorp, Peter M

    2018-01-01

    Bloom syndrome is a cancer predisposition disorder caused by mutations in the BLM helicase gene. Cells from persons with Bloom syndrome exhibit striking genomic instability characterized by excessive sister chromatid exchange events (SCEs). We applied single-cell DNA template strand sequencing

  4. Human RecQL4 helicase plays critical roles in prostate carcinogenesis

    DEFF Research Database (Denmark)

    Su, Yanrong; Meador, Jarah A; Calaf, Gloria M

    2010-01-01

    Prostate cancer is the second leading cause of cancer-associated deaths among men in the western countries. Here, we report that human RecQL4 helicase, which is implicated in the pathogenesis of a subset of cancer-prone Rothmund-Thomson syndrome, is highly elevated in metastatic prostate cancer c...

  5. MOV10 RNA helicase is a potent inhibitor of retrotransposition in cells.

    Directory of Open Access Journals (Sweden)

    John L Goodier

    Full Text Available MOV10 protein, a putative RNA helicase and component of the RNA-induced silencing complex (RISC, inhibits retrovirus replication. We show that MOV10 also severely restricts human LINE1 (L1, Alu, and SVA retrotransposons. MOV10 associates with the L1 ribonucleoprotein particle, along with other RNA helicases including DDX5, DHX9, DDX17, DDX21, and DDX39A. However, unlike MOV10, these other helicases do not strongly inhibit retrotransposition, an activity dependent upon intact helicase domains. MOV10 association with retrotransposons is further supported by its colocalization with L1 ORF1 protein in stress granules, by cytoplasmic structures associated with RNA silencing, and by the ability of MOV10 to reduce endogenous and ectopic L1 expression. The majority of the human genome is repetitive DNA, most of which is the detritus of millions of years of accumulated retrotransposition. Retrotransposons remain active mutagens, and their insertion can disrupt gene function. Therefore, the host has evolved defense mechanisms to protect against retrotransposition, an arsenal we are only beginning to understand. With homologs in other vertebrates, insects, and plants, MOV10 may represent an ancient and innate form of immunity against both infective viruses and endogenous retroelements.

  6. MRE11 complex links RECQ5 helicase to sites of DNA damage

    Czech Academy of Sciences Publication Activity Database

    Zheng, L.; Kanagaraj, R.; Mihaljevic, B.; Schwendener, S.; Sartori, A.A.; Gerrits, B.; Shevelev, Igor; Janščák, Pavel

    2009-01-01

    Roč. 37, č. 8 (2009), s. 2645-2657 ISSN 0305-1048 R&D Projects: GA ČR GA204/09/0565 Institutional research plan: CEZ:AV0Z50520514 Keywords : homologous recombination, * RECQ5 helicase * MRE11 * DNA repair Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 7.479, year: 2009

  7. TFIIH with inactive XPD helicase functions in transcription initiation but is defective in DNA repair

    NARCIS (Netherlands)

    G.S. Winkler (Sebastiaan); U. Fiedler; W. Vermeulen (Wim); F. Coin (Frédéric); R.D. Wood (Richard); H.T.M. Timmers (Marc); G. Weeda (Geert); J.H.J. Hoeijmakers (Jan); S.J. Araú jo; J-M. Egly (Jean-Marc)

    2000-01-01

    textabstractTFIIH is a multisubunit protein complex involved in RNA polymerase II transcription and nucleotide excision repair, which removes a wide variety of DNA lesions including UV-induced photoproducts. Mutations in the DNA-dependent ATPase/helicase subunits of TFIIH, XPB and

  8. FBH1 Helicase Disrupts RAD51 Filaments in Vitro and Modulates Homologous Recombination in Mammalian Cells

    Czech Academy of Sciences Publication Activity Database

    Šimandlová, Jitka; Zagelbaum, J.; Payne, M.J.; Chu, W.K.; Shevelev, Igor; Hanada, K.; Chatterjee, S.; Reid, D.A.; Liu, Y.; Janščák, Pavel; Rothenberg, E.; Hickson, I.D.

    2013-01-01

    Roč. 288, č. 47 (2013), s. 34168-34180 ISSN 0021-9258 R&D Projects: GA ČR GAP305/10/0281 Institutional support: RVO:68378050 Keywords : DNA damage * DNA helicase * DNA recombination * DNA repair * DNA replication Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 4.600, year: 2013

  9. Interaction between the helicases genetically linked to Fanconi anemia group J and Bloom's syndrome

    DEFF Research Database (Denmark)

    Suhasini, Avvaru N; Rawtani, Nina A; Wu, Yuliang

    2011-01-01

    Bloom's syndrome (BS) and Fanconi anemia (FA) are autosomal recessive disorders characterized by cancer and chromosomal instability. BS and FA group J arise from mutations in the BLM and FANCJ genes, respectively, which encode DNA helicases. In this work, FANCJ and BLM were found to interact...

  10. DNA binding polarity, dimerization, and ATPase ring remodeling in the CMG helicase of the eukaryotic replisome

    Science.gov (United States)

    Costa, Alessandro; Renault, Ludovic; Swuec, Paolo; Petojevic, Tatjana; Pesavento, James J; Ilves, Ivar; MacLellan-Gibson, Kirsty; Fleck, Roland A; Botchan, Michael R; Berger, James M

    2014-01-01

    The Cdc45/Mcm2-7/GINS (CMG) helicase separates DNA strands during replication in eukaryotes. How the CMG is assembled and engages DNA substrates remains unclear. Using electron microscopy, we have determined the structure of the CMG in the presence of ATPγS and a DNA duplex bearing a 3′ single-stranded tail. The structure shows that the MCM subunits of the CMG bind preferentially to single-stranded DNA, establishes the polarity by which DNA enters into the Mcm2-7 pore, and explains how Cdc45 helps prevent DNA from dissociating from the helicase. The Mcm2-7 subcomplex forms a cracked-ring, right-handed spiral when DNA and nucleotide are bound, revealing unexpected congruencies between the CMG and both bacterial DnaB helicases and the AAA+ motor of the eukaryotic proteasome. The existence of a subpopulation of dimeric CMGs establishes the subunit register of Mcm2-7 double hexamers and together with the spiral form highlights how Mcm2-7 transitions through different conformational and assembly states as it matures into a functional helicase. DOI: http://dx.doi.org/10.7554/eLife.03273.001 PMID:25117490

  11. Crystal structure of the FeS cluster-containing nucleotide excision repair helicase XPD.

    Directory of Open Access Journals (Sweden)

    Stefanie C Wolski

    2008-06-01

    Full Text Available DNA damage recognition by the nucleotide excision repair pathway requires an initial step identifying helical distortions in the DNA and a proofreading step verifying the presence of a lesion. This proofreading step is accomplished in eukaryotes by the TFIIH complex. The critical damage recognition component of TFIIH is the XPD protein, a DNA helicase that unwinds DNA and identifies the damage. Here, we describe the crystal structure of an archaeal XPD protein with high sequence identity to the human XPD protein that reveals how the structural helicase framework is combined with additional elements for strand separation and DNA scanning. Two RecA-like helicase domains are complemented by a 4Fe4S cluster domain, which has been implicated in damage recognition, and an alpha-helical domain. The first helicase domain together with the helical and 4Fe4S-cluster-containing domains form a central hole with a diameter sufficient in size to allow passage of a single stranded DNA. Based on our results, we suggest a model of how DNA is bound to the XPD protein, and can rationalize several of the mutations in the human XPD gene that lead to one of three severe diseases, xeroderma pigmentosum, Cockayne syndrome, and trichothiodystrophy.

  12. Enzymatic activities and DNA substrate specificity of Mycobacterium tuberculosis DNA helicase XPB.

    Science.gov (United States)

    Balasingham, Seetha V; Zegeye, Ephrem Debebe; Homberset, Håvard; Rossi, Marie L; Laerdahl, Jon K; Bohr, Vilhelm A; Tønjum, Tone

    2012-01-01

    XPB, also known as ERCC3 and RAD25, is a 3' → 5' DNA repair helicase belonging to the superfamily 2 of helicases. XPB is an essential core subunit of the eukaryotic basal transcription factor complex TFIIH. It has two well-established functions: in the context of damaged DNA, XPB facilitates nucleotide excision repair by unwinding double stranded DNA (dsDNA) surrounding a DNA lesion; while in the context of actively transcribing genes, XPB facilitates initiation of RNA polymerase II transcription at gene promoters. Human and other eukaryotic XPB homologs are relatively well characterized compared to conserved homologs found in mycobacteria and archaea. However, more insight into the function of bacterial helicases is central to understanding the mechanism of DNA metabolism and pathogenesis in general. Here, we characterized Mycobacterium tuberculosis XPB (Mtb XPB), a 3'→5' DNA helicase with DNA-dependent ATPase activity. Mtb XPB efficiently catalyzed DNA unwinding in the presence of significant excess of enzyme. The unwinding activity was fueled by ATP or dATP in the presence of Mg(2+)/Mn(2+). Consistent with the 3'→5' polarity of this bacterial XPB helicase, the enzyme required a DNA substrate with a 3' overhang of 15 nucleotides or more. Although Mtb XPB efficiently unwound DNA model substrates with a 3' DNA tail, it was not active on substrates containing a 3' RNA tail. We also found that Mtb XPB efficiently catalyzed ATP-independent annealing of complementary DNA strands. These observations significantly enhance our understanding of the biological roles of Mtb XPB.

  13. Enzymatic activities and DNA substrate specificity of Mycobacterium tuberculosis DNA helicase XPB.

    Directory of Open Access Journals (Sweden)

    Seetha V Balasingham

    Full Text Available XPB, also known as ERCC3 and RAD25, is a 3' → 5' DNA repair helicase belonging to the superfamily 2 of helicases. XPB is an essential core subunit of the eukaryotic basal transcription factor complex TFIIH. It has two well-established functions: in the context of damaged DNA, XPB facilitates nucleotide excision repair by unwinding double stranded DNA (dsDNA surrounding a DNA lesion; while in the context of actively transcribing genes, XPB facilitates initiation of RNA polymerase II transcription at gene promoters. Human and other eukaryotic XPB homologs are relatively well characterized compared to conserved homologs found in mycobacteria and archaea. However, more insight into the function of bacterial helicases is central to understanding the mechanism of DNA metabolism and pathogenesis in general. Here, we characterized Mycobacterium tuberculosis XPB (Mtb XPB, a 3'→5' DNA helicase with DNA-dependent ATPase activity. Mtb XPB efficiently catalyzed DNA unwinding in the presence of significant excess of enzyme. The unwinding activity was fueled by ATP or dATP in the presence of Mg(2+/Mn(2+. Consistent with the 3'→5' polarity of this bacterial XPB helicase, the enzyme required a DNA substrate with a 3' overhang of 15 nucleotides or more. Although Mtb XPB efficiently unwound DNA model substrates with a 3' DNA tail, it was not active on substrates containing a 3' RNA tail. We also found that Mtb XPB efficiently catalyzed ATP-independent annealing of complementary DNA strands. These observations significantly enhance our understanding of the biological roles of Mtb XPB.

  14. Chlorosis caused by two recessively interacting genes reveals a role of RNA helicase in hybrid breakdown in Arabidopsis thaliana.

    Science.gov (United States)

    Plötner, Björn; Nurmi, Markus; Fischer, Axel; Watanabe, Mutsumi; Schneeberger, Korbinian; Holm, Svante; Vaid, Neha; Schöttler, Mark Aurel; Walther, Dirk; Hoefgen, Rainer; Weigel, Detlef; Laitinen, Roosa A E

    2017-07-01

    Hybrids often differ in fitness from their parents. They may be superior, translating into hybrid vigour or heterosis, but they may also be markedly inferior, because of hybrid weakness or incompatibility. The underlying genetic causes for the latter can often be traced back to genes that evolve rapidly because of sexual or host-pathogen conflicts. Hybrid weakness may manifest itself only in later generations, in a phenomenon called hybrid breakdown. We have characterized a case of hybrid breakdown among two Arabidopsis thaliana accessions, Shahdara (Sha, Tajikistan) and Lövvik-5 (Lov-5, Northern Sweden). In addition to chlorosis, a fraction of the F 2 plants have defects in leaf and embryo development, and reduced photosynthetic efficiency. Hybrid chlorosis is due to two major-effect loci, of which one, originating from Lov-5, appears to encode an RNA helicase (AtRH18). To examine the role of the chlorosis allele in the Lövvik area, in addition to eight accessions collected in 2009, we collected another 240 accessions from 15 collections sites, including Lövvik, from Northern Sweden in 2015. Genotyping revealed that Lövvik collection site is separated from the rest. Crosses between 109 accessions from this area and Sha revealed 85 cases of hybrid chlorosis, indicating that the chlorosis-causing allele is common in this area. These results suggest that hybrid breakdown alleles not only occur at rapidly evolving loci, but also at genes that code for conserved processes. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

  15. 50 CFR 26.34 - What are the special regulations concerning public access, use, and recreation for individual...

    Science.gov (United States)

    2010-10-01

    ... chapter). (10) We prohibit the use or possession of glass food and beverage containers on lands within the... boat landing, access area, parking lot, structure, road, trail, or other recreation or management..., boat landings, access areas, parking lots, roads, trails, or any other recreation or management...

  16. DNA secondary structure of the released strand stimulates WRN helicase action on forked duplexes without coordinate action of WRN exonuclease

    Energy Technology Data Exchange (ETDEWEB)

    Ahn, Byungchan, E-mail: bbccahn@mail.ulsan.ac.kr [Department of Life Sciences, University of Ulsan, Ulsan (Korea, Republic of); Bohr, Vilhelm A. [Laboratory of Molecular Gerontology, Biomedical Research Center, National Institute on Aging, Baltimore, MD (United States)

    2011-08-12

    Highlights: {yields} In this study, we investigated the effect of a DNA secondary structure on the two WRN activities. {yields} We found that a DNA secondary structure of the displaced strand during unwinding stimulates WRN helicase without coordinate action of WRN exonuclease. {yields} These results imply that WRN helicase and exonuclease activities can act independently. -- Abstract: Werner syndrome (WS) is an autosomal recessive premature aging disorder characterized by aging-related phenotypes and genomic instability. WS is caused by mutations in a gene encoding a nuclear protein, Werner syndrome protein (WRN), a member of the RecQ helicase family, that interestingly possesses both helicase and exonuclease activities. Previous studies have shown that the two activities act in concert on a single substrate. We investigated the effect of a DNA secondary structure on the two WRN activities and found that a DNA secondary structure of the displaced strand during unwinding stimulates WRN helicase without coordinate action of WRN exonuclease. These results imply that WRN helicase and exonuclease activities can act independently, and we propose that the uncoordinated action may be relevant to the in vivo activity of WRN.

  17. Barriers to access to opioid medicines for patients with opioid dependence: a review of legislation and regulations in eleven central and eastern European countries.

    Science.gov (United States)

    Vranken, Marjolein J M; Mantel-Teeuwisse, Aukje K; Jünger, Saskia; Radbruch, Lukas; Scholten, Willem; Lisman, John A; Subataite, Marija; Schutjens, Marie-Hélène D B

    2017-06-01

    Barriers linked to drug control systems are considered to contribute to inequitable access to controlled medicines, leaving millions of people in pain and suffering. Most studies focus on access to opioids for the treatment of severe (cancer) pain. This study aims to identify specific access barriers for patients with opioid dependence in legislation and regulations of 11 central and eastern European countries. This study builds on a previous analysis of legislation and regulations as part of the EU 7th Framework Access To Opioid Medication in Europe (ATOME) project. An in-depth analysis was undertaken to determine specific barriers for patients with opioid dependence in need of opioid analgesics or opioid agonist therapy (OAT). For each country, the number and nature of specific potential barriers for these patients were assessed according to eight categories: prescribing; dispensing; manufacturing; usage; trade and distribution; affordability; penalties; and other. An additional keyword search was conducted to minimize the omission of barriers. Barriers in an additional category, language, were recorded qualitatively. Countries included Bulgaria, Cyprus, Estonia, Greece, Hungary, Latvia, Lithuania, Serbia, Slovakia, Slovenia and Turkey. Ten of the 11 countries (all except Estonia) showed specific potential barriers in their legislation and regulations. The total number of barriers varied from two (Slovenia) to 46 (Lithuania); the number of categories varied from one (Slovenia) to five (Lithuania). Most specific potential barriers were shown in the categories 'prescribing', 'usage' and 'other'. The total number in a single category varied from one to 18 (Lithuania, prescribing). Individual differences between countries in the same specific potential barrier were shown; for example, variation in minimum age criteria for admission to OAT ranging from 15 (Lithuania, in special cases) to 20 years (Greece). All countries had stigmatizing language in their legislation

  18. Comprehensive Protein Interactome Analysis of a Key RNA Helicase: Detection of Novel Stress Granule Proteins

    Directory of Open Access Journals (Sweden)

    Rebecca Bish

    2015-07-01

    Full Text Available DDX6 (p54/RCK is a human RNA helicase with central roles in mRNA decay and translation repression. To help our understanding of how DDX6 performs these multiple functions, we conducted the first unbiased, large-scale study to map the DDX6-centric protein-protein interactome using immunoprecipitation and mass spectrometry. Using DDX6 as bait, we identify a high-confidence and high-quality set of protein interaction partners which are enriched for functions in RNA metabolism and ribosomal proteins. The screen is highly specific, maximizing the number of true positives, as demonstrated by the validation of 81% (47/58 of the RNA-independent interactors through known functions and interactions. Importantly, we minimize the number of indirect interaction partners through use of a nuclease-based digestion to eliminate RNA. We describe eleven new interactors, including proteins involved in splicing which is an as-yet unknown role for DDX6. We validated and characterized in more detail the interaction of DDX6 with Nuclear fragile X mental retardation-interacting protein 2 (NUFIP2 and with two previously uncharacterized proteins, FAM195A and FAM195B (here referred to as granulin-1 and granulin-2, or GRAN1 and GRAN2. We show that NUFIP2, GRAN1, and GRAN2 are not P-body components, but re-localize to stress granules upon exposure to stress, suggesting a function in translation repression in the cellular stress response. Using a complementary analysis that resolved DDX6’s multiple complex memberships, we further validated these interaction partners and the presence of splicing factors. As DDX6 also interacts with the E3 SUMO ligase TIF1β, we tested for and observed a significant enrichment of sumoylation amongst DDX6’s interaction partners. Our results represent the most comprehensive screen for direct interaction partners of a key regulator of RNA life cycle and localization, highlighting new stress granule components and possible DDX6 functions

  19. Mycobacterium smegmatis SftH exemplifies a distinctive clade of superfamily II DNA-dependent ATPases with 3′ to 5′ translocase and helicase activities

    OpenAIRE

    Yakovleva, Lyudmila; Shuman, Stewart

    2012-01-01

    Bacterial DNA helicases are nucleic acid-dependent NTPases that play important roles in DNA replication, recombination and repair. We are interested in the DNA helicases of Mycobacteria, a genus of the phylum Actinobacteria, which includes the human pathogen Mycobacterium tuberculosis and its avirulent relative Mycobacterium smegmatis. Here, we identify and characterize M. smegmatis SftH, a superfamily II helicase with a distinctive domain structure, comprising an N-terminal NTPase domain and...

  20. hSSB1 associates with and promotes stability of the BLM helicase

    OpenAIRE

    O'BYRNE, KEN

    2017-01-01

    Background Maintenance of genome stability is critical in human cells. Mutations in or loss of genome stability pathways can lead to a number of pathologies including cancer. hSSB1 is a critical DNA repair protein functioning in the repair and signalling of stalled DNA replication forks, double strand DNA breaks and oxidised DNA lesions. The BLM helicase is central to the repair of both collapsed DNA replication forks and double strand DNA breaks by homologous recombination. Results In this s...

  1. The human RecQ helicases BLM and RECQL4 cooperate to preserve genome stability

    Czech Academy of Sciences Publication Activity Database

    Singh, D.K.; Popuri, V.; Kulikowicz, T.; Shevelev, Igor; Ghosh, A.K.; Ramamoorthy, M.; Rossi, M.L.; Janščák, Pavel; Croteau, D.L.; Bohr, V.A.

    2012-01-01

    Roč. 40, č. 14 (2012), s. 6632-6648 ISSN 0305-1048 R&D Projects: GA ČR GAP305/10/0281 Grant - others:NIH(US) Z01-AG000726-17 Institutional research plan: CEZ:AV0Z50520514 Institutional support: RVO:68378050 Keywords : RecQ helicase * genome stability * BLM * RECQL4 Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 8.278, year: 2012

  2. A Co-Opted DEAD-Box RNA helicase enhances tombusvirus plus-strand synthesis.

    Directory of Open Access Journals (Sweden)

    Nikolay Kovalev

    2012-02-01

    Full Text Available Replication of plus-strand RNA viruses depends on recruited host factors that aid several critical steps during replication. In this paper, we show that an essential translation factor, Ded1p DEAD-box RNA helicase of yeast, directly affects replication of Tomato bushy stunt virus (TBSV. To separate the role of Ded1p in viral protein translation from its putative replication function, we utilized a cell-free TBSV replication assay and recombinant Ded1p. The in vitro data show that Ded1p plays a role in enhancing plus-strand synthesis by the viral replicase. We also find that Ded1p is a component of the tombusvirus replicase complex and Ded1p binds to the 3'-end of the viral minus-stranded RNA. The data obtained with wt and ATPase deficient Ded1p mutants support the model that Ded1p unwinds local structures at the 3'-end of the TBSV (-RNA, rendering the RNA compatible for initiation of (+-strand synthesis. Interestingly, we find that Ded1p and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, which is another host factor for TBSV, play non-overlapping functions to enhance (+-strand synthesis. Altogether, the two host factors enhance TBSV replication synergistically by interacting with the viral (-RNA and the replication proteins. In addition, we have developed an in vitro assay for Flock house virus (FHV, a small RNA virus of insects, that also demonstrated positive effect on FHV replicase activity by the added Ded1p helicase. Thus, two small RNA viruses, which do not code for their own helicases, seems to recruit a host RNA helicase to aid their replication in infected cells.

  3. The eIF4AIII RNA helicase is a critical determinant of human cytomegalovirus replication

    International Nuclear Information System (INIS)

    Ziehr, Ben; Lenarcic, Erik; Cecil, Chad; Moorman, Nathaniel J.

    2016-01-01

    Human cytomegalovirus (HCMV) was recently shown to encode a large number of spliced mRNAs. While the nuclear export of unspliced viral transcripts has been extensively studied, the role of host mRNA export factors in HCMV mRNA trafficking remains poorly defined. We found that the eIF4AIII RNA helicase, a component of the exon junction complex, was necessary for efficient virus replication. Depletion of eIF4AIII limited viral DNA accumulation, export of viral mRNAs from the nucleus, and the production of progeny virus. However eIF4AIII was dispensable for the association of viral transcripts with ribosomes. We found that pateamine A, a natural compound that inhibits both eIF4AI/II and eIF4AIII, has potent antiviral activity and inhibits HCMV replication throughout the virus lytic cycle. Our results demonstrate that eIF4AIII is required for efficient HCMV replication, and suggest that eIF4A family helicases may be a new class of targets for the development of host-directed antiviral therapeutics. - Highlights: • The host eIF4AIII RNA helicase is required for efficient HCMV replication. • Depleting eIF4AIII inhibited the nuclear export of HCMV mRNAs. • HCMV mRNAs did not require eIF4AIII to associate with polyribosomes. • The eIF4A family helicases may be new targets for host-directed antiviral drugs.

  4. Evolution of the DEAD box helicase family in chicken: chickens have no DHX9 ortholog.

    Science.gov (United States)

    Sato, Haruko; Oshiumi, Hiroyuki; Takaki, Hiromi; Hikono, Hirokazu; Seya, Tsukasa

    2015-10-01

    Viral RNA represents a pattern molecule that can be recognized by RNA sensors in innate immunity. Humans and mice possess cytoplasmic DNA/RNA sensors for detecting viral replication. There are a number of DEAD (Asp-Glu-Ala-Asp; DExD/H) box-type helicases in mammals, among which retinoic acid-inducible gene 1 (RIG-I) and melanoma differentiation-associated protein 5 (MDA50) are indispensable for RNA sensing; however, they are functionally supported by a number of sensors that directly bind viral RNA or replicative RNA intermediates to convey signals to RIG-I and MDA5. Some DEAD box helicase members recognize DNA irrespective of the origin. These sensors transmit IFN-inducing signals through adaptors, including mitochondrial antiviral signaling. Viral double-stranded RNAs are reportedly sensed by the helicases DDX1, DDX21, DHX36, DHX9, DDX3, DDX41, LGP2 and DDX60, in addition to RIG-I and MDA5, and induce type I IFNs, thereby blocking viral replication. Humans and mice have all nucleic acid sensors listed here. In the RNA sensing system in chicken, it was found in the present study that most DEAD box helicases are conserved; however, DHX9 is genetically deficient in addition to reported RIG-I. Based on the current genome databases, similar DHX9 deficiency was observed in ducks and several other bird species. Because chicken, but not duck, was found to be deficient in RIG-I, the RNA-sensing system of chicken lacks RIG-I and DHX9 and is thus more fragile than that of duck or mammal. DHX9 may generally compensate for the function of RIG-I and deficiency of DHX9 possibly participates in exacerbations of viral infection such as influenza in chickens. © 2015 The Societies and Wiley Publishing Asia Pty Ltd.

  5. Physical interaction of RECQ5 helicase with RAD51 facilitates its anti-recombinase activity

    Czech Academy of Sciences Publication Activity Database

    Schwendener, S.; Raynard, S.; Paliwal, S.; Cheng, A.; Kanagaraj, R.; Shevelev, Igor; Stark, J.M.; Sung, P.; Janscak, P.

    2010-01-01

    Roč. 285, č. 21 (2010), s. 15739-15745 ISSN 0021-9258 Grant - others:NIH(US) R01CA120954; NIH(US) ES015632; SNSF(CH) 3100A0-116008 Institutional research plan: CEZ:AV0Z50520514 Keywords : DNA helicase * double-strand breaks * homologous recombination Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 5.328, year: 2010

  6. DEAD-box RNA helicase is dispensable for mitochondrial translation in Trypanosoma brucei

    Czech Academy of Sciences Publication Activity Database

    Richterová, Lenka; Vávrová, Zuzana; Lukeš, Julius

    2011-01-01

    Roč. 127, č. 1 (2011), 300-303 ISSN 0014-4894 R&D Projects: GA ČR GA204/09/1667; GA MŠk LC07032; GA MŠk 2B06129 Institutional research plan: CEZ:AV0Z60220518 Keywords : Trypanosoma * Mitochondrial translation * RNA helicase * Cytochrome c oxidase * Mitochondrion Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.122, year: 2011

  7. The eIF4AIII RNA helicase is a critical determinant of human cytomegalovirus replication

    Energy Technology Data Exchange (ETDEWEB)

    Ziehr, Ben; Lenarcic, Erik; Cecil, Chad; Moorman, Nathaniel J., E-mail: nmoorman@med.unc.edu

    2016-02-15

    Human cytomegalovirus (HCMV) was recently shown to encode a large number of spliced mRNAs. While the nuclear export of unspliced viral transcripts has been extensively studied, the role of host mRNA export factors in HCMV mRNA trafficking remains poorly defined. We found that the eIF4AIII RNA helicase, a component of the exon junction complex, was necessary for efficient virus replication. Depletion of eIF4AIII limited viral DNA accumulation, export of viral mRNAs from the nucleus, and the production of progeny virus. However eIF4AIII was dispensable for the association of viral transcripts with ribosomes. We found that pateamine A, a natural compound that inhibits both eIF4AI/II and eIF4AIII, has potent antiviral activity and inhibits HCMV replication throughout the virus lytic cycle. Our results demonstrate that eIF4AIII is required for efficient HCMV replication, and suggest that eIF4A family helicases may be a new class of targets for the development of host-directed antiviral therapeutics. - Highlights: • The host eIF4AIII RNA helicase is required for efficient HCMV replication. • Depleting eIF4AIII inhibited the nuclear export of HCMV mRNAs. • HCMV mRNAs did not require eIF4AIII to associate with polyribosomes. • The eIF4A family helicases may be new targets for host-directed antiviral drugs.

  8. Distinct functions of human RecQ helicases during DNA replication

    Czech Academy of Sciences Publication Activity Database

    Urban, Václav; Dobrovolná, Jana; Janščák, Pavel

    2017-01-01

    Roč. 225, červen (2017), s. 20-26 ISSN 0301-4622 R&D Projects: GA ČR(CZ) GA14-05743S; GA MŠk LH14037 Institutional support: RVO:68378050 Keywords : DNA replication * Replication stress * RecQ helicases * Genomic instability * Cancer Subject RIV: EB - Genetics ; Molecular Biology OBOR OECD: Biochemistry and molecular biology Impact factor: 2.402, year: 2016

  9. Relocalization of nuclear DNA helicase II during the growth period of bovine oocytes

    Czech Academy of Sciences Publication Activity Database

    Baran, V.; Kovářová, Hana; Klíma, Jiří; Hozák, Pavel; Motlík, Jan

    2006-01-01

    Roč. 125, 1-2 (2006), s. 155-164 ISSN 0948-6143 R&D Projects: GA ČR GA523/03/0857 Grant - others:Slovenská Akademie věd(SK) VEGA 2/3065/23 Institutional research plan: CEZ:AV0Z50450515; CEZ:AV0Z50390512 Keywords : DNA helicase II * fibroblasts * oocytes Subject RIV: EB - Genetics ; Molecular Biology Impact factor : 3.220, year: 2006

  10. Senataxin, the ortholog of a yeast RNA helicase, is mutant in ataxia-ocular apraxia 2.

    Science.gov (United States)

    Moreira, Maria-Céu; Klur, Sandra; Watanabe, Mitsunori; Németh, Andrea H; Le Ber, Isabelle; Moniz, José-Carlos; Tranchant, Christine; Aubourg, Patrick; Tazir, Meriem; Schöls, Lüdger; Pandolfo, Massimo; Schulz, Jörg B; Pouget, Jean; Calvas, Patrick; Shizuka-Ikeda, Masami; Shoji, Mikio; Tanaka, Makoto; Izatt, Louise; Shaw, Christopher E; M'Zahem, Abderrahim; Dunne, Eimear; Bomont, Pascale; Benhassine, Traki; Bouslam, Naïma; Stevanin, Giovanni; Brice, Alexis; Guimarães, João; Mendonça, Pedro; Barbot, Clara; Coutinho, Paula; Sequeiros, Jorge; Dürr, Alexandra; Warter, Jean-Marie; Koenig, Michel

    2004-03-01

    Ataxia-ocular apraxia 2 (AOA2) was recently identified as a new autosomal recessive ataxia. We have now identified causative mutations in 15 families, which allows us to clinically define this entity by onset between 10 and 22 years, cerebellar atrophy, axonal sensorimotor neuropathy, oculomotor apraxia and elevated alpha-fetoprotein (AFP). Ten of the fifteen mutations cause premature termination of a large DEAxQ-box helicase, the human ortholog of yeast Sen1p, involved in RNA maturation and termination.

  11. Role of the hydrophilic channels of simian virus 40 T-antigen helicase in DNA replication.

    Science.gov (United States)

    Wang, Weiping; Manna, David; Simmons, Daniel T

    2007-05-01

    The simian virus 40 (SV40) hexameric helicase consists of a central channel and six hydrophilic channels located between adjacent large tier domains within each hexamer. To study the function of the hydrophilic channels in SV40 DNA replication, a series of single-point substitutions were introduced at sites not directly involved in protein-protein contacts. The mutants were characterized biochemically in various ways. All mutants oligomerized normally in the absence of DNA. Interestingly, 8 of the 10 mutants failed to unwind an origin-containing DNA fragment and nine of them were totally unable to support SV40 DNA replication in vitro. The mutants fell into four classes based on their biochemical properties. Class A mutants bound DNA normally and had normal ATPase and helicase activities but failed to unwind origin DNA and support SV40 DNA replication. Class B mutants were compromised in single-stranded DNA and origin DNA binding at low protein concentrations. They were defective in helicase activity and unwinding of the origin and in supporting DNA replication. Class C and D mutants possessed higher-than-normal single-stranded DNA binding activity at low protein concentrations. The class C mutants failed to separate origin DNA and support DNA replication. The class D mutants unwound origin DNA normally but were compromised in their ability to support DNA replication. Taken together, these results suggest that the hydrophilic channels have an active role in the unwinding of SV40 DNA from the origin and the placement of the resulting single strands within the helicase.

  12. Archaeal MCM Proteins as an Analog for the Eukaryotic Mcm2–7 Helicase to Reveal Essential Features of Structure and Function

    Science.gov (United States)

    Miller, Justin M.; Enemark, Eric J.

    2015-01-01

    In eukaryotes, the replicative helicase is the large multisubunit CMG complex consisting of the Mcm2–7 hexameric ring, Cdc45, and the tetrameric GINS complex. The Mcm2–7 ring assembles from six different, related proteins and forms the core of this complex. In archaea, a homologous MCM hexameric ring functions as the replicative helicase at the replication fork. Archaeal MCM proteins form thermostable homohexamers, facilitating their use as models of the eukaryotic Mcm2–7 helicase. Here we review archaeal MCM helicase structure and function and how the archaeal findings relate to the eukaryotic Mcm2–7 ring. PMID:26539061

  13. The redefinition of the american and british gas industries: the regulation of the access load to the gas pipelines networks

    International Nuclear Information System (INIS)

    David, L.

    2000-10-01

    The transport and distribution networks regulation is the main stakes of the regulation reform of the gas industries. This thesis analyzes the models applied in The Usa and in the United Kingdom. The first part deals with the gas industries deregulation in these two countries, the impacts on the economy and the organization of the gas industries. The second part presents a theoretical approach of the regulation applied to the prices of the natural ags transport by gas pipelines. Regulation by the service cost price and by price cap are compared. (A.L.B.)

  14. Non-B DNA Secondary Structures and Their Resolution by RecQ Helicases

    Directory of Open Access Journals (Sweden)

    Sudha Sharma

    2011-01-01

    Full Text Available In addition to the canonical B-form structure first described by Watson and Crick, DNA can adopt a number of alternative structures. These non-B-form DNA secondary structures form spontaneously on tracts of repeat sequences that are abundant in genomes. In addition, structured forms of DNA with intrastrand pairing may arise on single-stranded DNA produced transiently during various cellular processes. Such secondary structures have a range of biological functions but also induce genetic instability. Increasing evidence suggests that genomic instabilities induced by non-B DNA secondary structures result in predisposition to diseases. Secondary DNA structures also represent a new class of molecular targets for DNA-interactive compounds that might be useful for targeting telomeres and transcriptional control. The equilibrium between the duplex DNA and formation of multistranded non-B-form structures is partly dependent upon the helicases that unwind (resolve these alternate DNA structures. With special focus on tetraplex, triplex, and cruciform, this paper summarizes the incidence of non-B DNA structures and their association with genomic instability and emphasizes the roles of RecQ-like DNA helicases in genome maintenance by resolution of DNA secondary structures. In future, RecQ helicases are anticipated to be additional molecular targets for cancer chemotherapeutics.

  15. Structural mechanisms of human RecQ helicases WRN and BLM

    Directory of Open Access Journals (Sweden)

    Ken eKitano

    2014-10-01

    Full Text Available The RecQ family DNA helicases WRN (Werner syndrome protein and BLM (Bloom syndrome protein play a key role in protecting the genome against deleterious changes. In humans, mutations in these proteins lead to rare genetic diseases associated with cancer predisposition and accelerated aging. WRN and BLM are distinguished from other helicases by possessing signature tandem domains toward the C terminus, referred to as the RecQ C-terminal (RQC and helicase-and-ribonuclease D-C-terminal (HRDC domains. Although the precise function of the HRDC domain remains unclear, the previous crystal structure of a WRN RQC-DNA complex visualized a central role for the RQC domain in recognizing, binding and unwinding DNA at branch points. In particular, a prominent hairpin structure (the β-wing within the RQC winged-helix motif acts as a scalpel to induce the unpairing of a Watson-Crick base pair at the DNA duplex terminus. A similar RQC-DNA interaction was also observed in the recent crystal structure of a BLM-DNA complex. I review the latest structures of WRN and BLM, and then provide a docking simulation of BLM with a Holliday junction. The model offers an explanation for the efficient branch migration activity of the RecQ family toward recombination and repair intermediates.

  16. Novel benzoxazole inhibitor of dengue virus replication that targets the NS3 helicase.

    Science.gov (United States)

    Byrd, Chelsea M; Grosenbach, Douglas W; Berhanu, Aklile; Dai, Dongcheng; Jones, Kevin F; Cardwell, Kara B; Schneider, Christine; Yang, Guang; Tyavanagimatt, Shanthakumar; Harver, Chris; Wineinger, Kristin A; Page, Jessica; Stavale, Eric; Stone, Melialani A; Fuller, Kathleen P; Lovejoy, Candace; Leeds, Janet M; Hruby, Dennis E; Jordan, Robert

    2013-04-01

    Dengue virus (DENV) is the predominant mosquito-borne viral pathogen that infects humans with an estimated 50 to 100 million infections per year worldwide. Over the past 50 years, the incidence of dengue disease has increased dramatically and the virus is now endemic in more than 100 countries. Moreover, multiple serotypes of DENV are now found in the same geographic region, increasing the likelihood of more severe forms of disease. Despite extensive research, there are still no approved vaccines or therapeutics commercially available to treat DENV infection. Here we report the results of a high-throughput screen of a chemical compound library using a whole-virus assay that identified a novel small-molecule inhibitor of DENV, ST-610, that potently and selectively inhibits all four serotypes of DENV replication in vitro. Sequence analysis of drug-resistant virus isolates has identified a single point mutation, A263T, in the NS3 helicase domain that confers resistance to this compound. ST-610 inhibits DENV NS3 helicase RNA unwinding activity in a molecular-beacon-based helicase assay but does not inhibit nucleoside triphosphatase activity based on a malachite green ATPase assay. ST-610 is nonmutagenic, is well tolerated (nontoxic) in mice, and has shown efficacy in a sublethal murine model of DENV infection with the ability to significantly reduce viremia and viral load compared to vehicle controls.

  17. Helicase properties of the Escherichia coli UvrAb protein complex

    International Nuclear Information System (INIS)

    Oh, E.Y.; Grossman, L.

    1987-01-01

    The Escherichia coli UvrA protein has an associated ATPase activity with a turnover number affected by the presence of UvrB protein as well as by DNA. Specifically, the structure of DNA significantly influences the turnover rate of the UvrAB ATPase activity. Double-stranded DNA maximally activates the turnover rate 10-fold whereas single-stranded DNA maximally activates the turnover rate 20-fold, suggesting that the mode of interaction of UvrAB protein with different DNAs is distinctive. We have previously shown that the UvrAB protein complex, driven by the binding energy of ATP, can locally unwind supercoiled DNA. The nature of the DNA unwinding activity and single-stranded DNA activation of ATPase activity suggest potential helicase activity. In the presence of a number of helicase substrates, the UvrAB complex, indeed, manifests a strand-displacement activity-unwinding short duplexes and D-loop DNA, thereby generating component DNA structures. The energy for the activity is derived from ATP or dATP hydrolysis. Unlike the E. coli DnaB, the UvrAB helicase is sensitive to UV-induced photoproducts

  18. Isolation and Characterization of Pepper Genes Interacting with the CMV-P1 Helicase Domain.

    Directory of Open Access Journals (Sweden)

    Yoomi Choi

    Full Text Available Cucumber mosaic virus (CMV is a destructive pathogen affecting Capsicum annuum (pepper production. The pepper Cmr1 gene confers resistance to most CMV strains, but is overcome by CMV-P1 in a process dependent on the CMV-P1 RNA1 helicase domain (P1 helicase. Here, to identify host factors involved in CMV-P1 infection in pepper, a yeast two-hybrid library derived from a C. annuum 'Bukang' cDNA library was screened, producing a total of 76 potential clones interacting with the P1 helicase. Beta-galactosidase filter lift assay, PCR screening, and sequencing analysis narrowed the candidates to 10 genes putatively involved in virus infection. The candidate host genes were silenced in Nicotiana benthamiana plants that were then inoculated with CMV-P1 tagged with the green fluorescent protein (GFP. Plants silenced for seven of the genes showed development comparable to N. benthamiana wild type, whereas plants silenced for the other three genes showed developmental defects including stunting and severe distortion. Silencing formate dehydrogenase and calreticulin-3 precursor led to reduced virus accumulation. Formate dehydrogenase-silenced plants showed local infection in inoculated leaves, but not in upper (systemic leaves. In the calreticulin-3 precursor-silenced plants, infection was not observed in either the inoculated or the upper leaves. Our results demonstrate that formate dehydrogenase and calreticulin-3 precursor are required for CMV-P1 infection.

  19. Staphylococcal SCCmec elements encode an active MCM-like helicase and thus may be replicative

    Energy Technology Data Exchange (ETDEWEB)

    Mir-Sanchis, Ignacio; Roman, Christina A.; Misiura, Agnieszka; Pigli, Ying Z.; Boyle-Vavra, Susan; Rice , Phoebe A. (UC)

    2016-08-29

    Methicillin-resistant Staphylococcus aureus (MRSA) is a public-health threat worldwide. Although the mobile genomic island responsible for this phenotype, staphylococcal cassette chromosome (SCC), has been thought to be nonreplicative, we predicted DNA-replication-related functions for some of the conserved proteins encoded by SCC. We show that one of these, Cch, is homologous to the self-loading initiator helicases of an unrelated family of genomic islands, that it is an active 3'-to-5' helicase and that the adjacent ORF encodes a single-stranded DNA–binding protein. Our 2.9-Å crystal structure of intact Cch shows that it forms a hexameric ring. Cch, like the archaeal and eukaryotic MCM-family replicative helicases, belongs to the pre–sensor II insert clade of AAA+ ATPases. Additionally, we found that SCC elements are part of a broader family of mobile elements, all of which encode a replication initiator upstream of their recombinases. Replication after excision would enhance the efficiency of horizontal gene transfer.

  20. A holistic evolutionary and structural study of flaviviridae provides insights into the function and inhibition of HCV helicase

    Directory of Open Access Journals (Sweden)

    Dimitrios Vlachakis

    2013-05-01

    Full Text Available Viral RNA helicases are involved in duplex unwinding during the RNA replication of the virus. It is suggested that these helicases represent very promising antiviral targets. Viruses of the flaviviridae family are the causative agents of many common and devastating diseases, including hepatitis, yellow fever and dengue fever. As there is currently no available anti-Flaviviridae therapy, there is urgent need for the development of efficient anti-viral pharmaceutical strategies. Herein, we report the complete phylogenetic analysis across flaviviridae alongside a more in-depth evolutionary study that revealed a series of conserved and invariant amino acids that are predicted to be key to the function of the helicase. Structural molecular modelling analysis revealed the strategic significance of these residues based on their relative positioning on the 3D structures of the helicase enzymes, which may be used as pharmacological targets. We previously reported a novel series of highly potent HCV helicase inhibitors, and we now re-assess their antiviral potential using the 3D structural model of the invariant helicase residues. It was found that the most active compound of the series, compound C4, exhibited an IC50 in the submicromolar range, whereas its stereoisomer (compound C12 was completely inactive. Useful insights were obtained from molecular modelling and conformational search studies via molecular dynamics simulations. C12 tends to bend and lock in an almost “U” shape conformation, failing to establish vital interactions with the active site of HCV. On the contrary, C4 spends most of its conformational time in a straight, more rigid formation that allows it to successfully block the passage of the oligonucleotide in the ssRNA channel of the HCV helicase. This study paves the way and provides the necessary framework for the in-depth analysis required to enable the future design of new and potent anti-viral agents.

  1. DNA unwinding by ring-shaped T4 helicase gp41 is hindered by tension on the occluded strand.

    Science.gov (United States)

    Ribeck, Noah; Saleh, Omar A

    2013-01-01

    The replicative helicase for bacteriophage T4 is gp41, which is a ring-shaped hexameric motor protein that achieves unwinding of dsDNA by translocating along one strand of ssDNA while forcing the opposite strand to the outside of the ring. While much study has been dedicated to the mechanism of binding and translocation along the ssDNA strand encircled by ring-shaped helicases, relatively little is known about the nature of the interaction with the opposite, 'occluded' strand. Here, we investigate the interplay between the bacteriophage T4 helicase gp41 and the ss/dsDNA fork by measuring, at the single-molecule level, DNA unwinding events on stretched DNA tethers in multiple geometries. We find that gp41 activity is significantly dependent on the geometry and tension of the occluded strand, suggesting an interaction between gp41 and the occluded strand that stimulates the helicase. However, the geometry dependence of gp41 activity is the opposite of that found previously for the E. coli hexameric helicase DnaB. Namely, tension applied between the occluded strand and dsDNA stem inhibits unwinding activity by gp41, while tension pulling apart the two ssDNA tails does not hinder its activity. This implies a distinct variation in helicase-occluded strand interactions among superfamily IV helicases, and we propose a speculative model for this interaction that is consistent with both the data presented here on gp41 and the data that had been previously reported for DnaB.

  2. Human Enterovirus Nonstructural Protein 2CATPase Functions as Both an RNA Helicase and ATP-Independent RNA Chaperone

    Science.gov (United States)

    Xia, Hongjie; Wang, Peipei; Wang, Guang-Chuan; Yang, Jie; Sun, Xianlin; Wu, Wenzhe; Qiu, Yang; Shu, Ting; Zhao, Xiaolu; Yin, Lei; Qin, Cheng-Feng; Hu, Yuanyang; Zhou, Xi

    2015-01-01

    RNA helicases and chaperones are the two major classes of RNA remodeling proteins, which function to remodel RNA structures and/or RNA-protein interactions, and are required for all aspects of RNA metabolism. Although some virus-encoded RNA helicases/chaperones have been predicted or identified, their RNA remodeling activities in vitro and functions in the viral life cycle remain largely elusive. Enteroviruses are a large group of positive-stranded RNA viruses in the Picornaviridae family, which includes numerous important human pathogens. Herein, we report that the nonstructural protein 2CATPase of enterovirus 71 (EV71), which is the major causative pathogen of hand-foot-and-mouth disease and has been regarded as the most important neurotropic enterovirus after poliovirus eradication, functions not only as an RNA helicase that 3′-to-5′ unwinds RNA helices in an adenosine triphosphate (ATP)-dependent manner, but also as an RNA chaperone that destabilizes helices bidirectionally and facilitates strand annealing and complex RNA structure formation independently of ATP. We also determined that the helicase activity is based on the EV71 2CATPase middle domain, whereas the C-terminus is indispensable for its RNA chaperoning activity. By promoting RNA template recycling, 2CATPase facilitated EV71 RNA synthesis in vitro; when 2CATPase helicase activity was impaired, EV71 RNA replication and virion production were mostly abolished in cells, indicating that 2CATPase-mediated RNA remodeling plays a critical role in the enteroviral life cycle. Furthermore, the RNA helicase and chaperoning activities of 2CATPase are also conserved in coxsackie A virus 16 (CAV16), another important enterovirus. Altogether, our findings are the first to demonstrate the RNA helicase and chaperoning activities associated with enterovirus 2CATPase, and our study provides both in vitro and cellular evidence for their potential roles during viral RNA replication. These findings increase our

  3. X-ray structure of the pestivirus NS3 helicase and its conformation in solution.

    Science.gov (United States)

    Tortorici, M Alejandra; Duquerroy, Stéphane; Kwok, Jane; Vonrhein, Clemens; Perez, Javier; Lamp, Benjamin; Bricogne, Gerard; Rümenapf, Till; Vachette, Patrice; Rey, Félix A

    2015-04-01

    Pestiviruses form a genus in the Flaviviridae family of small enveloped viruses with a positive-sense single-stranded RNA genome. Viral replication in this family requires the activity of a superfamily 2 RNA helicase contained in the C-terminal domain of nonstructural protein 3 (NS3). NS3 features two conserved RecA-like domains (D1 and D2) with ATPase activity, plus a third domain (D3) that is important for unwinding nucleic acid duplexes. We report here the X-ray structure of the pestivirus NS3 helicase domain (pNS3h) at a 2.5-Å resolution. The structure deviates significantly from that of NS3 of other genera in the Flaviviridae family in D3, as it contains two important insertions that result in a narrower nucleic acid binding groove. We also show that mutations in pNS3h that rescue viruses from which the core protein is deleted map to D3, suggesting that this domain may be involved in interactions that facilitate particle assembly. Finally, structural comparisons of the enzyme in different crystalline environments, together with the findings of small-angle X-ray-scattering studies in solution, show that D2 is mobile with respect to the rest of the enzyme, oscillating between closed and open conformations. Binding of a nonhydrolyzable ATP analog locks pNS3h in a conformation that is more compact than the closest apo-form in our crystals. Together, our results provide new insight and bring up new questions about pNS3h function during pestivirus replication. Although pestivirus infections impose an important toll on the livestock industry worldwide, little information is available about the nonstructural proteins essential for viral replication, such as the NS3 helicase. We provide here a comparative structural and functional analysis of pNS3h with respect to its orthologs in other viruses of the same family, the flaviviruses and hepatitis C virus. Our studies reveal differences in the nucleic acid binding groove that could have implications for understanding the

  4. Mms1 is an assistant for regulating G-quadruplex DNA structures.

    Science.gov (United States)

    Schwindt, Eike; Paeschke, Katrin

    2017-11-02

    The preservation of genome stability is fundamental for every cell. Genomic integrity is constantly challenged. Among those challenges are also non-canonical nucleic acid structures. In recent years, scientists became aware of the impact of G-quadruplex (G4) structures on genome stability. It has been shown that folded G4-DNA structures cause changes in the cell, such as transcriptional up/down-regulation, replication stalling, or enhanced genome instability. Multiple helicases have been identified to regulate G4 structures and by this preserve genome stability. Interestingly, although these helicases are mostly ubiquitous expressed, they show specificity for G4 regulation in certain cellular processes (e.g., DNA replication). To this date, it is not clear how this process and target specificity of helicases are achieved. Recently, Mms1, an ubiquitin ligase complex protein, was identified as a novel G4-DNA-binding protein that supports genome stability by aiding Pif1 helicase binding to these regions. In this perspective review, we discuss the question if G4-DNA interacting proteins are fundamental for helicase function and specificity at G4-DNA structures.

  5. Genome-wide comparative in silico analysis of the RNA helicase gene family in Zea mays and Glycine max: a comparison with Arabidopsis and Oryza sativa.

    Science.gov (United States)

    Xu, Ruirui; Zhang, Shizhong; Huang, Jinguang; Zheng, Chengchao

    2013-01-01

    RNA helicases are enzymes that are thought to unwind double-stranded RNA molecules in an energy-dependent fashion through the hydrolysis of NTP. RNA helicases are associated with all processes involving RNA molecules, including nuclear transcription, editing, splicing, ribosome biogenesis, RNA export, and organelle gene expression. The involvement of RNA helicase in response to stress and in plant growth and development has been reported previously. While their importance in Arabidopsis and Oryza sativa has been partially studied, the function of RNA helicase proteins is poorly understood in Zea mays and Glycine max. In this study, we identified a total of RNA helicase genes in Arabidopsis and other crop species genome by genome-wide comparative in silico analysis. We classified the RNA helicase genes into three subfamilies according to the structural features of the motif II region, such as DEAD-box, DEAH-box and DExD/H-box, and different species showed different patterns of alternative splicing. Secondly, chromosome location analysis showed that the RNA helicase protein genes were distributed across all chromosomes with different densities in the four species. Thirdly, phylogenetic tree analyses identified the relevant homologs of DEAD-box, DEAH-box and DExD/H-box RNA helicase proteins in each of the four species. Fourthly, microarray expression data showed that many of these predicted RNA helicase genes were expressed in different developmental stages and different tissues under normal growth conditions. Finally, real-time quantitative PCR analysis showed that the expression levels of 10 genes in Arabidopsis and 13 genes in Zea mays were in close agreement with the microarray expression data. To our knowledge, this is the first report of a comparative genome-wide analysis of the RNA helicase gene family in Arabidopsis, Oryza sativa, Zea mays and Glycine max. This study provides valuable information for understanding the classification and putative functions of

  6. Interactive Roles of DNA Helicases and Translocases with the Single-Stranded DNA Binding Protein RPA in Nucleic Acid Metabolism.

    Science.gov (United States)

    Awate, Sanket; Brosh, Robert M

    2017-06-08

    Helicases and translocases use the energy of nucleoside triphosphate binding and hydrolysis to unwind/resolve structured nucleic acids or move along a single-stranded or double-stranded polynucleotide chain, respectively. These molecular motors facilitate a variety of transactions including replication, DNA repair, recombination, and transcription. A key partner of eukaryotic DNA helicases/translocases is the single-stranded DNA binding protein Replication Protein A (RPA). Biochemical, genetic, and cell biological assays have demonstrated that RPA interacts with these human molecular motors physically and functionally, and their association is enriched in cells undergoing replication stress. The roles of DNA helicases/translocases are orchestrated with RPA in pathways of nucleic acid metabolism. RPA stimulates helicase-catalyzed DNA unwinding, enlists translocases to sites of action, and modulates their activities in DNA repair, fork remodeling, checkpoint activation, and telomere maintenance. The dynamic interplay between DNA helicases/translocases and RPA is just beginning to be understood at the molecular and cellular levels, and there is still much to be learned, which may inform potential therapeutic strategies.

  7. Access to essential medicines for sexual and reproductive health care: the role of the pharmaceutical industry and international regulation.

    Science.gov (United States)

    Cottingham, Jane; Berer, Marge

    2011-11-01

    The range of medicines and technologies that are essential for sexual and reproductive health care is well established, but access to them is far from universally assured, particularly in less developed countries. This paper shows how the pharmaceutical industry plays a major role in the lack of access to essential medicines for sexual and reproductive health care, by a) investing in products for profit-making reasons despite their negative health impact (e.g. hormone replacement therapy), b) marketing new essential medicines at prices beyond the reach of countries that most need them (e.g. HPV vaccines), and c) failing to invest in the development of new products (e.g. microbicides and medical abortion pills). Small companies, some of them non-profit-making, struggle to fill some of that demand (e.g. for female condoms). International patent protection contributes to high prices of medicines, and while international agreements such as compulsory licensing under TRIPS and the Medicines Patent Pool allow for mechanisms to enable poorer countries to get access to essential medicines, the obstacles created by "big pharma" are daunting. All these barriers have fostered a market in sub-standard medicines (e.g. fake medical abortion pills sold over the internet). An agenda driven by sexual and reproductive health needs, based on the right to health, must focus on universal access to essential medicines at prices developing countries can afford. We call for greater public investment in essential medicines, expanded production of affordable generic drugs, and the development of broad strategic plans, that include affordable medicines and technologies, for addressing identified public health problems, such as cervical cancer. Copyright © 2011 Reproductive Health Matters. Published by Elsevier Ltd. All rights reserved.

  8. Deregulation of the Building Code and the Norwegian Approach to Regulation of Accessibility in the Built Environment.

    Science.gov (United States)

    Lyngstad, Pål

    2016-01-01

    Deregulation is on the political agenda in the European countries. The Norwegian building code related to universal design and accessibility is challenged. To meet this, the Norwegian Building Authority have chosen to examine established truths and are basing their revised code on scientific research and field tests. But will this knowledge-based deregulation comply within the framework of the anti-discrimination act and, and if not: who suffers and to what extent?

  9. Regulations To Be Made In Urban Areas In Order To Improve Accessibility Of The “Visually-Impaired

    Directory of Open Access Journals (Sweden)

    Pelin Gökgür

    2014-06-01

    Full Text Available In our country, disabled people encounter many problems in integrating with the city and city life. The most important one is the "accessibility" in the physical space. Providing accessibility require disabled to move without being in need of any help, without any prevention and to participate in the life activities. It is seen in our country that the obstacle-free walkways for the disabled do not exist in the cities including the pedestrian areas, the legal arrangements with respect thereto are not yet able to achieve the required conditions in the physical environment, and the required sanctions and controls are not sufficient. Moreover, inability to present accurately the difference in the needs as based on the disability type is exposed in the problems that occur in the arrangements realized. The basic needs of "visually-impaired" people in the urban space, which constitutes the subject matter of the study can be listed as; safety, to understand where s/he is going, to know where s/he is and to obtain information about the surrounding. For the visually-impaired people to obtain such needs depends on their ability to move alone in the urban space. One of the most important issues in achieving this is "orientation". "Orientation" involves the process of determination of the person's own position and relation with the important objects around. It is known that the "accessibility" of a visually-impaired person is based on orientation and the arrangements in the physical environment in achieving this are very important. In this study, the purpose is to present the significance of "orientation" directed to increase the accessibility of the "visually-impaired" people in the urban space and as based on this, to discuss "the arrangements required to be realized in the physical environment”.

  10. Tet2 and Tet3 cooperate with B-lineage transcription factors to regulate DNA modification and chromatin accessibility.

    Science.gov (United States)

    Lio, Chan-Wang; Zhang, Jiayuan; González-Avalos, Edahí; Hogan, Patrick G; Chang, Xing; Rao, Anjana

    2016-11-21

    Ten-eleven translocation (TET) enzymes oxidize 5-methylcytosine, facilitating DNA demethylation and generating new epigenetic marks. Here we show that concomitant loss of Tet2 and Tet3 in mice at early B cell stage blocked the pro- to pre-B cell transition in the bone marrow, decreased Irf4 expression and impaired the germline transcription and rearrangement of the Igκ locus. Tet2/3-deficient pro-B cells showed increased CpG methylation at the Igκ 3' and distal enhancers that was mimicked by depletion of E2A or PU.1, as well as a global decrease in chromatin accessibility at enhancers. Importantly, re-expression of the Tet2 catalytic domain in Tet2/3-deficient B cells resulted in demethylation of the Igκ enhancers and restored their chromatin accessibility. Our data suggest that TET proteins and lineage-specific transcription factors cooperate to influence chromatin accessibility and Igκ enhancer function by modulating the modification status of DNA.

  11. The AAA-ATPase NVL2 is a component of pre-ribosomal particles that interacts with the DExD/H-box RNA helicase DOB1

    International Nuclear Information System (INIS)

    Nagahama, Masami; Yamazoe, Takeshi; Hara, Yoshimitsu; Tani, Katsuko; Tsuji, Akihiko; Tagaya, Mitsuo

    2006-01-01

    Nuclear VCP/p97-like protein 2 (NVL2) is a member of the chaperone-like AAA-ATPase family with two conserved ATP-binding modules. Our previous studies have shown that NVL2 is localized to the nucleolus by interacting with ribosomal protein L5 and may participate in ribosome synthesis, a process involving various non-ribosomal factors including chaperones and RNA helicases. Here, we show that NVL2 is associated with pre-ribosomal particles in the nucleus. Moreover, we used yeast two-hybrid and co-immunoprecipitation assays to identify an NVL2-interacting protein that could yield insights into NVL2 function in ribosome biogenesis. We found that NVL2 interacts with DOB1, a DExD/H-box RNA helicase, whose yeast homologue functions in a late stage of the 60S subunit synthesis. DOB1 can interact with a second ATP-binding module mutant of NVL2, which shows a dominant negative effect on ribosome synthesis. In contrast, it cannot interact with a first ATP-binding module mutant, which does not show the dominant negative effect. When the dominant negative mutant of NVL2 was overexpressed in cells, DOB1 appeared to remain associated with nuclear pre-ribosomal particles. Such accumulation was not observed upon overexpression of wild-type NVL2 or a nondominant-negative mutant. Taken together, our results suggest that NVL2 might regulate the association/dissociation reaction of DOB1 with pre-ribosomal particles by acting as a molecular chaperone

  12. Pea p68, a DEAD-box helicase, provides salinity stress tolerance in transgenic tobacco by reducing oxidative stress and improving photosynthesis machinery.

    Science.gov (United States)

    Tuteja, Narendra; Banu, Mst Sufara Akhter; Huda, Kazi Md Kamrul; Gill, Sarvajeet Singh; Jain, Parul; Pham, Xuan Hoi; Tuteja, Renu

    2014-01-01

    The DEAD-box helicases are required mostly in all aspects of RNA and DNA metabolism and they play a significant role in various abiotic stresses, including salinity. The p68 is an important member of the DEAD-box proteins family and, in animal system, it is involved in RNA metabolism including pre-RNA processing and splicing. In plant system, it has not been well characterized. Here we report the cloning and characterization of p68 from pea (Pisum sativum) and its novel function in salinity stress tolerance in plant. The pea p68 protein self-interacts and is localized in the cytosol as well as the surrounding of cell nucleus. The transcript of pea p68 is upregulated in response to high salinity stress in pea. Overexpression of p68 driven by constitutive cauliflower mosaic virus-35S promoter in tobacco transgenic plants confers enhanced tolerances to salinity stress by improving the growth, photosynthesis and antioxidant machinery. Under stress treatment, pea p68 overexpressing tobacco accumulated higher K+ and lower Na+ level than the wild-type plants. Reactive oxygen species (ROS) accumulation was remarkably regulated by the overexpression of pea p68 under salinity stress conditions, as shown from TBARS content, electrolyte leakage, hydrogen peroxide accumulation and 8-OHdG content and antioxidant enzyme activities. To the best of our knowledge this is the first direct report, which provides the novel function of pea p68 helicase in salinity stress tolerance. The results suggest that p68 can also be exploited for engineering abiotic stress tolerance in crop plants of economic importance.

  13. Pea p68, a DEAD-box helicase, provides salinity stress tolerance in transgenic tobacco by reducing oxidative stress and improving photosynthesis machinery.

    Directory of Open Access Journals (Sweden)

    Narendra Tuteja

    Full Text Available The DEAD-box helicases are required mostly in all aspects of RNA and DNA metabolism and they play a significant role in various abiotic stresses, including salinity. The p68 is an important member of the DEAD-box proteins family and, in animal system, it is involved in RNA metabolism including pre-RNA processing and splicing. In plant system, it has not been well characterized. Here we report the cloning and characterization of p68 from pea (Pisum sativum and its novel function in salinity stress tolerance in plant.The pea p68 protein self-interacts and is localized in the cytosol as well as the surrounding of cell nucleus. The transcript of pea p68 is upregulated in response to high salinity stress in pea. Overexpression of p68 driven by constitutive cauliflower mosaic virus-35S promoter in tobacco transgenic plants confers enhanced tolerances to salinity stress by improving the growth, photosynthesis and antioxidant machinery. Under stress treatment, pea p68 overexpressing tobacco accumulated higher K+ and lower Na+ level than the wild-type plants. Reactive oxygen species (ROS accumulation was remarkably regulated by the overexpression of pea p68 under salinity stress conditions, as shown from TBARS content, electrolyte leakage, hydrogen peroxide accumulation and 8-OHdG content and antioxidant enzyme activities.To the best of our knowledge this is the first direct report, which provides the novel function of pea p68 helicase in salinity stress tolerance. The results suggest that p68 can also be exploited for engineering abiotic stress tolerance in crop plants of economic importance.

  14. Conserved helicase domain of human RecQ4 is required for strand annealing-independent DNA unwinding

    DEFF Research Database (Denmark)

    Rossi, Marie L; Ghosh, Avik K; Kulikowicz, Tomasz

    2010-01-01

    Humans have five members of the well conserved RecQ helicase family: RecQ1, Bloom syndrome protein (BLM), Werner syndrome protein (WRN), RecQ4, and RecQ5, which are all known for their roles in maintaining genome stability. BLM, WRN, and RecQ4 are associated with premature aging and cancer...... provide the first evidence that human RecQ4's unwinding is independent of strand annealing, and that it does not require the presence of excess ssDNA. Moreover, we demonstrate that a point mutation of the conserved lysine in the Walker A motif abolished helicase activity, implying that not the N...... activities and protein partners of RecQ4 are conserved with those of the other RecQ helicases....

  15. MCM-BP regulates unloading of the MCM2–7 helicase in late S phase

    Science.gov (United States)

    Nishiyama, Atsuya; Frappier, Lori; Méchali, Marcel

    2011-01-01

    Origins of DNA replication are licensed by recruiting MCM2–7 to assemble the prereplicative complex (pre-RC). How MCM2–7 is inactivated or removed from chromatin at the end of S phase is still unclear. Here, we show that MCM-BP can disassemble the MCM2–7 complex and might function as an unloader of MCM2–7 from chromatin. In Xenopus egg extracts, MCM-BP exists in a stable complex with MCM7, but is not associated with the MCM2–7 hexameric complex. MCM-BP accumulates in nuclei in late S phase, well after the loading of MCM2–7 onto chromatin. MCM-BP immunodepletion in Xenopus egg extracts inhibits replication-dependent MCM dissociation without affecting pre-RC formation and DNA replication. When excess MCM-BP is incubated with Xenopus egg extracts or immunopurified MCM2–7, it binds to MCM proteins and promotes disassembly of the MCM2–7 complex. Recombinant MCM-BP also releases MCM2–7 from isolated late-S-phase chromatin, but this activity is abolished when DNA replication is blocked. MCM-BP silencing in human cells also delays MCM dissociation in late S phase. We propose that MCM-BP plays a key role in the mechanism by which pre-RC is cleared from replicated DNA in vertebrate cells. PMID:21196493

  16. The Q Motif Is Involved in DNA Binding but Not ATP Binding in ChlR1 Helicase.

    Directory of Open Access Journals (Sweden)

    Hao Ding

    Full Text Available Helicases are molecular motors that couple the energy of ATP hydrolysis to the unwinding of structured DNA or RNA and chromatin remodeling. The conversion of energy derived from ATP hydrolysis into unwinding and remodeling is coordinated by seven sequence motifs (I, Ia, II, III, IV, V, and VI. The Q motif, consisting of nine amino acids (GFXXPXPIQ with an invariant glutamine (Q residue, has been identified in some, but not all helicases. Compared to the seven well-recognized conserved helicase motifs, the role of the Q motif is less acknowledged. Mutations in the human ChlR1 (DDX11 gene are associated with a unique genetic disorder known as Warsaw Breakage Syndrome, which is characterized by cellular defects in genome maintenance. To examine the roles of the Q motif in ChlR1 helicase, we performed site directed mutagenesis of glutamine to alanine at residue 23 in the Q motif of ChlR1. ChlR1 recombinant protein was overexpressed and purified from HEK293T cells. ChlR1-Q23A mutant abolished the helicase activity of ChlR1 and displayed reduced DNA binding ability. The mutant showed impaired ATPase activity but normal ATP binding. A thermal shift assay revealed that ChlR1-Q23A has a melting point value similar to ChlR1-WT. Partial proteolysis mapping demonstrated that ChlR1-WT and Q23A have a similar globular structure, although some subtle conformational differences in these two proteins are evident. Finally, we found ChlR1 exists and functions as a monomer in solution, which is different from FANCJ, in which the Q motif is involved in protein dimerization. Taken together, our results suggest that the Q motif is involved in DNA binding but not ATP binding in ChlR1 helicase.

  17. Mutational analysis of an archaeal minichromosome maintenance protein exterior hairpin reveals critical residues for helicase activity and DNA binding

    Directory of Open Access Journals (Sweden)

    Brewster Aaron S

    2010-08-01

    Full Text Available Abstract Background The mini-chromosome maintenance protein (MCM complex is an essential replicative helicase for DNA replication in Archaea and Eukaryotes. While the eukaryotic complex consists of six homologous proteins (MCM2-7, the archaeon Sulfolobus solfataricus has only one MCM protein (ssoMCM, six subunits of which form a homohexamer. We have recently reported a 4.35Å crystal structure of the near full-length ssoMCM. The structure reveals a total of four β-hairpins per subunit, three of which are located within the main channel or side channels of the ssoMCM hexamer model generated based on the symmetry of the N-terminal Methanothermobacter thermautotrophicus (mtMCM structure. The fourth β-hairpin, however, is located on the exterior of the hexamer, near the exit of the putative side channels and next to the ATP binding pocket. Results In order to better understand this hairpin's role in DNA binding and helicase activity, we performed a detailed mutational and biochemical analysis of nine residues on this exterior β-hairpin (EXT-hp. We examined the activities of the mutants related to their helicase function, including hexamerization, ATPase, DNA binding and helicase activities. The assays showed that some of the residues on this EXT-hp play a role for DNA binding as well as for helicase activity. Conclusions These results implicate several current theories regarding helicase activity by this critical hexameric enzyme. As the data suggest that EXT-hp is involved in DNA binding, the results reported here imply that the EXT-hp located near the exterior exit of the side channels may play a role in contacting DNA substrate in a manner that affects DNA unwinding.

  18. Structural and functional analysis of the human spliceosomal DEAD-box helicase Prp28

    Energy Technology Data Exchange (ETDEWEB)

    Möhlmann, Sina [Georg-August-University Göttingen, Justus-von-Liebig Weg 11, 37077 Göttingen (Germany); Mathew, Rebecca [Max-Planck-Institute for Biophysical Chemistry, Am Fassberg, 37077 Göttingen (Germany); Neumann, Piotr; Schmitt, Andreas [Georg-August-University Göttingen, Justus-von-Liebig Weg 11, 37077 Göttingen (Germany); Lührmann, Reinhard [Max-Planck-Institute for Biophysical Chemistry, Am Fassberg, 37077 Göttingen (Germany); Ficner, Ralf, E-mail: rficner@uni-goettingen.de [Georg-August-University Göttingen, Justus-von-Liebig Weg 11, 37077 Göttingen (Germany)

    2014-06-01

    The crystal structure of the helicase domain of the human spliceosomal DEAD-box protein Prp28 was solved by SAD. The binding of ADP and ATP by Prp28 was studied biochemically and analysed with regard to the crystal structure. The DEAD-box protein Prp28 is essential for pre-mRNA splicing as it plays a key role in the formation of an active spliceosome. Prp28 participates in the release of the U1 snRNP from the 5′-splice site during association of the U5·U4/U6 tri-snRNP, which is a crucial step in the transition from a pre-catalytic spliceosome to an activated spliceosome. Here, it is demonstrated that the purified helicase domain of human Prp28 (hPrp28ΔN) binds ADP, whereas binding of ATP and ATPase activity could not be detected. ATP binding could not be observed for purified full-length hPrp28 either, but within an assembled spliceosomal complex hPrp28 gains ATP-binding activity. In order to understand the structural basis for the ATP-binding deficiency of isolated hPrp28, the crystal structure of hPrp28ΔN was determined at 2.0 Å resolution. In the crystal the helicase domain adopts a wide-open conformation, as the two RecA-like domains are extraordinarily displaced from the productive ATPase conformation. Binding of ATP is hindered by a closed conformation of the P-loop, which occupies the space required for the γ-phosphate of ATP.

  19. In vivo mapping of the functional regions of the DEAD-box helicase Vasa

    Directory of Open Access Journals (Sweden)

    Mehrnoush Dehghani

    2015-03-01

    Full Text Available The maternally expressed Drosophila melanogaster DEAD-box helicase Vasa (Vas is necessary for many cellular and developmental processes, including specification of primordial germ cells (pole cells, posterior patterning of the embryo, piRNA-mediated repression of transposon-encoded mRNAs, translational activation of gurken (grk mRNA, and completion of oogenesis itself. Vas protein accumulates in the perinuclear nuage in nurse cells soon after their specification, and then at stage 10 Vas translocates to the posterior pole plasm of the oocyte. We produced a series of transgenic constructs encoding eGFP-Vas proteins carrying mutations affecting different regions of the protein, and analyzed in vivo which Vas functions each could support. We identified novel domains in the N- and C-terminal regions of the protein that are essential for localization, transposon repression, posterior patterning, and pole cell specification. One such functional region, the most C-terminal seven amino acids, is specific to Vas orthologues and is thus critical to distinguishing Vas from other closely related DEAD-box helicases. Surprisingly, we also found that many eGFP-Vas proteins carrying mutations that would be expected to abrogate DEAD-box helicase function localized to the nuage and posterior pole, and retained the capacity to support oogenesis, although they did not function in embryonic patterning, pole cell specification, grk activation, or transposon repression. We conclude from these experiments that Vas, a multifunctional protein, uses different domains and different molecular associations to carry out its various cellular and developmental roles.

  20. Functional interaction between Smad, CREB binding protein, and p68 RNA helicase

    International Nuclear Information System (INIS)

    Warner, Dennis R.; Bhattacherjee, Vasker; Yin, Xiaolong; Singh, Saurabh; Mukhopadhyay, Partha; Pisano, M. Michele; Greene, Robert M.

    2004-01-01

    The transforming growth factors β control a diversity of biological processes including cellular proliferation, differentiation, apoptosis, and extracellular matrix production, and are critical effectors of embryonic patterning and development, including that of the orofacial region. TGFβ superfamily members signal through specific cell surface receptors that phosphorylate the cytoplasmic Smad proteins, resulting in their translocation to the nucleus and interaction with promoters of TGFβ-responsive genes. Subsequent alterations in transcription are cell type-specific and dependent on recruitment to the Smad/transcription factor complex of coactivators, such as CBP and p300, or corepressors, such as c-ski and SnoN. Since the affinity of Smads for DNA is generally low, additional accessory proteins that facilitate Smad/DNA binding are required, and are often cell- and tissue-specific. In order to identify novel Smad 3 binding proteins in developing orofacial tissue, a yeast two hybrid assay was employed in which the MH2 domain of Smad 3 was used to screen an expression library derived from mouse embryonic orofacial tissue. The RNA helicase, p68, was identified as a unique Smad binding protein, and the specificity of the interaction was confirmed through various in vitro and in vivo assays. Co-expression of Smad 3 and a CBP-Gal4 DNA binding domain fusion protein in a Gal4-luciferase reporter assay resulted in increased TGFβ-stimulated reporter gene transcription. Moreover, co-expression of p68 RNA helicase along with Smad 3 and CBP-Gal4 resulted in synergistic activation of Gal4-luciferase reporter expression. Collectively, these data indicate that the RNA helicase, p68, can directly interact with Smad 3 resulting in formation of a transcriptionally active ternary complex containing Smad 3, p68, and CBP. This offers a means of enhancing TGFβ-mediated cellular responses in developing orofacial tissue

  1. Deficiency of the Arabidopsis helicase RTEL1 triggers a SOG1-dependent replication checkpoint in response to DNA cross-links.

    Science.gov (United States)

    Hu, Zhubing; Cools, Toon; Kalhorzadeh, Pooneh; Heyman, Jefri; De Veylder, Lieven

    2015-01-01

    To maintain genome integrity, DNA replication is executed and regulated by a complex molecular network of numerous proteins, including helicases and cell cycle checkpoint regulators. Through a systematic screening for putative replication mutants, we identified an Arabidopsis thaliana homolog of human Regulator of Telomere Length 1 (RTEL1), which functions in DNA replication, DNA repair, and recombination. RTEL1 deficiency retards plant growth, a phenotype including a prolonged S-phase duration and decreased cell proliferation. Genetic analysis revealed that rtel1 mutant plants show activated cell cycle checkpoints, specific sensitivity to DNA cross-linking agents, and increased homologous recombination, but a lack of progressive shortening of telomeres, indicating that RTEL1 functions have only been partially conserved between mammals and plants. Surprisingly, RTEL1 deficiency induces tolerance to the deoxynucleotide-depleting drug hydroxyurea, which could be mimicked by DNA cross-linking agents. This resistance does not rely on the essential replication checkpoint regulator WEE1 but could be blocked by a mutation in the SOG1 transcription factor. Taken together, our data indicate that RTEL1 is required for DNA replication and that its deficiency activates a SOG1-dependent replication checkpoint. © 2015 American Society of Plant Biologists. All rights reserved.

  2. Yeast as a model system to study RecQ helicase function

    DEFF Research Database (Denmark)

    Ashton, Thomas M; Hickson, Ian David

    2010-01-01

    Mutations in the highly conserved RecQ helicase, BLM, cause the rare cancer predisposition disorder, Bloom's syndrome. The orthologues of BLM in Saccharomyces cerevisiae and Schizosaccharomyces pombe are SGS1 and rqh1(+), respectively. Studies in these yeast species have revealed a plethora...... of roles for the Sgs1 and Rqh1 proteins in repair of double strand breaks, restart of stalled replication forks, processing of aberrant intermediates that arise during meiotic recombination, and maintenance of telomeres. In this review, we focus on the known roles of Sgs1 and Rqh1 and how studies in yeast...

  3. New roles of the human Suv3 helicase in genome maintenance

    DEFF Research Database (Denmark)

    Venø, Susanne Trillingsgaard

    During her PhD studies, Susanne Trillingsgaard Venø carried out research into the role of the human Suv3 protein in stabilising the human genome – DNA. Suv3 is a helicase that separates the two strands of the DNA’s double helix. Throughout our lives, the DNA in our cells is constantly exposed...... maintenance. Based on these new research results, the Suv3 protein could be a valuable model for genome stability as an important factor in our understanding of why we get old....

  4. The helicase and ATPase activities of RECQL4 are compromised by mutations reported in three human patients

    DEFF Research Database (Denmark)

    Jensen, Martin Borch; Dunn, Christopher A; Keijzers, Guido

    2012-01-01

    RECQL4 is one of five members of the human RecQ helicase family, and is implicated in three syndromes displaying accelerating aging, developmental abnormalities and a predisposition to cancer. In this study, we purified three variants of RECQL4 carrying previously reported patient mutations....... These three mutant proteins were analyzed for the known biochemical activities of RECQL4: DNA binding, unwinding of duplex DNA, ATP hydrolysis and annealing of simplex DNA. Further, the mutant proteins were evaluated for stability and recruitment to sites of laser-induced DNA damage. One mutant was helicase...

  5. Structure of the US gas market, regulation and pricing of the third party access to the network

    International Nuclear Information System (INIS)

    David, L.; Mirabel, F.

    2000-06-01

    The opening of the US natural gas market to competition is followed by deep changes in the implemented regulations for an efficient organization and operation of the gas industry. The transformations of the industrial structures and the strategic behavior of the actors characterize the mutations of the gas markets in the USA. The aim of this paper is to transcribe in a synthetic form the characteristics of the gas market in North America and to model the structure of the industry in this sector opened to competition in its back-end. Two main questions are analyzed: the role and position of the former monopoly (utility) in this back-end market (should the local distribution companies continue to sell gas to end users?), and what kind of regulation should be implemented by the authorities in order to avoid the former monopoly to use its market power and make excessive profits. The analysis is made in two steps: the first part explains the hypotheses of the analysis using the characteristics of the North American market. It presents the general framework of the model and explains the two 'regulatory scenarios' retained in the study. The main steps of the resolution of the model are detailed in the second part. (J.S.)

  6. The adnAB Locus, Encoding a Putative Helicase-Nuclease Activity, Is Essential in Streptomyces

    Science.gov (United States)

    Zhang, Lingli; Nguyen, Hoang Chuong; Chipot, Ludovic; Piotrowski, Emilie; Bertrand, Claire

    2014-01-01

    Homologous recombination is a crucial mechanism that repairs a wide range of DNA lesions, including the most deleterious ones, double-strand breaks (DSBs). This multistep process is initiated by the resection of the broken DNA ends by a multisubunit helicase-nuclease complex exemplified by Escherichia coli RecBCD, Bacillus subtilis AddAB, and newly discovered Mycobacterium tuberculosis AdnAB. Here we show that in Streptomyces, neither recBCD nor addAB homologues could be detected. The only putative helicase-nuclease-encoding genes identified were homologous to M. tuberculosis adnAB genes. These genes are conserved as a single copy in all sequenced genomes of Streptomyces. The disruption of adnAB in Streptomyces ambofaciens and Streptomyces coelicolor could not be achieved unless an ectopic copy was provided, indicating that adnAB is essential for growth. Both adnA and adnB genes were shown to be inducible in response to DNA damage (mitomycin C) and to be independently transcribed. Introduction of S. ambofaciens adnAB genes in an E. coli recB mutant restored viability and resistance to UV light, suggesting that Streptomyces AdnAB could be a functional homologue of RecBCD and be involved in DNA damage resistance. PMID:24837284

  7. Real-time electrochemical monitoring of isothermal helicase-dependent amplification of nucleic acids.

    Science.gov (United States)

    Kivlehan, Francine; Mavré, François; Talini, Luc; Limoges, Benoît; Marchal, Damien

    2011-09-21

    We described an electrochemical method to monitor in real-time the isothermal helicase-dependent amplification of nucleic acids. The principle of detection is simple and well-adapted to the development of portable, easy-to-use and inexpensive nucleic acids detection technologies. It consists of monitoring a decrease in the electrochemical current response of a reporter DNA intercalating redox probe during the isothermal DNA amplification. The method offers the possibility to quantitatively analyze target nucleic acids in less than one hour at a single constant temperature, and to perform at the end of the isothermal amplification a DNA melt curve analysis for differentiating between specific and non-specific amplifications. To illustrate the potentialities of this approach for the development of a simple, robust and low-cost instrument with high throughput capability, the method was validated with an electrochemical system capable of monitoring up to 48 real-time isothermal HDA reactions simultaneously in a disposable microplate consisting of 48-electrochemical microwells. Results obtained with this approach are comparable to that obtained with a well-established but more sophisticated and expensive fluorescence-based method. This makes for a promising alternative detection method not only for real-time isothermal helicase-dependent amplification of nucleic acid, but also for other isothermal DNA amplification strategies.

  8. Characterization of the Caenorhabditis elegans HIM-6/BLM helicase: unwinding recombination intermediates.

    Science.gov (United States)

    Jung, Hana; Lee, Jin A; Choi, Seoyoon; Lee, Hyunwoo; Ahn, Byungchan

    2014-01-01

    Mutations in three human RecQ genes are implicated in heritable human syndromes. Mutations in BLM, a RecQ gene, cause Bloom syndrome (BS), which is characterized by short stature, cancer predisposition, and sensitivity to sunlight. BLM is a RecQ DNA helicase that, with interacting proteins, is able to dissolve various DNA structures including double Holliday junctions. A BLM ortholog, him-6, has been identified in Caenorhabditis elegans, but little is known about its enzymatic activities or its in vivo roles. By purifying recombinant HIM-6 and performing biochemical assays, we determined that the HIM-6 has DNA-dependent ATPase activity HIM-6 and helicase activity that proceeds in the 3'-5' direction and needs at least five 3' overhanging nucleotides. HIM-6 is also able to unwind DNA structures including D-loops and Holliday junctions. Worms with him-6 mutations were defective in recovering the cell cycle arrest after HU treatment. These activities strongly support in vivo roles for HIM-6 in processing recombination intermediates.

  9. Single molecule measurements of DNA helicase activity with magnetic tweezers and t-test based step-finding analysis

    Science.gov (United States)

    Seol, Yeonee; Strub, Marie-Paule; Neuman, Keir C.

    2016-01-01

    Magnetic tweezers is a versatile and easy to implement single-molecule technique that has become increasingly prevalent in the study of nucleic acid based molecular motors. Here, we provide a description of the magnetic tweezers instrument and guidelines for measuring and analyzing DNA helicase activity. Along with experimental methods, we describe a robust method of single-molecule trajectory analysis based on the Student’s t-test that accommodates continuous transitions in addition to the discrete transitions assumed in most widely employed analysis routines. To illustrate the single-molecule unwinding assay and the analysis routine, we provide DNA unwinding measurements of Escherichia coli RecQ helicase under a variety of conditions (Na+, ATP, temperature, and DNA substrate geometry). These examples reveal that DNA unwinding measurements under various conditions can aid in elucidating the unwinding mechanism of DNA helicase but also emphasize that environmental effects on DNA helicase activity must be considered in relation to in vivo activity and mechanism. PMID:27131595

  10. Acute inactivation of the replicative helicase in human cells triggers MCM8-9-dependent DNA synthesis

    DEFF Research Database (Denmark)

    Natsume, Toyoaki; Nishimura, Kohei; Minocherhomji, Sheroy

    2017-01-01

    stemming from replisome dissociation during DNA replication perturbation, we used a degron-based system for inducible proteolysis of a subunit of the replicative helicase. We show that MCM2-depleted cells activate a DNA damage response pathway and generate replication-associated DNA double-strand breaks...

  11. Circadian regulation of myocardial sarcomeric Titin-cap (Tcap, telethonin: identification of cardiac clock-controlled genes using open access bioinformatics data.

    Directory of Open Access Journals (Sweden)

    Peter S Podobed

    Full Text Available Circadian rhythms are important for healthy cardiovascular physiology and are regulated at the molecular level by a circadian clock mechanism. We and others previously demonstrated that 9-13% of the cardiac transcriptome is rhythmic over 24 h daily cycles; the heart is genetically a different organ day versus night. However, which rhythmic mRNAs are regulated by the circadian mechanism is not known. Here, we used open access bioinformatics databases to identify 94 transcripts with expression profiles characteristic of CLOCK and BMAL1 targeted genes, using the CircaDB website and JTK_Cycle. Moreover, 22 were highly expressed in the heart as determined by the BioGPS website. Furthermore, 5 heart-enriched genes had human/mouse conserved CLOCK:BMAL1 promoter binding sites (E-boxes, as determined by UCSC table browser, circadian mammalian promoter/enhancer database PEDB, and the European Bioinformatics Institute alignment tool (EMBOSS. Lastly, we validated findings by demonstrating that Titin cap (Tcap, telethonin was targeted by transcriptional activators CLOCK and BMAL1 by showing 1 Tcap mRNA and TCAP protein had a diurnal rhythm in murine heart; 2 cardiac Tcap mRNA was rhythmic in animals kept in constant darkness; 3 Tcap and control Per2 mRNA expression and cyclic amplitude were blunted in Clock(Δ19/Δ19 hearts; 4 BMAL1 bound to the Tcap promoter by ChIP assay; 5 BMAL1 bound to Tcap promoter E-boxes by biotinylated oligonucleotide assay; and 6 CLOCK and BMAL1 induced tcap expression by luciferase reporter assay. Thus this study identifies circadian regulated genes in silico, with validation of Tcap, a critical regulator of cardiac Z-disc sarcomeric structure and function.

  12. Study on the rules and practices in force abroad regarding the delimitation and access to regulated areas for radiation protection purposes. Final report - Appendices. Report no. 313

    International Nuclear Information System (INIS)

    Schieber, C.; Crouail, P.; Beltrami, L.A.; Reaud, C.

    2013-06-01

    Within the frame of a reviewing of European requirements in the field of radiation protection, this report presents a synthesis of rules which are applicable in seven countries (Belgium, Spain, United States, Finland, United-Kingdom, Sweden and Switzerland; a detailed report for each of these countries is provided in appendix) in the field of delimitation and access to regulated areas for radiation protection purposes. A synthesis is proposed for each country. Three countries are then selected (Finland, United Kingdom and Switzerland) and their rules and practices are applied to cases which correspond to representative exposure situations: a room with a glove box, a hall with hot spots, a parking of temporary warehousing (packages of used fuels), intermittent use of an X ray generator, use of an intense radiation beam, workshop being dismantled, laboratory in which radioactive iodine 131 is handled, and use of a mobile industrial radiography device

  13. Hydrology in Lichens: How Biological Architecture is Used to Regulate Water Access to Support Drought Resilience and Nutrient Transport

    Science.gov (United States)

    Ten Veldhuis, M. C.; Dismukes, G. C.; Ananyev, G.

    2017-12-01

    Lichens are Nature's masters at controlling water and air flux within a symbiotic organism comprised of an algal photobiont and its fungal host. Here we investigated the equilibrium partitioning and kinetic transport of water between the symbionts in the lichen flavoparmelia species. Lichens have developed a unique strategy to recover after deep dehydration, that otherwise would kill the majority of free living phototrophs. By measuring both kinetics of water content and chlorophyll fluorescence emission (indicative of algal charge separation and water oxidation) during dehydration, we identified 3 distinct temporal stages and mapped these to physical zones by confocal microscopy using a combination of hydro-philic/-phobic dyes. Below a critical level of water content, controlled by the greater hydrophilicity of fungal tissues, algal photosynthesis rapidly turns off. We show that the distinct stages in dehydration mirror the 3D architecture of lichen tissue (the thallus). We provide evidence that control of water distribution is achieved by capillary forces within ordered zones of physical space possessing different hydro-phobic/-philic characteristics. This strategy ensures that photosynthetic capacity is protected from and can quickly recover after desiccation. The fungal host controls the onset and extent of photosynthesis in the enslaved alga, presumably to ensure transport of algal derived sugars and oxygen (O2) to the fungal host only when sufficient water exists for transport. Lichen architecture provides Nature's solution to gas-water transport that is self-regulated by humidity. It offers novel lessons for designing practical devices such as fuel cell membranes and dialysis membranes. Supported by the US Dept of Energy, Basic Energy Sciences, Physical Biosciences Division.

  14. Regulation

    International Nuclear Information System (INIS)

    Ballereau, P.

    1999-01-01

    The different regulations relative to nuclear energy since the first of January 1999 are given here. Two points deserve to be noticed: the decree of the third august 1999 authorizing the national Agency for the radioactive waste management to install and exploit on the commune of Bures (Meuse) an underground laboratory destined to study the deep geological formations where could be stored the radioactive waste. The second point is about the uranium residues and the waste notion. The judgment of the administrative tribunal of Limoges ( 9. july 1998) forbidding the exploitation of a storage installation of depleted uranium considered as final waste and qualifying it as an industrial waste storage facility has been annulled bu the Court of Appeal. It stipulated that, according to the law number 75663 of the 15. july 1965, no criteria below can be applied to depleted uranium: production residue (possibility of an ulterior enrichment), abandonment of a personal property or simple intention to do it ( future use aimed in the authorization request made in the Prefecture). This judgment has devoted the primacy of the waste notion on this one of final waste. (N.C.)

  15. Substrate-assisted mechanism of RNP disruption by the spliceosomal Brr2 RNA helicase

    Science.gov (United States)

    Theuser, Matthias; Höbartner, Claudia; Wahl, Markus C.; Santos, Karine F.

    2016-01-01

    The Brr2 RNA helicase disrupts the U4/U6 di-small nuclear RNA–protein complex (di-snRNP) during spliceosome activation via ATP-driven translocation on the U4 snRNA strand. However, it is unclear how bound proteins influence U4/U6 unwinding, which regions of the U4/U6 duplex the helicase actively unwinds, and whether U4/U6 components are released as individual molecules or as subcomplexes. Here, we set up a recombinant Brr2-mediated U4/U6 di-snRNP disruption system, showing that sequential addition of the U4/U6 proteins small nuclear ribonucleoprotein-associated protein 1 (Snu13), pre-mRNA processing factor 31 (Prp31), and Prp3 to U4/U6 di-snRNA leads to a stepwise decrease of Brr2-mediated U4/U6 unwinding, but that unwinding is largely restored by a Brr2 cofactor, the C-terminal Jab1/MPN domain of the Prp8 protein. Brr2-mediated U4/U6 unwinding was strongly inhibited by mutations in U4/U6 di-snRNAs that diminish the ability of U6 snRNA to adopt an alternative conformation but leave the number and kind of U4/U6 base pairs unchanged. Irrespective of the presence of the cofactor, the helicase segregated a Prp3-Prp31-Snu13-U4/U6 RNP into an intact Prp31-Snu13-U4 snRNA particle, free Prp3, and free U6 snRNA. Together, these observations suggest that Brr2 translocates only a limited distance on the U4 snRNA strand and does not actively release RNA-bound proteins. Unwinding is then completed by the partially displaced U6 snRNA adopting an alternative conformation, which leads to dismantling of the Prp3-binding site on U4/U6 di-snRNA but leaves the Prp31- and Snu13-binding sites on U4 snRNA unaffected. In this fashion, Brr2 can activate the spliceosome by stripping U6 snRNA of all precatalytic binding partners, while minimizing logistic requirements for U4/U6 di-snRNP reassembly after splicing. PMID:27354531

  16. FBH1 helicase disrupts RAD51 filaments in vitro and modulates homologous recombination in mammalian cells

    DEFF Research Database (Denmark)

    Simandlova, Jitka; Zagelbaum, Jennifer; Payne, Miranda J

    2013-01-01

    Efficient repair of DNA double strand breaks and interstrand cross-links requires the homologous recombination (HR) pathway, a potentially error-free process that utilizes a homologous sequence as a repair template. A key player in HR is RAD51, the eukaryotic ortholog of bacterial RecA protein. RAD......51 can polymerize on DNA to form a nucleoprotein filament that facilitates both the search for the homologous DNA sequences and the subsequent DNA strand invasion required to initiate HR. Because of its pivotal role in HR, RAD51 is subject to numerous positive and negative regulatory influences...... filaments on DNA through its ssDNA translocase function. Consistent with this, a mutant mouse embryonic stem cell line with a deletion in the FBH1 helicase domain fails to limit RAD51 chromatin association and shows hyper-recombination. Our data are consistent with FBH1 restraining RAD51 DNA binding under...

  17. Retinitis Pigmentosa Mutations in Bad Response to Refrigeration 2 (Brr2) Impair ATPase and Helicase Activity.

    Science.gov (United States)

    Ledoux, Sarah; Guthrie, Christine

    2016-06-03

    Brr2 is an RNA-dependent ATPase required to unwind the U4/U6 snRNA duplex during spliceosome assembly. Mutations within the ratchet helix of the Brr2 RNA binding channel result in a form of degenerative human blindness known as retinitis pigmentosa (RP). The biochemical consequences of these mutations on Brr2's RNA binding, helicase, and ATPase activity have not yet been characterized. Therefore, we identified the largest construct of Brr2 that is soluble in vitro, which truncates the first 247 amino acids of the N terminus (Δ247-Brr2), to characterize the effects of the RP mutations on Brr2 activity. The Δ247-Brr2 RP mutants exhibit a gradient of severity of weakened RNA binding, reduced helicase activity, and reduced ATPase activity compared with wild type Δ247-Brr2. The globular C-terminal Jab1/Mpn1-like domain of Prp8 increases the ability of Δ247-Brr2 to bind the U4/U6 snRNA duplex at high pH and increases Δ247-Brr2's RNA-dependent ATPase activity and the extent of RNA unwinding. However, this domain of Prp8 does not differentially affect the Δ247-Brr2 RP mutants compared with the wild type Δ247-Brr2. When stimulated by Prp8, wild type Δ247-Brr2 is able to unwind long stable duplexes in vitro, and even the RP mutants capable of binding RNA with tight affinity are incapable of fully unwinding short duplex RNAs. Our data suggest that the RP mutations within the ratchet helix impair Brr2 translocation through RNA helices. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  18. Identification and Biochemical Characterization of Halisulfate 3 and Suvanine as Novel Inhibitors of Hepatitis C Virus NS3 Helicase from a Marine Sponge

    Directory of Open Access Journals (Sweden)

    Atsushi Furuta

    2014-01-01

    Full Text Available Hepatitis C virus (HCV is an important etiological agent that is responsible for the development of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. HCV nonstructural protein 3 (NS3 helicase is a possible target for novel drug development due to its essential role in viral replication. In this study, we identified halisulfate 3 (hal3 and suvanine as novel NS3 helicase inhibitors, with IC50 values of 4 and 3 µM, respectively, from a marine sponge by screening extracts of marine organisms. Both hal3 and suvanine inhibited the ATPase, RNA binding, and serine protease activities of NS3 helicase with IC50 values of 8, 8, and 14 µM, and 7, 3, and 34 µM, respectively. However, the dengue virus (DENV NS3 helicase, which shares a catalytic core (consisting mainly of ATPase and RNA binding sites with HCV NS3 helicase, was not inhibited by hal3 and suvanine, even at concentrations of 100 µM. Therefore, we conclude that hal3 and suvanine specifically inhibit HCV NS3 helicase via an interaction with an allosteric site in NS3 rather than binding to the catalytic core. This led to the inhibition of all NS3 activities, presumably by inducing conformational changes.

  19. SAD-3, a Putative Helicase Required for Meiotic Silencing by Unpaired DNA, Interacts with Other Components of the Silencing Machinery

    Science.gov (United States)

    Hammond, Thomas M.; Xiao, Hua; Boone, Erin C.; Perdue, Tony D.; Pukkila, Patricia J.; Shiu, Patrick K. T.

    2011-01-01

    In Neurospora crassa, genes lacking a pairing partner during meiosis are suppressed by a process known as meiotic silencing by unpaired DNA (MSUD). To identify novel MSUD components, we have developed a high-throughput reverse-genetic screen for use with the N. crassa knockout library. Here we describe the screening method and the characterization of a gene (sad-3) subsequently discovered. SAD-3 is a putative helicase required for MSUD and sexual spore production. It exists in a complex with other known MSUD proteins in the perinuclear region, a center for meiotic silencing activity. Orthologs of SAD-3 include Schizosaccharomyces pombe Hrr1, a helicase required for RNAi-induced heterochromatin formation. Both SAD-3 and Hrr1 interact with an RNA-directed RNA polymerase and an Argonaute, suggesting that certain aspects of silencing complex formation may be conserved between the two fungal species. PMID:22384347

  20. Mutations of the RTEL1 Helicase in a Hoyeraal-Hreidarsson Syndrome Patient Highlight the Importance of the ARCH Domain.

    Science.gov (United States)

    Jullien, Laurent; Kannengiesser, Caroline; Kermasson, Laetitia; Cormier-Daire, Valérie; Leblanc, Thierry; Soulier, Jean; Londono-Vallejo, Arturo; de Villartay, Jean-Pierre; Callebaut, Isabelle; Revy, Patrick

    2016-05-01

    The DNA helicase RTEL1 participates in telomere maintenance and genome stability. Biallelic mutations in the RTEL1 gene account for the severe telomere biology disorder characteristic of the Hoyeraal-Hreidarsson syndrome (HH). Here, we report a HH patient (P4) carrying two novel compound heterozygous mutations in RTEL1: a premature stop codon (c.949A>T, p.Lys317*) and an intronic deletion leading to an exon skipping and an in-frame deletion of 25 amino-acids (p.Ile398_Lys422). P4's cells exhibit short and dysfunctional telomeres similarly to other RTEL1-deficient patients. 3D structure predictions indicated that the p.Ile398_Lys422 deletion affects a part of the helicase ARCH domain, which lines the pore formed with the core HD and the iron-sulfur cluster domains and is highly specific of sequences from the eukaryotic XPD family members. © 2016 WILEY PERIODICALS, INC.

  1. Identification of human genes involved in cellular responses to ionizing radiation: molecular and cellular studies of gene encoding the p68 helicase in mammalian cells

    International Nuclear Information System (INIS)

    Menaa, F.

    2003-12-01

    Cells submitted to genotoxic factors -like IR- activate several and important mechanisms such as repair, cell cycle arrest or 'apoptosis' to maintain genetic integrity. So, the damaged cells will induce many and different genes. The human transcriptome analysis by 'SSH' method in a human breast carcinoma cell line MCF7 γ-irradiated versus not irradiated, allowed to identify about one hundred genes. Among of these genes, we have focused our study on a radio-induced gene encoding the p68 helicase. In the conditions of irradiation used, our results show that the kinetic and the regulation of this gene expression differs between the nature of radiations used. Indeed, in γ-irradiated mammalian cells, ATM, a protein kinase activated by DSB and IR, is required to induce quickly P68 gene via the important transcription factor p53 stabilized by IR. In the case of UVC-irradiated cells, the P68 gene induction is late and the intracellular signalling pathway that lead to this induction is independent from the p53 protein. Finally, we show that the p68 protein under-expression is responsible for an increased radiosensitivity of MCF7 cells. Consequently, we can postulate that the p68 protein is involved in cellular responses to radiations to reduce the increased radiosensitivity of cells exposed to γ-rays. (author)

  2. The RNA-mediated, asymmetric ring regulatory mechanism of the transcription termination Rho helicase decrypted by time-resolved nucleotide analog interference probing (trNAIP).

    Science.gov (United States)

    Soares, Emilie; Schwartz, Annie; Nollmann, Marcello; Margeat, Emmanuel; Boudvillain, Marc

    2014-08-01

    Rho is a ring-shaped, ATP-dependent RNA helicase/translocase that dissociates transcriptional complexes in bacteria. How RNA recognition is coupled to ATP hydrolysis and translocation in Rho is unclear. Here, we develop and use a new combinatorial approach, called time-resolved Nucleotide Analog Interference Probing (trNAIP), to unmask RNA molecular determinants of catalytic Rho function. We identify a regulatory step in the translocation cycle involving recruitment of the 2'-hydroxyl group of the incoming 3'-RNA nucleotide by a Rho subunit. We propose that this step arises from the intrinsic weakness of one of the subunit interfaces caused by asymmetric, split-ring arrangement of primary RNA tethers around the Rho hexamer. Translocation is at highest stake every seventh nucleotide when the weak interface engages the incoming 3'-RNA nucleotide or breaks, depending on RNA threading constraints in the Rho pore. This substrate-governed, 'test to run' iterative mechanism offers a new perspective on how a ring-translocase may function or be regulated. It also illustrates the interest and versatility of the new trNAIP methodology to unveil the molecular mechanisms of complex RNA-based systems. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

  3. Essential and distinct roles of the F-box and helicase domains of Fbh1 in DNA damage repair

    Directory of Open Access Journals (Sweden)

    Shinagawa Hideo

    2008-03-01

    Full Text Available Abstract Background DNA double-strand breaks (DSBs are induced by exogenous insults such as ionizing radiation and chemical exposure, and they can also arise as a consequence of stalled or collapsed DNA replication forks. Failure to repair DSBs can lead to genomic instability or cell death and cancer in higher eukaryotes. The Schizosaccharomyces pombe fbh1 gene encodes an F-box DNA helicase previously described to play a role in the Rhp51 (an orthologue of S. cerevisiae RAD51-dependent recombinational repair of DSBs. Fbh1 fused to GFP localizes to discrete nuclear foci following DNA damage. Results To determine the functional roles of the highly conserved F-box and helicase domains, we have characterized fbh1 mutants carrying specific mutations in these domains. We show that the F-box mutation fbh1-fb disturbs the nuclear localization of Fbh1, conferring an fbh1 null-like phenotype. Moreover, nuclear foci do not form in fbh1-fb cells with DNA damage even if Fbh1-fb is targeted to the nucleus by fusion to a nuclear localization signal sequence. In contrast, the helicase mutation fbh1-hl causes the accumulation of Fbh1 foci irrespective of the presence of DNA damage and confers damage sensitivity greater than that conferred by the null allele. Additional mutation of the F-box alleviates the hypermorphic phenotype of the fbh1-hl mutant. Conclusion These results suggest that the F-box and DNA helicase domains play indispensable but distinct roles in Fbh1 function. Assembly of the SCFFbh1 complex is required for both the nuclear localization and DNA damage-induced focus formation of Fbh1 and is therefore prerequisite for the Fbh1 recombination function.

  4. DEAD-Box RNA Helicases are among the Constituents of the Tobacco Pollen mRNA Storing Bodies

    Czech Academy of Sciences Publication Activity Database

    Hafidh, Said; Potěšil, D.; Zdráhal, Z.; Honys, David

    2013-01-01

    Roč. 1, č. 3 (2013) ISSN 2329-9029 R&D Projects: GA ČR GPP501/11/P321; GA ČR(CZ) GAP501/11/1462; GA MŠk(CZ) ED1.1.00/02.0068; GA MŠk(CZ) LD13049 Institutional support: RVO:61389030 Keywords : Translation * mRNA storage * RNA helicase Subject RIV: EB - Genetics ; Molecular Biology

  5. Structural Studies of RNA Helicases Involved in Eukaryotic Pre-mRNA Splicing, Ribosome Biogenesis, and Translation Initiation

    DEFF Research Database (Denmark)

    He, Yangzi

    and ligates the neighbouring exons to generate mature mRNAs. Prp43 is an RNA helicase of the DEAH/RHA family. In yeast, once mRNAs are released, Prp43 catalyzes the disassembly of spliceosomes. The 18S, 5.8S and 25S rRNAs are transcribed as a single polycistronic transcript—the 35S pre......-rRNA. It is nucleolytically cleaved and chemically modified to generate mature rRNAs, which assemble with ribosomal proteins to form the ribosome. Prp43 is required for the processing of the 18S rRNA. Using X-ray crystallography, I determined a high resolution structure of Prp43 bound to ADP, the first structure of a DEAH....../RHA helicase. It defined the conserved structural features of all DEAH/RHA helicases, and unveiled a novel nucleotide binding site. Additionally a preliminary low resolution structure of a ternary complex comprising Prp43, a non-hydrolyzable ATP analogue, and a single-stranded RNA, was obtained. The ribosome...

  6. Zebrafish P54 RNA helicases are cytoplasmic granule residents that are required for development and stress resilience

    Directory of Open Access Journals (Sweden)

    Cecilia Zampedri

    2016-10-01

    Full Text Available Stress granules are cytoplasmic foci that directly respond to the protein synthesis status of the cell. Various environmental insults, such as oxidative stress or extreme heat, block protein synthesis; consequently, mRNA will stall in translation, and stress granules will immediately form and become enriched with mRNAs. P54 DEAD box RNA helicases are components of RNA granules such as P-bodies and stress granules. We studied the expression, in cytoplasmic foci, of both zebrafish P54 RNA helicases (P54a and P54b during development and found that they are expressed in cytoplasmic granules under both normal conditions and stress conditions. In zebrafish embryos exposed to heat shock, some proportion of P54a and P54b helicases move to larger granules that exhibit the properties of genuine stress granules. Knockdown of P54a and/or P54b in zebrafish embryos produces developmental abnormalities restricted to the posterior trunk; further, these embryos do not form stress granules, and their survival upon exposure to heat-shock conditions is compromised. Our observations fit the model that cells lacking stress granules have no resilience or ability to recover once the stress has ended, indicating that stress granules play an essential role in the way organisms adapt to a changing environment.

  7. Towards a behavioral vaccine: Exposure to accessible temptation when self-regulation is endorsed enhances future resistance to similar temptations in children

    NARCIS (Netherlands)

    Boer, de C.; Ridder, de D.T.D.; Vet, de E.W.M.L.; Grubliauskiene, A.; Dewitte, S.

    2015-01-01

    Background: Access to temptation is blamed for the rising prevalence of obesity in children. A popular way to counter this is to restrict physical access to temptation. As restrictions cannot be widely applied and may have adverse long-term effects, we examine whether accessible temptations in

  8. RSPO1/β-catenin signaling pathway regulates oogonia differentiation and entry into meiosis in the mouse fetal ovary

    NARCIS (Netherlands)

    Chassot, Anne-Amandine; Gregoire, Elodie P.; Lavery, Rowena; Taketo, Makoto M.; de Rooij, Dirk G.; Adams, Ian R.; Chaboissier, Marie-Christine

    2011-01-01

    Differentiation of germ cells into male gonocytes or female oocytes is a central event in sexual reproduction. Proliferation and differentiation of fetal germ cells depend on the sex of the embryo. In male mouse embryos, germ cell proliferation is regulated by the RNA helicase Mouse Vasa homolog

  9. RSPO1/beta-Catenin Signaling Pathway Regulates Oogonia Differentiation and Entry into Meiosis in the Mouse Fetal Ovary

    NARCIS (Netherlands)

    Chassot, A.A.; Gregoire, E.P.; Lavery, R.; Taketo, M.M.; de Rooij, D.G.; Adams, I.R.; Chaboissier, M.C.

    2011-01-01

    Differentiation of germ cells into male gonocytes or female oocytes is a central event in sexual reproduction. Proliferation and differentiation of fetal germ cells depend on the sex of the embryo. In male mouse embryos, germ cell proliferation is regulated by the RNA helicase Mouse Vasa homolog

  10. A conserved helicase processivity factor is needed for conjugation and replication of an integrative and conjugative element.

    Directory of Open Access Journals (Sweden)

    Jacob Thomas

    Full Text Available Integrative and conjugative elements (ICEs are agents of horizontal gene transfer and have major roles in evolution and acquisition of new traits, including antibiotic resistances. ICEs are found integrated in a host chromosome and can excise and transfer to recipient bacteria via conjugation. Conjugation involves nicking of the ICE origin of transfer (oriT by the ICE-encoded relaxase and transfer of the nicked single strand of ICE DNA. For ICEBs1 of Bacillus subtilis, nicking of oriT by the ICEBs1 relaxase NicK also initiates rolling circle replication. This autonomous replication of ICEBs1 is critical for stability of the excised element in growing cells. We found a conserved and previously uncharacterized ICE gene that is required for conjugation and replication of ICEBs1. Our results indicate that this gene, helP (formerly ydcP, encodes a helicase processivity factor that enables the host-encoded helicase PcrA to unwind the double-stranded ICEBs1 DNA. HelP was required for both conjugation and replication of ICEBs1, and HelP and NicK were the only ICEBs1 proteins needed for replication from ICEBs1 oriT. Using chromatin immunoprecipitation, we measured association of HelP, NicK, PcrA, and the host-encoded single-strand DNA binding protein Ssb with ICEBs1. We found that NicK was required for association of HelP and PcrA with ICEBs1 DNA. HelP was required for association of PcrA and Ssb with ICEBs1 regions distal, but not proximal, to oriT, indicating that PcrA needs HelP to progress beyond nicked oriT and unwind ICEBs1. In vitro, HelP directly stimulated the helicase activity of the PcrA homologue UvrD. Our findings demonstrate that HelP is a helicase processivity factor needed for efficient unwinding of ICEBs1 for conjugation and replication. Homologues of HelP and PcrA-type helicases are encoded on many known and putative ICEs. We propose that these factors are essential for ICE conjugation, replication, and genetic stability.

  11. The roles of WRN and BLM RecQ helicases in the Alternative Lengthening of Telomeres.

    Science.gov (United States)

    Mendez-Bermudez, Aaron; Hidalgo-Bravo, Alberto; Cotton, Victoria E; Gravani, Athanasia; Jeyapalan, Jennie N; Royle, Nicola J

    2012-11-01

    Approximately 10% of all cancers, but a higher proportion of sarcomas, use the recombination-based alternative lengthening of telomeres (ALT) to maintain telomeres. Two RecQ helicase genes, BLM and WRN, play important roles in homologous recombination repair and they have been implicated in telomeric recombination activity, but their precise roles in ALT are unclear. Using analysis of sequence variation present in human telomeres, we found that a WRN- ALT+ cell line lacks the class of complex telomere mutations attributed to inter-telomeric recombination in other ALT+ cell lines. This suggests that WRN facilitates inter-telomeric recombination when there are sequence differences between the donor and recipient molecules or that sister-telomere interactions are suppressed in the presence of WRN and this promotes inter-telomeric recombination. Depleting BLM in the WRN- ALT+ cell line increased the mutation frequency at telomeres and at the MS32 minisatellite, which is a marker of ALT. The absence of complex telomere mutations persisted in BLM-depleted clones, and there was a clear increase in sequence homogenization across the telomere and MS32 repeat arrays. These data indicate that BLM suppresses unequal sister chromatid interactions that result in excessive homogenization at MS32 and at telomeres in ALT+ cells.

  12. A rapid Salmonella detection method involving thermophilic helicase-dependent amplification and a lateral flow assay.

    Science.gov (United States)

    Du, Xin-Jun; Zhou, Tian-Jiao; Li, Ping; Wang, Shuo

    2017-08-01

    Salmonella is a major foodborne pathogen that is widespread in the environment and can cause serious human and animal disease. Since conventional culture methods to detect Salmonella are time-consuming and laborious, rapid and accurate techniques to detect this pathogen are critically important for food safety and diagnosing foodborne illness. In this study, we developed a rapid, simple and portable Salmonella detection strategy that combines thermophilic helicase-dependent amplification (tHDA) with a lateral flow assay to provide a detection result based on visual signals within 90 min. Performance analyses indicated that the method had detection limits for DNA and pure cultured bacteria of 73.4-80.7 fg and 35-40 CFU, respectively. Specificity analyses showed no cross reactions with Escherichia coli, Staphylococcus aureus, Listeria monocytogenes, Enterobacter aerogenes, Shigella and Campylobacter jejuni. The results for detection in real food samples showed that 1.3-1.9 CFU/g or 1.3-1.9 CFU/mL of Salmonella in contaminated chicken products and infant nutritional cereal could be detected after 2 h of enrichment. The same amount of Salmonella in contaminated milk could be detected after 4 h of enrichment. This tHDA-strip can be used for the rapid detection of Salmonella in food samples and is particularly suitable for use in areas with limited equipment. Copyright © 2017 Elsevier Ltd. All rights reserved.

  13. Antiviral drug resistance and helicase-primase inhibitors of herpes simplex virus.

    Science.gov (United States)

    Field, Hugh J; Biswas, Subhajit

    2011-02-01

    A new class of chemical inhibitors has been discovered that interferes with the process of herpesvirus DNA replication. To date, the majority of useful herpesvirus antivirals are nucleoside analogues that block herpesvirus DNA replication by targeting the DNA polymerase. The new helicase-primase inhibitors (HPI) target a different enzyme complex that is also essential for herpesvirus DNA replication. This review will place the HPI in the context of previous work on the nucleoside analogues. Several promising highly potent HPI will be described with a particular focus on the identification of drug-resistance mutations. Several HPI have good pharmacological profiles and are now at the outset of phase II clinical trials. Provided there are no safety issues to stop their progress, this new class of compound will be a major advance in the herpesvirus antiviral field. Furthermore, HPI are likely to have a major impact on the therapy and prevention of herpes simplex virus and varicella zoster in both immunocompetent and immunocompromised patients alone or in combination with current nucleoside analogues. The possibility of acquired drug-resistance to HPI will then become an issue of great practical importance. Copyright © 2010 Elsevier Ltd. All rights reserved.

  14. Mutation and Methylation Analysis of the Chromodomain-Helicase-DNA Binding 5 Gene in Ovarian Cancer

    Directory of Open Access Journals (Sweden)

    Kylie L. Gorringe

    2008-11-01

    Full Text Available Chromodomain, helicase, DNA binding 5 (CHD5 is a member of a subclass of the chromatin remodeling Swi/Snf proteins and has recently been proposed as a tumor suppressor in a diverse range of human cancers. We analyzed all 41 coding exons of CHD5 for somatic mutations in 123 primary ovarian cancers as well as 60 primary breast cancers using high-resolution melt analysis. We also examined methylation of the CHD5 promoter in 48 ovarian cancer samples by methylation-specific single-stranded conformation polymorphism and bisulfite sequencing. In contrast to previous studies, no mutations were identified in the breast cancers, but somatic heterozygous missense mutations were identified in 3 of 123 ovarian cancers. We identified promoter methylation in 3 of 45 samples with normal CHD5 and in 2 of 3 samples with CHD5 mutation, suggesting these tumors may have biallelic inactivation of CHD5. Hemizygous copy number loss at CHD5 occurred in 6 of 85 samples as assessed by single nucleotide polymorphism array. Tumors with CHD5 mutation or methylation were more likely to have mutation of KRAS or BRAF (P = .04. The aggregate frequency of CHD5 haploinsufficiency or inactivation is 16.2% in ovarian cancer. Thus, CHD5 may play a role as a tumor suppressor gene in ovarian cancer; however, it is likely that there is another target of the frequent copy number neutral loss of heterozygosity observed at 1p36.

  15. Helicase Dependent Isothermal Amplification of DNA and RNA using Self-Avoiding Molecular Recognition Systems

    Science.gov (United States)

    Yang, Zunyi; McLendon, Chris; Hutter, Daniel; Bradley, Kevin M.; Hoshika, Shuichi; Frye, Carole; Benner, Steven A.

    2015-01-01

    Assays that target DNA or RNA (xNA) are highly sensitive, as small amounts of xNA can be amplified by PCR. Unfortunately, PCR is inconvenient in low resource environments, requiring equipment and power that may not be available in these environments. However, isothermal procedures that avoid thermal cycling are often confounded by primer dimers, off-target priming, and other artifacts. Here, we show how a “self avoiding molecular recognition system” (SAMRS) eliminates these artifacts to give clean amplicons in a helicase-dependent isothermal amplification (SAMRS-HDA). We also show that incorporating SAMRS into the 3′-ends of primers facilitates the design and screening of primers for HDA assays. Finally, we show that SAMRS-HDA can be twofold multiplexed, something difficult to achieve with HDA using standard primers. This shows that SAMRS-HDA is a more versatile approach than standard HDA with a broader applicability for xNA-targeted diagnostics and research. PMID:25953623

  16. The DEAD box helicase RDE-12 promotes amplification of RNAi in cytoplasmic foci in C. elegans.

    Science.gov (United States)

    Yang, Huan; Vallandingham, Jim; Shiu, Philip; Li, Hua; Hunter, Craig P; Mak, Ho Yi

    2014-04-14

    RNAi is a potent mechanism for downregulating gene expression. Conserved RNAi pathway components are found in animals, plants, fungi, and other eukaryotes. In C. elegans, the RNAi response is greatly amplified by the synthesis of abundant secondary small interfering RNAs (siRNAs). Exogenous double-stranded RNA is processed by Dicer and RDE-1/Argonaute into primary siRNA that guides target mRNA recognition. The RDE-10/RDE-11 complex and the RNA-dependent RNA polymerase RRF-1 then engage the target mRNA for secondary siRNA synthesis. However, the molecular link between primary siRNA production and secondary siRNA synthesis remains largely unknown. Furthermore, it is unclear whether the subcellular sites for target mRNA recognition and degradation coincide with sites where siRNA synthesis and amplification occur. In the C. elegans germline, cytoplasmic P granules at the nuclear pores and perinuclear Mutator foci contribute to target mRNA surveillance and siRNA amplification, respectively. We report that RDE-12, a conserved phenylalanine-glycine (FG) domain-containing DEAD box helicase, localizes in P granules and cytoplasmic foci that are enriched in RSD-6 but are excluded from the Mutator foci. Our results suggest that RDE-12 promotes secondary siRNA synthesis by orchestrating the recruitment of RDE-10 and RRF-1 to primary siRNA-targeted mRNA in distinct cytoplasmic compartments. Copyright © 2014 Elsevier Ltd. All rights reserved.

  17. Phosphopeptide binding by Sld3 links Dbf4-dependent kinase to MCM replicative helicase activation.

    Science.gov (United States)

    Deegan, Tom D; Yeeles, Joseph Tp; Diffley, John Fx

    2016-05-02

    The initiation of eukaryotic DNA replication requires the assembly of active CMG (Cdc45-MCM-GINS) helicases at replication origins by a set of conserved and essential firing factors. This process is controlled during the cell cycle by cyclin-dependent kinase (CDK) and Dbf4-dependent kinase (DDK), and in response to DNA damage by the checkpoint kinase Rad53/Chk1. Here we show that Sld3, previously shown to be an essential CDK and Rad53 substrate, is recruited to the inactive MCM double hexamer in a DDK-dependent manner. Sld3 binds specifically to DDK-phosphorylated peptides from two MCM subunits (Mcm4, 6) and then recruits Cdc45. MCM mutants that cannot bind Sld3 or Sld3 mutants that cannot bind phospho-MCM or Cdc45 do not support replication. Moreover, phosphomimicking mutants in Mcm4 and Mcm6 bind Sld3 without DDK and facilitate DDK-independent replication. Thus, Sld3 is an essential "reader" of DDK phosphorylation, integrating signals from three distinct protein kinase pathways to coordinate DNA replication during S phase. © 2016 The Authors. Published under the terms of the CC BY 4.0 license.

  18. Functional Dynamics of Hexameric Helicase Probed by Hydrogen Exchange and Simulation

    Science.gov (United States)

    Radou, Gaël; Dreyer, Frauke N.; Tuma, Roman; Paci, Emanuele

    2014-01-01

    The biological function of large macromolecular assemblies depends on their structure and their dynamics over a broad range of timescales; for this reason, it is a significant challenge to investigate these assemblies using conventional experimental techniques. One of the most promising experimental techniques is hydrogen-deuterium exchange detected by mass spectrometry. Here, we describe to our knowledge a new computational method for quantitative interpretation of deuterium exchange kinetics and apply it to a hexameric viral helicase P4 that unwinds and translocates RNA into a virus capsid at the expense of ATP hydrolysis. Room-temperature dynamics probed by a hundred nanoseconds of all-atom molecular dynamics simulations is sufficient to predict the exchange kinetics of most sequence fragments and provide a residue-level interpretation of the low-resolution experimental results. The strategy presented here is also a valuable tool to validate experimental data, e.g., assignments, and to probe mechanisms that cannot be observed by x-ray crystallography, or that occur over timescales longer than those that can be realistically simulated, such as the opening of the hexameric ring. PMID:25140434

  19. Helicase-primase inhibitor amenamevir for herpesvirus infection: Towards practical application for treating herpes zoster.

    Science.gov (United States)

    Shiraki, K

    2017-11-01

    Valacyclovir and famciclovir enabled successful systemic therapy for treating herpes simplex virus (HSV) and varicella zoster virus (VZV) infection by their phosphorylation with viral thymidine kinase. Helicase-primase inhibitors (HPIs) inhibit the progression of the replication fork, an initial step in DNA synthesis to separate the double strand into two single strands. The HPIs amenamevir and pritelivir have a novel mechanism of action, once-daily administration with nonrenal excretory characteristics, and clinical efficacy for genital herpes. Amenamevir exhibits anti-VZV and anti-HSV activity while pritelivir only has anti-HSV activity. A clinical trial of amenamevir for herpes zoster has been completed, and amenamevir has been licensed and successfully used in 20,000 patients with herpes zoster so far in Japan. We have characterized the features of the antiviral action of amenamevir and, unlike acyclovir, the drug's antiviral activity is not influenced by the viral replication cycle. Amenamevir is opening a new era of antiherpes therapy. Copyright 2017 Clarivate Analytics.

  20. Broadband Access

    Indian Academy of Sciences (India)

    First page Back Continue Last page Overview Graphics. Broadband Access. Worldwide market for broadband access $30 Billion! Over 200 million broadband subscribers worldwide! Various Competing Broadband access. Digital Subscriber line; Wireless; Optical Fiber.

  1. Mycobacterium smegmatis Lhr Is a DNA-dependent ATPase and a 3'-to-5' DNA translocase and helicase that prefers to unwind 3'-tailed RNA:DNA hybrids.

    Science.gov (United States)

    Ordonez, Heather; Shuman, Stewart

    2013-05-17

    We are interested in the distinctive roster of helicases of Mycobacterium, a genus of the phylum Actinobacteria that includes the human pathogen Mycobacterium tuberculosis and its avirulent relative Mycobacterium smegmatis. Here, we identify and characterize M. smegmatis Lhr as the exemplar of a novel clade of superfamily II helicases, by virtue of its biochemical specificities and signature domain organization. Lhr is a 1507-amino acid monomeric nucleic acid-dependent ATPase that uses the energy of ATP hydrolysis to drive unidirectional 3'-to-5' translocation along single strand DNA and to unwind duplexes en route. The ATPase is more active in the presence of calcium than magnesium. ATP hydrolysis is triggered by either single strand DNA or single strand RNA, yet the apparent affinity for a DNA activator is 11-fold higher than for an RNA strand of identical size and nucleobase sequence. Lhr is 8-fold better at unwinding an RNA:DNA hybrid than it is at displacing a DNA:DNA duplex of identical nucleobase sequence. The truncated derivative Lhr-(1-856) is an autonomous ATPase, 3'-to-5' translocase, and RNA:DNA helicase. Lhr-(1-856) is 100-fold better RNA:DNA helicase than DNA:DNA helicase. Lhr homologs are found in bacteria representing eight different phyla, being especially prevalent in Actinobacteria (including M. tuberculosis) and Proteobacteria (including Escherichia coli).

  2. The pipeline transportation of natural gas and the regulation of open access in Brazil: historical perspectives and current; Os dutos de transporte do gas natural e a regulacao do livre acesso no Brasil: perspectivas historicas e atuais

    Energy Technology Data Exchange (ETDEWEB)

    Siqueira, Mariana de; Xavier, Yanko Marcius de Alencar [Universidade Federal do Rio Grande do Norte (UFRN), Natal, RN (Brazil)

    2008-07-01

    The present research, attempting for the economic relevance of the natural gas sector, for the inexistence of a law that disciplines it and, still, for the structural question of the natural gas ducts activity; it analyzes the monopoly of the natural gas ducts activity and the mechanism of competition chosen to brighten up it: the open access. The regulation of the open access and the viability that the insert of this practice has to Brazil nowadays are very important points to be discussed and, in that way, the present research also analyses them. (author)

  3. The SMC-5/6 Complex and the HIM-6 (BLM Helicase Synergistically Promote Meiotic Recombination Intermediate Processing and Chromosome Maturation during Caenorhabditis elegans Meiosis.

    Directory of Open Access Journals (Sweden)

    Ye Hong

    2016-03-01

    Full Text Available Meiotic recombination is essential for the repair of programmed double strand breaks (DSBs to generate crossovers (COs during meiosis. The efficient processing of meiotic recombination intermediates not only needs various resolvases but also requires proper meiotic chromosome structure. The Smc5/6 complex belongs to the structural maintenance of chromosome (SMC family and is closely related to cohesin and condensin. Although the Smc5/6 complex has been implicated in the processing of recombination intermediates during meiosis, it is not known how Smc5/6 controls meiotic DSB repair. Here, using Caenorhabditis elegans we show that the SMC-5/6 complex acts synergistically with HIM-6, an ortholog of the human Bloom syndrome helicase (BLM during meiotic recombination. The concerted action of the SMC-5/6 complex and HIM-6 is important for processing recombination intermediates, CO regulation and bivalent maturation. Careful examination of meiotic chromosomal morphology reveals an accumulation of inter-chromosomal bridges in smc-5; him-6 double mutants, leading to compromised chromosome segregation during meiotic cell divisions. Interestingly, we found that the lethality of smc-5; him-6 can be rescued by loss of the conserved BRCA1 ortholog BRC-1. Furthermore, the combined deletion of smc-5 and him-6 leads to an irregular distribution of condensin and to chromosome decondensation defects reminiscent of condensin depletion. Lethality conferred by condensin depletion can also be rescued by BRC-1 depletion. Our results suggest that SMC-5/6 and HIM-6 can synergistically regulate recombination intermediate metabolism and suppress ectopic recombination by controlling chromosome architecture during meiosis.

  4. The SMC-5/6 Complex and the HIM-6 (BLM) Helicase Synergistically Promote Meiotic Recombination Intermediate Processing and Chromosome Maturation during Caenorhabditis elegans Meiosis.

    Science.gov (United States)

    Hong, Ye; Sonneville, Remi; Agostinho, Ana; Meier, Bettina; Wang, Bin; Blow, J Julian; Gartner, Anton

    2016-03-01

    Meiotic recombination is essential for the repair of programmed double strand breaks (DSBs) to generate crossovers (COs) during meiosis. The efficient processing of meiotic recombination intermediates not only needs various resolvases but also requires proper meiotic chromosome structure. The Smc5/6 complex belongs to the structural maintenance of chromosome (SMC) family and is closely related to cohesin and condensin. Although the Smc5/6 complex has been implicated in the processing of recombination intermediates during meiosis, it is not known how Smc5/6 controls meiotic DSB repair. Here, using Caenorhabditis elegans we show that the SMC-5/6 complex acts synergistically with HIM-6, an ortholog of the human Bloom syndrome helicase (BLM) during meiotic recombination. The concerted action of the SMC-5/6 complex and HIM-6 is important for processing recombination intermediates, CO regulation and bivalent maturation. Careful examination of meiotic chromosomal morphology reveals an accumulation of inter-chromosomal bridges in smc-5; him-6 double mutants, leading to compromised chromosome segregation during meiotic cell divisions. Interestingly, we found that the lethality of smc-5; him-6 can be rescued by loss of the conserved BRCA1 ortholog BRC-1. Furthermore, the combined deletion of smc-5 and him-6 leads to an irregular distribution of condensin and to chromosome decondensation defects reminiscent of condensin depletion. Lethality conferred by condensin depletion can also be rescued by BRC-1 depletion. Our results suggest that SMC-5/6 and HIM-6 can synergistically regulate recombination intermediate metabolism and suppress ectopic recombination by controlling chromosome architecture during meiosis.

  5. Barriers to access to opioid medicines for patients with opioid dependence : a review of legislation and regulations in eleven central and eastern European countries

    NARCIS (Netherlands)

    Vranken, Marjolein J M; Mantel-Teeuwisse, Aukje K; Jünger, Saskia; Radbruch, Lukas; Scholten, Willem; Lisman, John A; Subataite, Marija; Schutjens, Marie-Hélène D B

    BACKGROUND AND AIMS: Barriers linked to drug control systems are considered to contribute to inequitable access to controlled medicines, leaving millions of people in pain and suffering. Most studies focus on access to opioids for the treatment of severe (cancer) pain. This study aims to identify

  6. Ion channel regulation by phosphoinositides analyzed with VSPs – PI(4,5P2 affinity, phosphoinositide selectivity, and PI(4,5P2 pool accessibility

    Directory of Open Access Journals (Sweden)

    Alexandra eRjasanow

    2015-06-01

    Full Text Available The activity of many proteins depends on the phosphoinositide (PI content of the membrane. E.g., dynamic changes of the concentration of PI(4,5P2 are cellular signals that regulate ion channels. The susceptibility of a channel to such dynamics depends on its affinity for PI(4,5P2. Yet, measuring affinities for endogenous PIs has not been possible directly, but has relied largely on the response to soluble analogs, which may not quantitatively reflect binding to native lipids.Voltage-sensitive phosphatases (VSPs turn over PI(4,5P2 to PI(4P when activated by depolarization. In combination with voltage-clamp electrophysiology VSPs are useful tools for rapid and reversible depletion of PI(4,5P2. Because cellular PI(4,5P2 is resynthesized rapidly, steady state PI(4,5P2 changes with the degree of VSP activation and thus depends on membrane potential.Here we show that titration of endogenous PI(4,5P2 with Ci-VSP allows for the quantification of relative PI(4,5P2 affinities of ion channels. The sensitivity of inward rectifier and voltage-gated K+ channels to Ci-VSP allowed for comparison of PI(4,5P2 affinities within and across channel subfamilies and detected changes of affinity in mutant channels. The results also reveal that VSPs are useful only for PI effectors with high binding specificity among PI isoforms, because PI(4,5P2 depletion occurs at constant overall PI level. Thus, Kir6.2, a channel activated by PI(4,5P2 and PI(4P was insensitive to VSP.Surprisingly, despite comparable PI(4,5P2 affinity as determined by Ci-VSP, the Kv7 and Kir channel families strongly differed in their sensitivity to receptor-mediated depletion of PI(4,5P2. While Kv7 members were highly sensitive to activation of PLC by Gq-coupled receptors, Kir channels were insensitive even when PI(4,5P2 affinity was lowered by mutation. We hypothesize that different channels may be associated with distinct pools of PI(4,5P2 that differ in their accessibility to PLC and VSPs.

  7. Mechanisms and regulation of DNA replication initiation in eukaryotes.

    Science.gov (United States)

    Parker, Matthew W; Botchan, Michael R; Berger, James M

    2017-04-01

    Cellular DNA replication is initiated through the action of multiprotein complexes that recognize replication start sites in the chromosome (termed origins) and facilitate duplex DNA melting within these regions. In a typical cell cycle, initiation occurs only once per origin and each round of replication is tightly coupled to cell division. To avoid aberrant origin firing and re-replication, eukaryotes tightly regulate two events in the initiation process: loading of the replicative helicase, MCM2-7, onto chromatin by the origin recognition complex (ORC), and subsequent activation of the helicase by its incorporation into a complex known as the CMG. Recent work has begun to reveal the details of an orchestrated and sequential exchange of initiation factors on DNA that give rise to a replication-competent complex, the replisome. Here, we review the molecular mechanisms that underpin eukaryotic DNA replication initiation - from selecting replication start sites to replicative helicase loading and activation - and describe how these events are often distinctly regulated across different eukaryotic model organisms.

  8. Nanomechanical microcantilever operated in vibration modes with use of RNA aptamer as receptor molecules for label-free detection of HCV helicase.

    Science.gov (United States)

    Hwang, Kyo Seon; Lee, Sang-Myung; Eom, Kilho; Lee, Jeong Hoon; Lee, Yoon-Sik; Park, Jung Ho; Yoon, Dae Sung; Kim, Tae Song

    2007-11-30

    We report the nanomechanical microcantilevers operated in vibration modes (oscillation) with use of RNA aptamers as receptor molecules for label-free detection of hepatitis C virus (HCV) helicase. The nanomechanical detection principle is that the ligand-receptor binding on the microcantilever surface induces the dynamic response change of microcantilevers. We implemented the label-free detection of HCV helicase in the low concentration as much as 100 pg/ml from measuring the dynamic response change of microcantilevers. Moreover, from the recent studies showing that the ligand-receptor binding generates the surface stress on the microcantilever, we estimate the surface stress, on the oscillating microcantilevers, induced by ligand-receptor binding, i.e. binding between HCV helicase and RNA aptamer. In this article, it is suggested that the oscillating microcantilevers with use of RNA aptamers as receptor molecules may enable one to implement the sensitive label-free detection of very small amount of small-scale proteins.

  9. CMG helicase and DNA polymerase ε form a functional 15-subunit holoenzyme for eukaryotic leading-strand DNA replication.

    Science.gov (United States)

    Langston, Lance D; Zhang, Dan; Yurieva, Olga; Georgescu, Roxana E; Finkelstein, Jeff; Yao, Nina Y; Indiani, Chiara; O'Donnell, Mike E

    2014-10-28

    DNA replication in eukaryotes is asymmetric, with separate DNA polymerases (Pol) dedicated to bulk synthesis of the leading and lagging strands. Pol α/primase initiates primers on both strands that are extended by Pol ε on the leading strand and by Pol δ on the lagging strand. The CMG (Cdc45-MCM-GINS) helicase surrounds the leading strand and is proposed to recruit Pol ε for leading-strand synthesis, but to date a direct interaction between CMG and Pol ε has not been demonstrated. While purifying CMG helicase overexpressed in yeast, we detected a functional complex between CMG and native Pol ε. Using pure CMG and Pol ε, we reconstituted a stable 15-subunit CMG-Pol ε complex and showed that it is a functional polymerase-helicase on a model replication fork in vitro. On its own, the Pol2 catalytic subunit of Pol ε is inefficient in CMG-dependent replication, but addition of the Dpb2 protein subunit of Pol ε, known to bind the Psf1 protein subunit of CMG, allows stable synthesis with CMG. Dpb2 does not affect Pol δ function with CMG, and thus we propose that the connection between Dpb2 and CMG helps to stabilize Pol ε on the leading strand as part of a 15-subunit leading-strand holoenzyme we refer to as CMGE. Direct binding between Pol ε and CMG provides an explanation for specific targeting of Pol ε to the leading strand and provides clear mechanistic evidence for how strand asymmetry is maintained in eukaryotes.

  10. Assessment of Dengue virus helicase and methyltransferase as targets for fragment-based drug discovery.

    Science.gov (United States)

    Coutard, Bruno; Decroly, Etienne; Li, Changqing; Sharff, Andrew; Lescar, Julien; Bricogne, Gérard; Barral, Karine

    2014-06-01

    Seasonal and pandemic flaviviruses continue to be leading global health concerns. With the view to help drug discovery against Dengue virus (DENV), a fragment-based experimental approach was applied to identify small molecule ligands targeting two main components of the flavivirus replication complex: the NS3 helicase (Hel) and the NS5 mRNA methyltransferase (MTase) domains. A library of 500 drug-like fragments was first screened by thermal-shift assay (TSA) leading to the identification of 36 and 32 fragment hits binding Hel and MTase from DENV, respectively. In a second stage, we set up a fragment-based X-ray crystallographic screening (FBS-X) in order to provide both validated fragment hits and structural binding information. No fragment hit was confirmed for DENV Hel. In contrast, a total of seven fragments were identified as DENV MTase binders and structures of MTase-fragment hit complexes were solved at resolution at least 2.0Å or better. All fragment hits identified contain either a five- or six-membered aromatic ring or both, and three novel binding sites were located on the MTase. To further characterize the fragment hits identified by TSA and FBS-X, we performed enzymatic assays to assess their inhibition effect on the N7- and 2'-O-MTase enzymatic activities: five of these fragment hits inhibit at least one of the two activities with IC50 ranging from 180μM to 9mM. This work validates the FBS-X strategy for identifying new anti-flaviviral hits targeting MTase, while Hel might not be an amenable target for fragment-based drug discovery (FBDD). This approach proved to be a fast and efficient screening method for FBDD target validation and discovery of starting hits for the development of higher affinity molecules that bind to novel allosteric sites. Copyright © 2014 Elsevier B.V. All rights reserved.

  11. The roles of the Saccharomyces cerevisiae RecQ helicase SGS1 in meiotic genome surveillance.

    Directory of Open Access Journals (Sweden)

    Amit Dipak Amin

    2010-11-01

    Full Text Available The Saccharomyces cerevisiae RecQ helicase Sgs1 is essential for mitotic and meiotic genome stability. The stage at which Sgs1 acts during meiosis is subject to debate. Cytological experiments showed that a deletion of SGS1 leads to an increase in synapsis initiation complexes and axial associations leading to the proposal that it has an early role in unwinding surplus strand invasion events. Physical studies of recombination intermediates implicate it in the dissolution of double Holliday junctions between sister chromatids.In this work, we observed an increase in meiotic recombination between diverged sequences (homeologous recombination and an increase in unequal sister chromatid events when SGS1 is deleted. The first of these observations is most consistent with an early role of Sgs1 in unwinding inappropriate strand invasion events while the second is consistent with unwinding or dissolution of recombination intermediates in an Mlh1- and Top3-dependent manner. We also provide data that suggest that Sgs1 is involved in the rejection of 'second strand capture' when sequence divergence is present. Finally, we have identified a novel class of tetrads where non-sister spores (pairs of spores where each contains a centromere marker from a different parent are inviable. We propose a model for this unusual pattern of viability based on the inability of sgs1 mutants to untangle intertwined chromosomes. Our data suggest that this role of Sgs1 is not dependent on its interaction with Top3. We propose that in the absence of SGS1 chromosomes may sometimes remain entangled at the end of pre-meiotic replication. This, combined with reciprocal crossing over, could lead to physical destruction of the recombined and entangled chromosomes. We hypothesise that Sgs1, acting in concert with the topoisomerase Top2, resolves these structures.This work provides evidence that Sgs1 interacts with various partner proteins to maintain genome stability throughout

  12. Mcm3 replicative helicase mutation impairs neuroblast proliferation and memory in Drosophila.

    Science.gov (United States)

    Blumröder, R; Glunz, A; Dunkelberger, B S; Serway, C N; Berger, C; Mentzel, B; de Belle, J S; Raabe, T

    2016-09-01

    In the developing Drosophila brain, a small number of neural progenitor cells (neuroblasts) generate in a co-ordinated manner a high variety of neuronal cells by integration of temporal, spatial and cell-intrinsic information. In this study, we performed the molecular and phenotypic characterization of a structural brain mutant called small mushroom bodies (smu), which was isolated in a screen for mutants with altered brain structure. Focusing on the mushroom body neuroblast lineages we show that failure of neuroblasts to generate the normal number of mushroom body neurons (Kenyon cells) is the major cause of the smu phenotype. In particular, the premature loss of mushroom body neuroblasts caused a pronounced effect on the number of late-born Kenyon cells. Neuroblasts showed no obvious defects in processes controlling asymmetric cell division, but generated less ganglion mother cells. Cloning of smu uncovered a single amino acid substitution in an evolutionarily conserved protein interaction domain of the Minichromosome maintenance 3 (Mcm3) protein. Mcm3 is part of the multimeric Cdc45/Mcm/GINS (CMG) complex, which functions as a helicase during DNA replication. We propose that at least in the case of mushroom body neuroblasts, timely replication is not only required for continuous proliferation but also for their survival. The absence of Kenyon cells in smu reduced learning and early phases of conditioned olfactory memory. Corresponding to the absence of late-born Kenyon cells projecting to α'/β' and α/β lobes, smu is profoundly defective in later phases of persistent memory. © 2016 John Wiley & Sons Ltd and International Behavioural and Neural Genetics Society.

  13. Frequency of Werner helicase 1367 polymorphism and age-related morbidity in an elderly Brazilian population

    Directory of Open Access Journals (Sweden)

    M.A.C. Smith

    2005-07-01

    Full Text Available Werner syndrome (WS is a premature aging disease caused by a mutation in the WRN gene. The gene was identified in 1996 and its product acts as a DNA helicase and exonuclease. Some specific WRN polymorphic variants were associated with increased risk for cardiovascular diseases. The identification of genetic polymorphisms as risk factors for complex diseases affecting older people can improve their prevention, diagnosis and prognosis. We investigated WRN codon 1367 polymorphism in 383 residents in a district of the city of São Paulo, who were enrolled in an Elderly Brazilian Longitudinal Study. Their mean age was 79.70 ± 5.32 years, ranging from 67 to 97. This population was composed of 262 females (68.4% and 121 males (31.6% of European (89.2%, Japanese (3.3%, Middle Eastern (1.81%, and mixed and/or other origins (5.7%. There are no studies concerning this polymorphism in Brazilian population. These subjects were evaluated clinically every two years. The major health problems and morbidities affecting this cohort were cardiovascular diseases (21.7%, hypertension (83.7%, diabetes (63.3%, obesity (41.23%, dementia (8.0%, depression (20.0%, and neoplasia (10.8%. Their prevalence is similar to some urban elderly Brazilian samples. DNA was isolated from blood cells, amplified by PCR and digested with PmaCI. Allele frequencies were 0.788 for the cysteine and 0.211 for the arginine. Genotype distributions were within that expected for the Hardy-Weinberg equilibrium. Female gender was associated with hypertension and obesity. Logistic regression analysis did not detect significant association between the polymorphism and morbidity. These findings confirm those from Europeans and differ from Japanese population.

  14. RECQ5 Helicase Cooperates with MUS81 Endonuclease in Processing Stalled Replication Forks at Common Fragile Sites during Mitosis

    DEFF Research Database (Denmark)

    Di Marco, Stefano; Hasanova, Zdenka; Kanagaraj, Radhakrishnan

    2017-01-01

    The MUS81-EME1 endonuclease cleaves late replication intermediates at common fragile sites (CFSs) during early mitosis to trigger DNA-repair synthesis that ensures faithful chromosome segregation. Here, we show that these DNA transactions are promoted by RECQ5 DNA helicase in a manner dependent...... on its Ser727 phosphorylation by CDK1. Upon replication stress, RECQ5 associates with CFSs in early mitosis through its physical interaction with MUS81 and promotes MUS81-dependent mitotic DNA synthesis. RECQ5 depletion or mutational inactivation of its ATP-binding site, RAD51-interacting domain...

  15. Human RECQ5 helicase promotes repair of DNA double-strand breaks by synthesis-dependent strand annealing

    Czech Academy of Sciences Publication Activity Database

    Paliwal, S.; Kanagaraj, R.; Sturzenegger, A.; Burdová, Kamila; Janščák, Pavel

    2014-01-01

    Roč. 42, č. 4 (2014), s. 2380-2390 ISSN 0305-1048 R&D Projects: GA ČR GA204/09/0565; GA ČR GAP305/10/0281 Grant - others:Swiss National Science Foundation(CH) 31003A-129747; Swiss National Science Foundation(CH) 31003A_146206 Institutional support: RVO:68378050 Keywords : Human RECQ5 helicase * DNA double-strand breaks * mitotic homologous recombination Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 9.112, year: 2014

  16. Binding of DEAD-box helicase Dhh1 to the 5'-untranslated region of ASH1 mRNA represses localized translation of ASH1 in yeast cells.

    Science.gov (United States)

    Zhang, Qianjun; Meng, Xiuhua; Li, Delin; Chen, Shaoyin; Luo, Jianmin; Zhu, Linjie; Singer, Robert H; Gu, Wei

    2017-06-09

    Local translation of specific mRNAs is regulated by dynamic changes in their subcellular localization, and these changes are due to complex mechanisms controlling cytoplasmic mRNA transport. The budding yeast Saccharomyces cerevisiae is well suited to studying these mechanisms because many of its transcripts are transported from the mother cell to the budding daughter cell. Here, we investigated the translational control of ASH1 mRNA after transport and localization. We show that although ASH1 transcripts were translated after they reached the bud tip, some mRNAs were bound by the RNA-binding protein Puf6 and were non-polysomal. We also found that the DEAD-box helicase Dhh1 complexed with the untranslated ASH1 mRNA and Puf6. Loss of Dhh1 affected local translation of ASH1 mRNA and resulted in delocalization of ASH1 transcript in the bud. Forcibly shifting the non-polysomal ASH1 mRNA into polysomes was associated with Dhh1 dissociation. We further demonstrated that Dhh1 is not recruited to ASH1 mRNA co-transcriptionally, suggesting that it could bind to ASH1 mRNA within the cytoplasm. Of note, Dhh1 bound to the 5'-UTR of ASH1 mRNA and inhibited its translation in vitro These results suggest that after localization to the bud tip, a portion of the localized ASH1 mRNA becomes translationally inactive because of binding of Dhh1 and Puf6 to the 5'- and 3'-UTRs of ASH1 mRNA. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  17. DNA helicase HIM-6/BLM both promotes MutSγ-dependent crossovers and antagonizes MutSγ-independent interhomolog associations during caenorhabditis elegans meiosis.

    Science.gov (United States)

    Schvarzstein, Mara; Pattabiraman, Divya; Libuda, Diana E; Ramadugu, Ajit; Tam, Angela; Martinez-Perez, Enrique; Roelens, Baptiste; Zawadzki, Karl A; Yokoo, Rayka; Rosu, Simona; Severson, Aaron F; Meyer, Barbara J; Nabeshima, Kentaro; Villeneuve, Anne M

    2014-09-01

    Meiotic recombination is initiated by the programmed induction of double-strand DNA breaks (DSBs), lesions that pose a potential threat to the genome. A subset of the DSBs induced during meiotic prophase become designated to be repaired by a pathway that specifically yields interhomolog crossovers (COs), which mature into chiasmata that temporarily connect the homologs to ensure their proper segregation at meiosis I. The remaining DSBs must be repaired by other mechanisms to restore genomic integrity prior to the meiotic divisions. Here we show that HIM-6, the Caenorhabditis elegans ortholog of the RecQ family DNA helicase BLM, functions in both of these processes. We show that him-6 mutants are competent to load the MutSγ complex at multiple potential CO sites, to generate intermediates that fulfill the requirements of monitoring mechanisms that enable meiotic progression, and to accomplish and robustly regulate CO designation. However, recombination events at a subset of CO-designated sites fail to mature into COs and chiasmata, indicating a pro-CO role for HIM-6/BLM that manifests itself late in the CO pathway. Moreover, we find that in addition to promoting COs, HIM-6 plays a role in eliminating and/or preventing the formation of persistent MutSγ-independent associations between homologous chromosomes. We propose that HIM-6/BLM enforces biased outcomes of recombination events to ensure that both (a) CO-designated recombination intermediates are reliably resolved as COs and (b) other recombination intermediates reliably mature into noncrossovers in a timely manner. Copyright © 2014 by the Genetics Society of America.

  18. Wireless Access

    Indian Academy of Sciences (India)

    First page Back Continue Last page Overview Graphics. Wireless Access. Wireless connect to the Base station. Easy and Convenient access. Costlier as compared to the wired technology. Reliability challenges. We see it as a complementary technology to the DSL.

  19. Open access

    NARCIS (Netherlands)

    Valkenburg, P.M.

    2015-01-01

    Open access week Van 19 tot en met 25 oktober 2015 vond wereldwijd de Open Access Week plaats. Tijdens deze week werden er over de hele wereld evenementen georganiseerd waar open access een rol speelt. Ook in Nederland zijn er diverse symposia, workshops en debatten georganiseerd zoals het debat in

  20. An ATR-dependent function for the Ddx19 RNA helicase in nuclear R-loop metabolism.

    Science.gov (United States)

    Hodroj, Dana; Recolin, Bénédicte; Serhal, Kamar; Martinez, Susan; Tsanov, Nikolay; Abou Merhi, Raghida; Maiorano, Domenico

    2017-05-02

    Coordination between transcription and replication is crucial in the maintenance of genome integrity. Disturbance of these processes leads to accumulation of aberrant DNA:RNA hybrids (R-loops) that, if unresolved, generate DNA damage and genomic instability. Here we report a novel, unexpected role for the nucleopore-associated mRNA export factor Ddx19 in removing nuclear R-loops formed upon replication stress or DNA damage. We show, in live cells, that Ddx19 transiently relocalizes from the nucleopore to the nucleus upon DNA damage, in an ATR/Chk1-dependent manner, and that Ddx19 nuclear relocalization is required to clear R-loops. Ddx19 depletion induces R-loop accumulation, proliferation-dependent DNA damage and defects in replication fork progression. Further, we show that Ddx19 resolves R-loops in vitro via its helicase activity. Furthermore, mutation of a residue phosphorylated by Chk1 in Ddx19 disrupts its interaction with Nup214 and allows its nuclear relocalization. Finally, we show that Ddx19 operates in resolving R-loops independently of the RNA helicase senataxin. Altogether these observations put forward a novel, ATR-dependent function for Ddx19 in R-loop metabolism to preserve genome integrity in mammalian cells. © 2017 The Authors.

  1. TbPIF5 is a Trypanosoma brucei mitochondrial DNA helicase involved in processing of minicircle Okazaki fragments.

    Directory of Open Access Journals (Sweden)

    Beiyu Liu

    2009-09-01

    Full Text Available Trypanosoma brucei's mitochondrial genome, kinetoplast DNA (kDNA, is a giant network of catenated DNA rings. The network consists of a few thousand 1 kb minicircles and several dozen 23 kb maxicircles. Here we report that TbPIF5, one of T. brucei's six mitochondrial proteins related to Saccharomyces cerevisiae mitochondrial DNA helicase ScPIF1, is involved in minicircle lagging strand synthesis. Like its yeast homolog, TbPIF5 is a 5' to 3' DNA helicase. Together with other enzymes thought to be involved in Okazaki fragment processing, TbPIF5 localizes in vivo to the antipodal sites flanking the kDNA. Minicircles in wild type cells replicate unidirectionally as theta-structures and are unusual in that Okazaki fragments are not joined until after the progeny minicircles have segregated. We now report that overexpression of TbPIF5 causes premature removal of RNA primers and joining of Okazaki fragments on theta structures. Further elongation of the lagging strand is blocked, but the leading strand is completed and the minicircle progeny, one with a truncated H strand (ranging from 0.1 to 1 kb, are segregated. The minicircles with a truncated H strand electrophorese on an agarose gel as a smear. This replication defect is associated with kinetoplast shrinkage and eventual slowing of cell growth. We propose that TbPIF5 unwinds RNA primers after lagging strand synthesis, thus facilitating processing of Okazaki fragments.

  2. Dissociation from DNA of Type III Restriction–Modification enzymes during helicase-dependent motion and following endonuclease activity

    Science.gov (United States)

    Tóth, Júlia; van Aelst, Kara; Salmons, Hannah; Szczelkun, Mark D.

    2012-01-01

    DNA cleavage by the Type III Restriction–Modification (RM) enzymes requires the binding of a pair of RM enzymes at two distant, inversely orientated recognition sequences followed by helicase-catalysed ATP hydrolysis and long-range communication. Here we addressed the dissociation from DNA of these enzymes at two stages: during long-range communication and following DNA cleavage. First, we demonstrated that a communicating species can be trapped in a DNA domain without a recognition site, with a non-specific DNA association lifetime of ∼200 s. If free DNA ends were present the lifetime became too short to measure, confirming that ends accelerate dissociation. Secondly, we observed that Type III RM enzymes can dissociate upon DNA cleavage and go on to cleave further DNA molecules (they can ‘turnover’, albeit inefficiently). The relationship between the observed cleavage rate and enzyme concentration indicated independent binding of each site and a requirement for simultaneous interaction of at least two enzymes per DNA to achieve cleavage. In light of various mechanisms for helicase-driven motion on DNA, we suggest these results are most consistent with a thermally driven random 1D search model (i.e. ‘DNA sliding’). PMID:22523084

  3. Cdc45 (cell division cycle protein 45) guards the gate of the Eukaryote Replisome helicase stabilizing leading strand engagement

    Science.gov (United States)

    Petojevic, Tatjana; Pesavento, James J.; Costa, Alessandro; Liang, Jingdan; Wang, Zhijun; Berger, James M.; Botchan, Michael R.

    2015-01-01

    DNA replication licensing is now understood to be the pathway that leads to the assembly of double hexamers of minichromosome maintenance (Mcm2–7) at origin sites. Cell division control protein 45 (Cdc45) and GINS proteins activate the latent Mcm2–7 helicase by inducing allosteric changes through binding, forming a Cdc45/Mcm2-7/GINS (CMG) complex that is competent to unwind duplex DNA. The CMG has an active gate between subunits Mcm2 and Mcm5 that opens and closes in response to nucleotide binding. The consequences of inappropriate Mcm2/5 gate actuation and the role of a side channel formed between GINS/Cdc45 and the outer edge of the Mcm2–7 ring for unwinding have remained unexplored. Here we uncover a novel function for Cdc45. Cross-linking studies trace the path of the DNA with the CMG complex at a fork junction between duplex and single strands with the bound CMG in an open or closed gate conformation. In the closed state, the lagging strand does not pass through the side channel, but in the open state, the leading strand surprisingly interacts with Cdc45. Mutations in the recombination protein J fold of Cdc45 that ablate this interaction diminish helicase activity. These data indicate that Cdc45 serves as a shield to guard against occasional slippage of the leading strand from the core channel. PMID:25561522

  4. Structural insights into RISC assembly facilitated by dsRNA-binding domains of human RNA helicase A (DHX9).

    Science.gov (United States)

    Fu, Qinqin; Yuan, Y Adam

    2013-03-01

    Intensive research interest has focused on small RNA-processing machinery and the RNA-induced silencing complex (RISC), key cellular machines in RNAi pathways. However, the structural mechanism regarding RISC assembly, the primary step linking small RNA processing and RNA-mediated gene silencing, is largely unknown. Human RNA helicase A (DHX9) was reported to function as an RISC-loading factor, and such function is mediated mainly by its dsRNA-binding domains (dsRBDs). Here, we report the crystal structures of human RNA helicase A (RHA) dsRBD1 and dsRBD2 domains in complex with dsRNAs, respectively. Structural analysis not only reveals higher siRNA duplex-binding affinity displayed by dsRBD1, but also identifies a crystallographic dsRBD1 pair of physiological significance in cooperatively recognizing dsRNAs. Structural observations are further validated by isothermal titration calorimetric (ITC) assay. Moreover, co-immunoprecipitation (co-IP) assay coupled with mutagenesis demonstrated that both dsRBDs are required for RISC association, and such association is mediated by dsRNA. Hence, our structural and functional efforts have revealed a potential working model for siRNA recognition by RHA tandem dsRBDs, and together they provide direct structural insights into RISC assembly facilitated by RHA.

  5. The helicase, DDX3X, interacts with poly(A)-binding protein 1 (PABP1) and caprin-1 at the leading edge of migrating fibroblasts and is required for efficient cell spreading.

    Science.gov (United States)

    Copsey, Alice C; Cooper, Simon; Parker, Robert; Lineham, Ella; Lapworth, Cuzack; Jallad, Deema; Sweet, Steve; Morley, Simon J

    2017-08-30

    DDX3X, a helicase, can interact directly with mRNA and translation initiation factors, regulating the selective translation of mRNAs that contain a structured 5' untranslated region. This activity modulates the expression of mRNAs controlling cell cycle progression and mRNAs regulating actin dynamics, contributing to cell adhesion and motility. Previously, we have shown that ribosomes and translation initiation factors localise to the leading edge of migrating fibroblasts in loci enriched with actively translating ribosomes, thereby promoting steady-state levels of ArpC2 and Rac1 proteins at the leading edge of cells during spreading. As DDX3X can regulate Rac1 levels, cell motility and metastasis, we have examined DDX3X protein interactions and localisation using many complementary approaches. We now show that DDX3X can physically interact and co-localise with poly(A)-binding protein 1 and caprin-1 at the leading edge of spreading cells. Furthermore, as depletion of DDX3X leads to decreased cell motility, this provides a functional link between DDX3X, caprin-1 and initiation factors at the leading edge of migrating cells to promote cell migration and spreading. © 2017 The Author(s).

  6. Limited Access to Food and Physiological Trade-Offs in a Long-Distance Migrant Shorebird. I. Energy Metabolism, Behavior, and Body-Mass Regulation

    NARCIS (Netherlands)

    Vezina, Francois; Petit, Magali; Buehler, Deborah M.; Dekinga, Anne; Piersma, Theunis

    2009-01-01

    Previous experiments showed reduction of basal metabolic rate (BMR) in birds facing energetic challenges. We alternately exposed two groups of red knots (Calidris canutus) to either 6 h or 22 h of food availability for periods of 22 d. Six h of access to food led to a 6%-10% loss of body mass over

  7. Mycobacterium smegmatis SftH exemplifies a distinctive clade of superfamily II DNA-dependent ATPases with 3' to 5' translocase and helicase activities.

    Science.gov (United States)

    Yakovleva, Lyudmila; Shuman, Stewart

    2012-08-01

    Bacterial DNA helicases are nucleic acid-dependent NTPases that play important roles in DNA replication, recombination and repair. We are interested in the DNA helicases of Mycobacteria, a genus of the phylum Actinobacteria, which includes the human pathogen Mycobacterium tuberculosis and its avirulent relative Mycobacterium smegmatis. Here, we identify and characterize M. smegmatis SftH, a superfamily II helicase with a distinctive domain structure, comprising an N-terminal NTPase domain and a C-terminal DUF1998 domain (containing a putative tetracysteine metal-binding motif). We show that SftH is a monomeric DNA-dependent ATPase/dATPase that translocates 3' to 5' on single-stranded DNA and has 3' to 5' helicase activity. SftH homologs are found in bacteria representing 12 different phyla, being especially prevalent in Actinobacteria (including M. tuberculosis). SftH homologs are evident in more than 30 genera of Archaea. Among eukarya, SftH homologs are present in plants and fungi.

  8. Arabidopsis RecQsim, a plant-specific member of the RecQ helicase family, can suppress the MMS hypersensitivity of the yeast sgs1 mutant

    NARCIS (Netherlands)

    Bagherieh-Najjar, MB; de Vries, OMH; Kroon, JTM; Wright, EL; Elborough, KM; Hille, J; Dijkwel, PP

    The Arabidopsis genome contains seven genes that belong to the RecQ family of ATP-dependent DNA helicases. RecQ members in Saccharomyces cerevisiae (SGS1) and man (WRN, BLM and RecQL4) are involved in DNA recombination, repair and genome stability maintenance, but little is known about the function

  9. Unique Helicase Determinants in the Essential Conjugative TraI Factor from Salmonella enterica Serovar Typhimurium Plasmid pCU1

    Energy Technology Data Exchange (ETDEWEB)

    McLaughlin, Krystle J.; Nash, Rebekah P.; Redinbo, Mathew R. (UNC)

    2014-06-16

    The widespread development of multidrug-resistant bacteria is a major health emergency. Conjugative DNA plasmids, which harbor a wide range of antibiotic resistance genes, also encode the protein factors necessary to orchestrate the propagation of plasmid DNA between bacterial cells through conjugative transfer. Successful conjugative DNA transfer depends on key catalytic components to nick one strand of the duplex DNA plasmid and separate the DNA strands while cell-to-cell transfer occurs. The TraI protein from the conjugative Salmonella plasmid pCU1 fulfills these key catalytic roles, as it contains both single-stranded DNA-nicking relaxase and ATP-dependent helicase domains within a single, 1,078-residue polypeptide. In this work, we unraveled the helicase determinants of Salmonella pCU1 TraI through DNA binding, ATPase, and DNA strand separation assays. TraI binds DNA substrates with high affinity in a manner influenced by nucleic acid length and the presence of a DNA hairpin structure adjacent to the nick site. TraI selectively hydrolyzes ATP, and mutations in conserved helicase motifs eliminate ATPase activity. Surprisingly, the absence of a relatively short (144-residue) domain at the extreme C terminus of the protein severely diminishes ATP-dependent strand separation. Collectively, these data define the helicase motifs of the conjugative factor TraI from Salmonella pCU1 and reveal a previously uncharacterized C-terminal functional domain that uncouples ATP hydrolysis from strand separation activity.

  10. Robust translocation along a molecular monorail: the NS3 helicase from hepatitis C virus traverses unusually large disruptions in its track.

    Science.gov (United States)

    Beran, Rudolf K F; Bruno, Michael M; Bowers, Heath A; Jankowsky, Eckhard; Pyle, Anna Marie

    2006-05-12

    The NS3 helicase is essential for replication of the hepatitis C virus. This multifunctional Superfamily 2 helicase protein unwinds nucleic acid duplexes in a stepwise, ATP-dependent manner. Although kinetic features of its mechanism are beginning to emerge, little is known about the physical determinants for NS3 translocation along a strand of nucleic acid. For example, it is not known whether NS3 can traverse covalent or physical discontinuities on the tracking strand. Here we provide evidence that NS3 translocates with a mechanism that is different from its well-studied relative, the Vaccinia helicase NPH-II. Like NPH-II, NS3 translocates along the loading strand (the strand bearing the 3'-overhang) and it fails to unwind substrates that contain nicks, or covalent discontinuities in the loading strand. However, unlike NPH-II, NS3 readily unwinds RNA duplexes that contain long stretches of polyglycol, which are moieties that bear no resemblance to nucleic acid. Whether located on the tracking strand, the top strand, or both, long polyglycol regions fail to disrupt the function of NS3. This suggests that NS3 does not require the continuous formation of specific contacts with the ribose-phosphate backbone as it translocates along an RNA duplex, which is an observation consistent with the large NS3 kinetic step size (18 base-pairs). Rather, once NS3 loads onto a substrate, the helicase can translocate along the loading strand of an RNA duplex like a monorail train following a track. Bumps in the track do not significantly disturb NS3 unwinding, but a break in the track de-rails the helicase.

  11. Mathematic Model of Digital Control System with PID Regulator and Regular Step of Quantization with Information Transfer via the Channel of Plural Access

    Science.gov (United States)

    Abramov, G. V.; Emeljanov, A. E.; Ivashin, A. L.

    Theoretical bases for modeling a digital control system with information transfer via the channel of plural access and a regular quantization cycle are submitted. The theory of dynamic systems with random changes of the structure including elements of the Markov random processes theory is used for a mathematical description of a network control system. The characteristics of similar control systems are received. Experimental research of the given control systems is carried out.

  12. Open Access

    Science.gov (United States)

    Suber, Peter

    2012-01-01

    The Internet lets us share perfect copies of our work with a worldwide audience at virtually no cost. We take advantage of this revolutionary opportunity when we make our work "open access": digital, online, free of charge, and free of most copyright and licensing restrictions. Open access is made possible by the Internet and copyright-holder…

  13. Open access

    African Journals Online (AJOL)

    Prof. Dennis Ocholla

    The argument that access to information is an instrumental and individual as well as ... and Dean School of Information Studies, University of Wisconsin, Milwaukee, USA. ... to scholarly publications and can be in any digital format, including text, movies and ... language barriers, censorship, lack of access to the Internet and ...

  14. HTLV-1 Tax plugs and freezes UPF1 helicase leading to nonsense-mediated mRNA decay inhibition.

    Science.gov (United States)

    Fiorini, Francesca; Robin, Jean-Philippe; Kanaan, Joanne; Borowiak, Malgorzata; Croquette, Vincent; Le Hir, Hervé; Jalinot, Pierre; Mocquet, Vincent

    2018-01-30

    Up-Frameshift Suppressor 1 Homolog (UPF1) is a key factor for nonsense-mediated mRNA decay (NMD), a cellular process that can actively degrade mRNAs. Here, we study NMD inhibition during infection by human T-cell lymphotropic virus type I (HTLV-1) and characterise the influence of the retroviral Tax factor on UPF1 activity. Tax interacts with the central helicase core domain of UPF1 and might plug the RNA channel of UPF1, reducing its affinity for nucleic acids. Furthermore, using a single-molecule approach, we show that the sequential interaction of Tax with a RNA-bound UPF1 freezes UPF1: this latter is less sensitive to the presence of ATP and shows translocation defects, highlighting the importance of this feature for NMD. These mechanistic insights reveal how HTLV-1 hijacks the central component of NMD to ensure expression of its own genome.

  15. RECQ HELICASE RECQL4 PARTICIPATES IN NON-HOMOLOGOUS END JOINING AND INTERACTS WITH THE KU COMPLEX

    DEFF Research Database (Denmark)

    Shamanna, Raghavendra A; Singh, Dharmendra Kumar; Lu, Huiming

    2014-01-01

    -irradiation and resulted in accumulation of 53BP1 foci after irradiation, indicating defects in the processing of DSB. We find that RECQL4 interacts with the Ku70/Ku80 heterodimer, part of the DNA-dependent protein kinase (DNA-PK) complex, via its N-terminal domain. Further, RECQL4 stimulates higher order DNA binding...... of Ku70/Ku80 to a blunt end DNA substrate. Taken together, these results implicate that RECQL4 participates in the NHEJ pathway of DSB repair via a functional interaction with the Ku70/Ku80 complex. This is the first study to provide both in vitro and in vivo evidence for a role of a RecQ helicase...

  16. Disintegration of cruciform and G-quadruplex structures during the course of helicase-dependent amplification (HDA).

    Science.gov (United States)

    Li, Dawei; Lv, Bei; Zhang, Hao; Lee, Jasmine Yiqin; Li, Tianhu

    2015-04-15

    Unlike chemical damages on DNA, physical alterations of B-form of DNA occur commonly in organisms that serve as signals for specified cellular events. Although the modes of action for repairing of chemically damaged DNA have been well studied nowadays, the repairing mechanisms for physically altered DNA structures have not yet been understood. Our current in vitro studies show that both breakdown of stable non-B DNA structures and resumption of canonical B-conformation of DNA can take place during the courses of isothermal helicase-dependent amplification (HDA). The pathway that makes the non-B DNA structures repairable is presumably the relieving of the accumulated torsional stress that was caused by the positive supercoiling. Our new findings suggest that living organisms might have evolved this distinct and economical pathway for repairing their physically altered DNA structures. Copyright © 2015 Elsevier Ltd. All rights reserved.

  17. Mutations in the putative zinc-binding motif of UL52 demonstrate a complex interdependence between the UL5 and UL52 subunits of the human herpes simplex virus type 1 helicase/primase complex.

    Science.gov (United States)

    Chen, Yan; Carrington-Lawrence, Stacy D; Bai, Ping; Weller, Sandra K

    2005-07-01

    Herpes simplex virus type 1 (HSV-1) encodes a heterotrimeric helicase-primase (UL5/8/52) complex. UL5 contains seven motifs found in helicase superfamily 1, and UL52 contains conserved motifs found in primases. The contributions of each subunit to the biochemical activities of the complex, however, remain unclear. We have previously demonstrated that a mutation in the putative zinc finger at UL52 C terminus abrogates not only primase but also ATPase, helicase, and DNA-binding activities of a UL5/UL52 subcomplex, indicating a complex interdependence between the two subunits. To test this hypothesis and to further investigate the role of the zinc finger in the enzymatic activities of the helicase-primase, a series of mutations were constructed in this motif. They differed in their ability to complement a UL52 null virus: totally defective, partial complementation, and potentiating. In this study, four of these mutants were studied biochemically after expression and purification from insect cells infected with recombinant baculoviruses. All mutants show greatly reduced primase activity. Complementation-defective mutants exhibited severe defects in ATPase, helicase, and DNA-binding activities. Partially complementing mutants displayed intermediate levels of these activities, except that one showed a wild-type level of helicase activity. These data suggest that the UL52 zinc finger motif plays an important role in the activities of the helicase-primase complex. The observation that mutations in UL52 affected helicase, ATPase, and DNA-binding activities indicates that UL52 binding to DNA via the zinc finger may be necessary for loading UL5. Alternatively, UL5 and UL52 may share a DNA-binding interface.

  18. Access Contested

    International Development Research Centre (IDRC) Digital Library (Canada)

    Transforming Global Information and Communication Markets: The Political Economy of ... 8 Control and Resistance: Attacks on Burmese Opposition Media 153 ...... “Reluctant Gatekeepers: Corporate Ethics on a Filtered Internet,” in Access ...

  19. Accessing memory

    Science.gov (United States)

    Yoon, Doe Hyun; Muralimanohar, Naveen; Chang, Jichuan; Ranganthan, Parthasarathy

    2017-09-26

    A disclosed example method involves performing simultaneous data accesses on at least first and second independently selectable logical sub-ranks to access first data via a wide internal data bus in a memory device. The memory device includes a translation buffer chip, memory chips in independently selectable logical sub-ranks, a narrow external data bus to connect the translation buffer chip to a memory controller, and the wide internal data bus between the translation buffer chip and the memory chips. A data access is performed on only the first independently selectable logical sub-rank to access second data via the wide internal data bus. The example method also involves locating a first portion of the first data, a second portion of the first data, and the second data on the narrow external data bus during separate data transfers.

  20. Forbidden Access

    CERN Multimedia

    C. Colloca TS/FM

    2004-01-01

    TS/FM group informs you that, for the replacement of the door of the main entrance at bldg. 500, the access will be closed to the public between 19 and 30 July 2004. Access to the Main Building complex will be assured at any time through both of the side doors and from bldg. 64. For more information, please contact 73273. C. Colloca TS/FM

  1. Crucial role of dynamic linker histone binding and divalent ions for DNA accessibility and gene regulation revealed by mesoscale modeling of oligonucleosomes

    Science.gov (United States)

    Collepardo-Guevara, Rosana; Schlick, Tamar

    2012-01-01

    Monte Carlo simulations of a mesoscale model of oligonucleosomes are analyzed to examine the role of dynamic-linker histone (LH) binding/unbinding in high monovalent salt with divalent ions, and to further interpret noted chromatin fiber softening by dynamic LH in monovalent salt conditions. We find that divalent ions produce a fiber stiffening effect that competes with, but does not overshadow, the dramatic softening triggered by dynamic-LH behavior. Indeed, we find that in typical in vivo conditions, dynamic-LH binding/unbinding reduces fiber stiffening dramatically (by a factor of almost 5, as measured by the elasticity modulus) compared with rigidly fixed LH, and also the force needed to initiate chromatin unfolding, making it consistent with those of molecular motors. Our data also show that, during unfolding, divalent ions together with LHs induce linker-DNA bending and DNA–DNA repulsion screening, which guarantee formation of heteromorphic superbeads-on-a-string structures that combine regions of loose and compact fiber independently of the characteristics of the LH–core bond. These structures might be important for gene regulation as they expose regions of the DNA selectively. Dynamic control of LH binding/unbinding, either globally or locally, in the presence of divalent ions, might constitute a mechanism for regulation of gene expression. PMID:22790986

  2. Accessible Knowledge - Knowledge on Accessibility

    DEFF Research Database (Denmark)

    Kirkeby, Inge Mette

    2015-01-01

    Although serious efforts are made internationally and nationally, it is a slow process to make our physical environment accessible. In the actual design process, architects play a major role. But what kinds of knowledge, including research-based knowledge, do practicing architects make use of when...... designing accessible environments? The answer to the question is crucially important since it affects how knowledge is distributed and how accessibility can be ensured. In order to get first-hand knowledge about the design process and the sources from which they gain knowledge, 11 qualitative interviews...... were conducted with architects with experience of designing for accessibility. The analysis draws on two theoretical distinctions. The first is research-based knowledge versus knowledge used by architects. The second is context-independent knowledge versus context-dependent knowledge. The practitioners...

  3. Open access

    CERN Document Server

    Suber, Peter

    2012-01-01

    The Internet lets us share perfect copies of our work with a worldwide audience at virtually no cost. We take advantage of this revolutionary opportunity when we make our work "open access": digital, online, free of charge, and free of most copyright and licensing restrictions. Open access is made possible by the Internet and copyright-holder consent, and many authors, musicians, filmmakers, and other creators who depend on royalties are understandably unwilling to give their consent. But for 350 years, scholars have written peer-reviewed journal articles for impact, not for money, and are free to consent to open access without losing revenue. In this concise introduction, Peter Suber tells us what open access is and isn't, how it benefits authors and readers of research, how we pay for it, how it avoids copyright problems, how it has moved from the periphery to the mainstream, and what its future may hold. Distilling a decade of Suber's influential writing and thinking about open access, this is the indispe...

  4. Concerted action of the PHD, chromo and motor domains regulates the human chromatin remodelling ATPase CHD4

    OpenAIRE

    Morra, Rosa; Lee, Benjamin M; Shaw, Heather; Tuma, Roman; Mancini, Erika J

    2012-01-01

    CHD4, the core subunit of the Nucleosome Remodelling and Deacetylase (NuRD) complex, is a chromatin remodelling ATPase that, in addition to a helicase domain, harbors tandem plant homeo finger and chromo domains. By using a panel of domain constructs we dissect their roles and demonstrate that DNA binding, histone binding and ATPase activities are allosterically regulated. Molecular shape reconstruction from small-angle X-ray scattering reveals extensive domain-domain interactions, which prov...

  5. p300-mediated acetylation of the Rothmund-Thomson-syndrome gene product RECQL4 regulates its subcellular localization

    DEFF Research Database (Denmark)

    Dietschy, Tobias; Shevelev, Igor; Pena Diaz, Javier

    2009-01-01

    RECQL4 belongs to the conserved RecQ family of DNA helicases, members of which play important roles in the maintenance of genome stability in all organisms that have been examined. Although genetic alterations in the RECQL4 gene are reported to be associated with three autosomal recessive disorde...... by p300 regulates the trafficking of RECQL4 between the nucleus and the cytoplasm....

  6. Heteroduplex DNA position defines the roles of the Sgs1, Srs2, and Mph1 helicases in promoting distinct recombination outcomes.

    Directory of Open Access Journals (Sweden)

    Katrina Mitchel

    Full Text Available The contributions of the Sgs1, Mph1, and Srs2 DNA helicases during mitotic double-strand break (DSB repair in yeast were investigated using a gap-repair assay. A diverged chromosomal substrate was used as a repair template for the gapped plasmid, allowing mismatch-containing heteroduplex DNA (hDNA formed during recombination to be monitored. Overall DSB repair efficiencies and the proportions of crossovers (COs versus noncrossovers (NCOs were determined in wild-type and helicase-defective strains, allowing the efficiency of CO and NCO production in each background to be calculated. In addition, the products of individual NCO events were sequenced to determine the location of hDNA. Because hDNA position is expected to differ depending on whether a NCO is produced by synthesis-dependent-strand-annealing (SDSA or through a Holliday junction (HJ-containing intermediate, its position allows the underlying molecular mechanism to be inferred. Results demonstrate that each helicase reduces the proportion of CO recombinants, but that each does so in a fundamentally different way. Mph1 does not affect the overall efficiency of gap repair, and its loss alters the CO-NCO by promoting SDSA at the expense of HJ-containing intermediates. By contrast, Sgs1 and Srs2 are each required for efficient gap repair, strongly promoting NCO formation and having little effect on CO efficiency. hDNA analyses suggest that all three helicases promote SDSA, and that Sgs1 and Srs2 additionally dismantle HJ-containing intermediates. The hDNA data are consistent with the proposed role of Sgs1 in the dissolution of double HJs, and we propose that Srs2 dismantles nicked HJs.

  7. Synthesis and SAR studies of 5-(pyridin-4-yl)-1,3,4-thiadiazol-2-amine derivatives as potent inhibitors of Bloom helicase

    DEFF Research Database (Denmark)

    Rosenthal, Andrew S; Dexheimer, Thomas S; Gileadi, Opher

    2013-01-01

    complementary strands of duplex DNA as well as atypical DNA structures such as Holliday junctions. Mutations of the BLM gene can result in Bloom syndrome, an autosomal recessive disorder associated with cancer predisposition. BLM-deficient cells exhibit increased sensitivity to DNA damaging agents indicating...... and related analogs, which possess potent BLM inhibition and exhibit selectivity over related helicases. Moreover, these compounds demonstrated cellular activity by inducing sister chromatid exchanges, a hallmark of Bloom syndrome....

  8. RECQ5 helicase associates with the C-terminal repeat domain of RNA polymerase II during productive elongation phase of transcription

    Czech Academy of Sciences Publication Activity Database

    Kanagaraj, R.; Huehn, D.; Mackellar, A.; Menigatti, M.; Zheng, L.; Urban, Václav; Shevelev, Igor; Greenleaf, A.L.; Janščák, Pavel

    2010-01-01

    Roč. 38, č. 22 (2010), s. 8131-8140 ISSN 0305-1048 R&D Projects: GA ČR GA204/09/0565 Grant - others:SNSF(CH) 3100A0-116008; NIH(US) GM040505 Institutional research plan: CEZ:AV0Z50520514 Keywords : RECQ5 DNA helicase * transcription * genome stability Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 7.836, year: 2010

  9. Cloning and expression of NS3 helicase fragment of hepatitis C virus and the study of its immunoreactivity in HCV infected patients

    Directory of Open Access Journals (Sweden)

    Mahrou Sadri

    2015-02-01

    Full Text Available Objective(s: Hepatitis C is a major cause of liver failure worldwide. Current therapies applied for this disease are not fully effective and produce side effects in most cases. Non-structural protein 3 helicase (NS3 of HCV is one of the key enzymes in viral replication and infection. Therefore, this region is a promising target to design new drugs and therapies against HCV infection. The aim of this study was cloning and expression of HCV NS3 helicase fragment in Escherichia coli BL21 (DE3 using pET102/D-TOPO expression vector and studying immunoreactivity of the expressed antigen in Iranian infected with hepatitis C. Materials and Methods: The viral RNA was extracted from the serum of HCV infected patient. The NS3 helicase region was amplified by RT-PCR. The PCR product was directionally cloned into the expression vector pET102/D-TOPO and transformed into the BL21 strain of E. coli (DE3. The transformed bacteria were then induced by adding 1mM isopropyl-β-D-thiogalactopyranoside (IPTG into the culture medium to enhance the protein expression. SDS-PAGE and western blotting were carried out to identify the protein under investigation, and finally purified recombinant fusion protein was used as the antigen for ELISA method. Results: Theinsertion of theDNA fragment of the NS3 regioninto the expression vectorwas further confirmed by PCR and sequencing. SDS-PAGE analysis showed the successful expression of the recombinant protein of interest. Furthermore, immunoreactivity of fusion NS3 helicase was confirmed by ELISA and western blotting. Conclusion: It seems that this recombinant protein could be a useful source of antigen for future studies on HCV diagnosis and therapy.

  10. Current economic cost, the ARENH (Regulated Access to the Historic (EDF) Nuclear Energy Supplier) price, the differential rent and the scarcity rent of nuclear power: some observations

    International Nuclear Information System (INIS)

    Percebois, J.

    2012-01-01

    This article sets out to explain the ARENH mechanism, the regulated price at which the EDF (France's historic monopoly supplier) must sell part of its production to its competitors in the framework of the Nome Act (New Organisation of the Electricity Market). This price by its nature is different from the Current Economic Cost (CEC) of nuclear power, as estimated by France's Government Accounting Office in its report submitted in January 2012. These two approaches revert to the problem of the determination of the scarcity rent of nuclear power in the context of the liberalised European market, in which nuclear power benefits from a 'cost' advantage relative to thermal electricity generated from fossil fuels. Furthermore, scarcity rent is not the same thing as differential rent. Selling a nuclear kWh at the price of a 'gas' kWh at certain times results in a differential rent enabling nuclear generated power to cover fixed costs. One can only speak of scarcity rent for nuclear power when the price at which the kWh is sold allows the recovery of more than is necessary to cover overall costs of nuclear power and it is this scarcity rent and its allocation that is the subject of debate. (author)

  11. Medical tourism's impact on health care equity and access in low- and middle-income countries: making the case for regulation.

    Science.gov (United States)

    Chen, Y Y Brandon; Flood, Colleen M

    2013-01-01

    There is currently an evidentiary gap in the scholarship concerning medical tourism's impact on low- and middle-income destination countries (LMICs). This article reviews relevant evidence that exists and concludes that there are signs of correlation between medical tourism and the expansion of private, technology- intensive health care in LMICs, which has largely remained out of reach for the majority of the local patients. In light of this health care inequity between local residents and medical tourists in LMICs, we argue that the presumption should not be in favor of medical tourism and that governments have a legitimate interest in seeking to regulate this industry to ensure that the net effects for their citizens is positive. Moreover, sending countries, particularly those in the developed world, have the responsibility to adopt public policies to diminish demand on the part of their citizens for medical tourism and to work with LMICs to ensure that the growth of medical tourism does not occur at the expense of the poorest of the poor. © 2013 American Society of Law, Medicine & Ethics, Inc.

  12. Access French

    CERN Document Server

    Grosz, Bernard

    2014-01-01

    Access is the major new language series designed with the needs of today's generation of students firmly in mind. Whether learning for leisure or business purposes or working towards a curriculum qualification, Access French is specially designed for adults of all ages and gives students a thorough grounding in all the skills required to understand, speak, read and write contemporary French from scratch. The coursebook consists of 10 units covering different topic areas, each of which includes Language Focus panels explaining the structures covered and a comprehensive glossary. Learning tips

  13. Open access to transmission

    International Nuclear Information System (INIS)

    Keith, D.M.

    1996-01-01

    For the past 12 to 15 years, the US electric power and energy industry and its federal regulators have been going through a prolonged exercise leading to opening up the national interconnected transmission grid for all qualified wholesale users to have open and equal access. The debates have been painful in a sense that not all parties - especially some of the transmission system owning utilities - believe that the concept of Open Access is achievable, due to technical constraints on the systems. The present Open Access activity is limited to wholesales transaction under the federal jurisdiction, but several states are either experimenting with or considering retail wheeling. In fact, the FERC - Federal Energy Regulatory Commission - has already expanded its view to embrace retail transmission, if the retail transaction involves the use of the interstate transmission systems which are under FERC's jurisdiction. This paper delves into some of the results of the technical cost and pricing analysis for open access. The statutes and resulting regulations are not addressed herein. (author). 1 fig

  14. Interplay between chromatin modulators and histone acetylation regulates the formation of accessible chromatin in the upstream regulatory region of fission yeast fbp1.

    Science.gov (United States)

    Adachi, Akira; Senmatsu, Satoshi; Asada, Ryuta; Abe, Takuya; Hoffman, Charles S; Ohta, Kunihiro; Hirota, Kouji

    2018-05-03

    Numerous noncoding RNA transcripts are detected in eukaryotic cells. Noncoding RNAs transcribed across gene promoters are involved in the regulation of mRNA transcription via chromatin modulation. This function of noncoding RNA transcription was first demonstrated for the fission yeast fbp1 gene, where a cascade of noncoding RNA transcription events induces chromatin remodeling to facilitate transcription factor binding. We recently demonstrated that the noncoding RNAs from the fbp1 upstream region facilitate binding of the transcription activator Atf1 and thereby promote histone acetylation. Histone acetylation by histone acetyl transferases (HATs) and ATP-dependent chromatin remodelers (ADCRs) are implicated in chromatin remodeling, but the interplay between HATs and ADCRs in this process has not been fully elucidated. Here, we examine the roles played by two distinct ADCRs, Snf22 and Hrp3, and by the HAT Gcn5 in the transcriptional activation of fbp1. Snf22 and Hrp3 redundantly promote disassembly of chromatin in the fbp1 upstream region. Gcn5 critically contributes to nucleosome eviction in the absence of either Snf22 or Hrp3, presumably by recruiting Hrp3 in snf22∆ cells and Snf22 in hrp3∆ cells. Conversely, Gcn5-dependent histone H3 acetylation is impaired in snf22∆/hrp3∆ cells, suggesting that both redundant ADCRs induce recruitment of Gcn5 to the chromatin array in the fbp1 upstream region. These results reveal a previously unappreciated interplay between ADCRs and histone acetylation in which histone acetylation facilitates recruitment of ADCRs, while ADCRs are required for histone acetylation.

  15. Gaining Access.

    Science.gov (United States)

    Wand, Sean; Thermos, Adam C.

    1998-01-01

    Explains the issues to consider before a college decides to purchase a card-access system. The benefits of automation, questions involving implementation, the criteria for technology selection, what typical card technology involves, privacy concerns, and the placement of card readers are discussed. (GR)

  16. Unwinding after high salinity stress: Pea DNA helicase 45 over- expression in tobacco confers high salinity tolerance without affecting yield (abstract)

    International Nuclear Information System (INIS)

    Tuteja, N.

    2005-01-01

    Soil salinity is an increasing threat for agriculture and is a major factor in reducing plant productivity; therefore, it is necessary to obtain salinity-tolerant varieties. A typical characteristic of soil salinity is the induction of multiple stress- inducible genes. Some of the genes encoding osmolytes, ion channels or enzymes are able to confer salinity-tolerant phenotypes when transferred to sensitive plants. As salinity stress affects the cellular gene-expression machinery, it is evident that molecules involved in nucleic acid processing including helicases, are likely to be affected as well. DNA helicases unwind duplex DNA and are involved in replication, repair, recombination and transcription while RNA helicases unfold the secondary structures in RNA and are involved in transcription, ribosome biogenesis and translation initiation. We have earlier reported the isolation of a pea DNA helicase 45 (PDH45) that exhibits striking homology with eIF-4A (Plant J. 24:219-230,2000). Here we report that PDH45 mRNA is induced in pea seedlings in response to high salt and its over- expression driven by a constitutive CAMV-355-promoter in tobacco plants confers salinity tolerance, thus suggesting a new pathway for manipulating stress tolerance in crop plants. The T0 transgenic plants showed high-levels of PDH45 protein in normal and stress conditions, as compared to wild type (WT) plants. The T0 transgenics also showed tolerance to high salinity as tested by a leaf disc senescence assay. The T1 transgenics were able to grow to maturity and set normal viable seeds under continuous salinity stress, without any reduction in plant yield, in terms of seed weight. Measurement of Na/sup +/ ions in different parts of the plant showed higher accumulation in the old leaves and negligible in seeds of T1 transgenic lines as compared with the WT plants. The possible mechanism of salinity tolerance will be discussed. Over-expression of PDH45 provides a possible example of the

  17. Intrinsic and extrinsic predictors of video-gaming behaviour and adolescent bedtimes: the relationship between flow states, self-perceived risk-taking, device accessibility, parental regulation of media and bedtime.

    Science.gov (United States)

    Smith, Lisa J; Gradisar, Michael; King, Daniel L; Short, Michelle

    2017-02-01

    How computer games affect the time at which adolescents go to bed is of growing research interest; however, the intrinsic individual and extrinsic sociocultural factors mediating the relationship between gaming and sleep have received minimal attention. This paper investigates how gaming duration mediates the relationship between intrinsic factors (perception of risky events and flow) and extrinsic factors (parental regulation and media accessibility) and adolescent bedtime. Adolescents (N = 422; age = 16.3 ± 2.02 years, 41% M) from six metropolitan schools and the Flinders University completed a questionnaire battery. More flow states (r = .34, p relationship between flow and bedtime during adolescence was fully mediated by gaming duration (b = .142, p Flow and parental regulation of media were identified as the key points for clinical intervention to decrease the duration of gaming of adolescents, thus promoting earlier bedtimes. Crown Copyright © 2016. Published by Elsevier B.V. All rights reserved.

  18. Structure based modification of Bluetongue virus helicase protein VP6 to produce a viable VP6-truncated BTV

    Energy Technology Data Exchange (ETDEWEB)

    Matsuo, Eiko [Microbiology and Immunology, Division of Animal Science, Department of Bioresource Science, Graduate School of Agricultural Science, Kobe University, 1-1, Rokkodai, Nada-ku, Kobe-City 657-8501 (Japan); Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT (United Kingdom); Leon, Esther; Matthews, Steve J. [Division of Molecular Biosciences, Centre for Structural Biology, Imperial College London, South Kensington, London SW7 2AZ (United Kingdom); Roy, Polly, E-mail: polly.roy@lshtm.ac.uk [Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT (United Kingdom)

    2014-09-05

    Highlights: • NMR analysis on BTV VP6 reveals two large loop regions. • The loss of a loop (aa 34–130) does not affect the overall fold of the protein. • A region of VP6 (aa 34–92) is not required for BTV replication. • A region of VP6 (aa 93–130) plays an essential role in the virus replication. - Abstract: Bluetongue virus core protein VP6 is an ATP hydrolysis dependent RNA helicase. However, despite much study, the precise role of VP6 within the viral capsid and its structure remain unclear. To investigate the requirement of VP6 in BTV replication, we initiated a structural and biological study. Multinuclear nuclear magnetic resonance spectra were assigned on his-tagged full-length VP6 (329 amino acid residues) as well as several truncated VP6 variants. The analysis revealed a large structured domain with two large loop regions that exhibit significant conformational exchange. One of the loops (amino acid position 34–130) could be removed without affecting the overall fold of the protein. Moreover, using a BTV reverse genetics system, it was possible to demonstrate that the VP6-truncated BTV was viable in BHK cells in the absence of any helper VP6 protein, suggesting that a large portion of this loop region is not absolutely required for BTV replication.

  19. Relationship between osteosarcoma and ionizing radiation hypersensitive human B lymphocyte cells lacking RecQL4 helicase

    International Nuclear Information System (INIS)

    Kohzaki, Masaoki; Moritake, Takashi; Okazaki, Ryuji; Ootsuyama, Akira

    2015-01-01

    Japanese society is now facing a transition period from aging society to super aging society. Concomitant with this situation, it is estimated that number of cancer patients and the requirement of less invasive Radiation Therapy (RT) for cancers will increase. Therefore, understanding of mechanisms without delay on second cancers caused by RT is indispensable. Osteosarcoma, an aggressive bone tumor frequently occurring 5% of cancers in young adult and children, increase statistically after RT for cancers. Although, mutation in p53, Rb and RecQL4 genes statistically relate with osteosarcoma incidence, precise mechanisms of osteosarcoma development by ionizing Radiation (IR) remain to be elucidated. Genome instability is one of the tumor promoting factors and we focused on RecQL4 in RecQ helicase family, which is involved in aging and cancer. We established RecQL4 knock-in human B lymphocyte Nalm-6 cells and found their hypersensitivity to IR, replication fork stall/collapses after IR. In this review, we summarize recently published studies on genetic cancer-predisposing syndrome and possible origins of bone cancers induced by IR. Then, we discuss what and how we address molecular mechanisms on osteosarcoma induced by IR in the future. (author)

  20. Non-Watson–Crick interactions between PNA and DNA inhibit the ATPase activity of bacteriophage T4 Dda helicase

    Science.gov (United States)

    Tackett, Alan J.; Corey, David R.; Raney, Kevin D.

    2002-01-01

    Peptide nucleic acid (PNA) is a DNA mimic in which the nucleobases are linked by an N-(2-aminoethyl) glycine backbone. Here we report that PNA can interact with single-stranded DNA (ssDNA) in a non-sequence-specific fashion. We observed that a 15mer PNA inhibited the ssDNA-stimulated ATPase activity of a bacteriophage T4 helicase, Dda. Surprisingly, when a fluorescein-labeled 15mer PNA was used in binding studies no interaction was observed between PNA and Dda. However, fluorescence polarization did reveal non-sequence-specific interactions between PNA and ssDNA. Thus, the inhibition of ATPase activity of Dda appears to result from depletion of the available ssDNA due to non-Watson–Crick binding of PNA to ssDNA. Inhibition of the ssDNA-stimulated ATPase activity was observed for several PNAs of varying length and sequence. To study the basis for this phenomenon, we examined self-aggregation by PNAs. The 15mer PNA readily self-aggregates to the point of precipitation. Since PNAs are hydrophobic, they aggregate more than DNA or RNA, making the study of this phenomenon essential for understanding the properties of PNA. Non-sequence-specific interactions between PNA and ssDNA were observed at moderate concentrations of PNA, suggesting that such interactions should be considered for antisense and antigene applications. PMID:11842106

  1. Characterization of papillomavirus E1 helicase mutants defective for interaction with the SUMO-conjugating enzyme Ubc9

    International Nuclear Information System (INIS)

    Fradet-Turcotte, Amelie; Brault, Karine; Titolo, Steve; Howley, Peter M.; Archambault, Jacques

    2009-01-01

    The E1 helicase from BPV and HPV16 interacts with Ubc9 to facilitate viral genome replication. We report that HPV11 E1 also interacts with Ubc9 in vitro and in the yeast two-hybrid system. Residues in E1 involved in oligomerization (353-435) were sufficient for binding to Ubc9 in vitro, but the origin-binding and ATPase domains were additionally required in yeast. Nuclear accumulation of BPV E1 was shown previously to depend on its interaction with Ubc9 and sumoylation on lysine 514. In contrast, HPV11 and HPV16 E1 mutants defective for Ubc9 binding remained nuclear even when the SUMO pathway was inhibited. Furthermore, we found that K514 in BPV E1 and the analogous K559 in HPV11 E1 are not essential for nuclear accumulation of E1. These results suggest that the interaction of E1 with Ubc9 is not essential for its nuclear accumulation but, rather, depends on its oligomerization and binding to DNA and ATP.

  2. DNA unwinding by ASCC3 helicase is coupled to ALKBH3 dependent DNA alkylation repair and cancer cell proliferation

    Science.gov (United States)

    Dango, Sebastian; Mosammaparast, Nima; Sowa, Mathew E.; Xiong, Li-Jun; Wu, Feizhen; Park, Keyjung; Rubin, Mark; Gygi, Steve; Harper, J. Wade; Shi, Yang

    2011-01-01

    Summary Demethylation by the AlkB dioxygenases represents an important mechanism for repair of N-alkylated nucleotides. However, little is known about their functions in mammalian cells. We report the purification of the ALKBH3 complex and demonstrate its association with the Activating Signal Co-integrator Complex (ASCC). ALKBH3 is overexpressed in various cancers, and both ALKBH3 and ASCC are important for alkylation damage resistance in these tumor cell lines. ASCC3, the largest subunit of ASCC, encodes a 3′-5′ DNA helicase, whose activity is crucial for the generation of single-stranded DNA upon which ALKBH3 preferentially functions for dealkylation. In cell lines that are dependent on ALKBH3 and ASCC3 for alkylation damage resistance, loss of ALKBH3 or ASCC3 leads to increased 3-methylcytosine and reduced cell proliferation, which correlates with pH2A.X and 53BP1 foci formation. Our data provide a molecular mechanism by which ALKBH3 collaborates with ASCC to maintain genomic integrity in a cell type specific manner. PMID:22055184

  3. Development of Reverse Transcription Thermostable Helicase-Dependent DNA Amplification for the Detection of Tomato Spotted Wilt Virus.

    Science.gov (United States)

    Wu, Xinghai; Chen, Chanfa; Xiao, Xizhi; Deng, Ming Jun

    2016-11-01

    A protocol for the reverse transcription-helicase-dependent amplification (RT-HDA) of isothermal DNA was developed for the detection of tomato spotted wilt virus (TSWV). Specific primers, which were based on the highly conserved region of the N gene sequence in TSWV, were used for the amplification of virus's RNA. The LOD of RT-HDA, reverse transcriptase-loop-mediated isothermal amplification (RT-LAMP), and reverse transcriptase-polymerase chain reaction (RT-PCR) assays were conducted using 10-fold serial dilution of RNA eluates. TSWV sensitivity in RT-HDA and RT-LAMP was 4 pg RNA compared with 40 pg RNA in RT-PCR. The specificity of RT-HDA for TSWV was high, showing no cross-reactivity with other tomato and Tospovirus viruses including cucumber mosaic virus (CMV), tomato black ring virus (TBRV), tomato mosaic virus (ToMV), or impatiens necrotic spot virus (INSV). The RT-HDA method is effective for the detection of TSWV in plant samples and is a potential tool for early and rapid detection of TSWV.

  4. Helicase-Dependent Isothermal Amplification of DNA and RNA by Using Self-Avoiding Molecular Recognition Systems.

    Science.gov (United States)

    Yang, Zunyi; McLendon, Chris; Hutter, Daniel; Bradley, Kevin M; Hoshika, Shuichi; Frye, Carole B; Benner, Steven A

    2015-06-15

    Assays that detect DNA or RNA (xNA) are highly sensitive, as small amounts of xNA can be amplified by PCR. Unfortunately, PCR is inconvenient in low-resource environments, and requires equipment and power that might not be available in these environments. Isothermal procedures, which avoid thermal cycling, are often confounded by primer dimers, off-target priming, and other artifacts. Here, we show how a "self avoiding molecular recognition system" (SAMRS) eliminates these artifacts and gives clean amplicons in a helicase-dependent isothermal amplification (SAMRS-HDA). We also show that incorporating SAMRS into the 3'-ends of primers facilitates the design and screening of primers for HDA assays. Finally, we show that SAMRS-HDA can be twofold multiplexed, difficult to achieve with HDA using standard primers. Thus, SAMRS-HDA is a more versatile approach than standard HDA, with a broader applicability for xNA-targeted diagnostics and research. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. DHX9 helicase is involved in preventing genomic instability induced by alternatively structured DNA in human cells.

    Science.gov (United States)

    Jain, Aklank; Bacolla, Albino; Del Mundo, Imee M; Zhao, Junhua; Wang, Guliang; Vasquez, Karen M

    2013-12-01

    Sequences that have the capacity to adopt alternative (i.e. non-B) DNA structures in the human genome have been implicated in stimulating genomic instability. Previously, we found that a naturally occurring intra-molecular triplex (H-DNA) caused genetic instability in mammals largely in the form of DNA double-strand breaks. Thus, it is of interest to determine the mechanism(s) involved in processing H-DNA. Recently, we demonstrated that human DHX9 helicase preferentially unwinds inter-molecular triplex DNA in vitro. Herein, we used a mutation-reporter system containing H-DNA to examine the relevance of DHX9 activity on naturally occurring H-DNA structures in human cells. We found that H-DNA significantly increased mutagenesis in small-interfering siRNA-treated, DHX9-depleted cells, affecting mostly deletions. Moreover, DHX9 associated with H-DNA in the context of supercoiled plasmids. To further investigate the role of DHX9 in the recognition/processing of H-DNA, we performed binding assays in vitro and chromatin immunoprecipitation assays in U2OS cells. DHX9 recognized H-DNA, as evidenced by its binding to the H-DNA structure and enrichment at the H-DNA region compared with a control region in human cells. These composite data implicate DHX9 in processing H-DNA structures in vivo and support its role in the overall maintenance of genomic stability at sites of alternatively structured DNA.

  6. A Listeria monocytogenes RNA helicase essential for growth and ribosomal maturation at low temperatures uses its C terminus for appropriate interaction with the ribosome.

    Science.gov (United States)

    Netterling, Sakura; Vaitkevicius, Karolis; Nord, Stefan; Johansson, Jörgen

    2012-08-01

    Listeria monocytogenes, a Gram-positive food-borne human pathogen, is able to grow at temperatures close to 0°C and is thus of great concern for the food industry. In this work, we investigated the physiological role of one DExD-box RNA helicase in Listeria monocytogenes. The RNA helicase Lmo1722 was required for optimal growth at low temperatures, whereas it was dispensable at 37°C. A Δlmo1722 strain was less motile due to downregulation of the major subunit of the flagellum, FlaA, caused by decreased flaA expression. By ribosomal fractionation experiments, it was observed that Lmo1722 was mainly associated with the 50S subunit of the ribosome. Absence of Lmo1722 decreased the fraction of 50S ribosomal subunits and mature 70S ribosomes and affected the processing of the 23S precursor rRNA. The ribosomal profile could be restored to wild-type levels in a Δlmo1722 strain expressing Lmo1722. Interestingly, the C-terminal part of Lmo1722 was redundant for low-temperature growth, motility, 23S rRNA processing, and appropriate ribosomal maturation. However, Lmo1722 lacking the C terminus showed a reduced affinity for the 50S and 70S fractions, suggesting that the C terminus is important for proper guidance of Lmo1722 to the 50S subunit. Taken together, our results show that the Listeria RNA helicase Lmo1722 is essential for growth at low temperatures, motility, and rRNA processing and is important for ribosomal maturation, being associated mainly with the 50S subunit of the ribosome.

  7. Identification of unique interactions between the flexible linker and the RecA-like domains of DEAD-box helicase Mss116

    International Nuclear Information System (INIS)

    Zhang, Yuan; Palla, Mirkó; Liao, Jung-Chi; Sun, Andrew

    2013-01-01

    DEAD-box RNA helicases are ATP-dependent proteins implicated in nearly all aspects of RNA metabolism. The yeast DEAD-box helicase Mss116 is unique in its functions of splicing group I and group II introns and activating mRNA translation, but the structural understanding of why it performs these unique functions remains unclear. Here we used sequence analysis and molecular dynamics simulation to identify residues in the flexible linker specific for yeast Mss116, potentially associated with its unique functions. We first identified residues that are 100% conserved in Mss116 of different species of the Saccharomycetaceae family. The amino acids of these conserved residues were then compared with the amino acids of the corresponding residue positions of other RNA helicases to identify residues that have distinct amino acids from other DEAD-box proteins. Four residues in the flexible linker, i.e. N334, E335, P336 and H339, are conserved and Mss116-specific. Molecular dynamics simulation was conducted for the wild-type Mss116 structure and mutant models to examine mutational effects of the linker on the conformational equilibrium. Relatively short MD simulation runs (within 20 ns) were enough for us to observe mutational effects, suggesting serious structural perturbations by these mutations. The mutation of E335 depletes the interactions between E335 and K95 in domain 1. The interactions between N334/P336 and N496/I497 of domain 2 are also abolished by mutation. Our results suggest that tight interactions between the Mss116-specific flexible linker and the two RecA-like domains may be mechanically required to crimp RNA for the unique RNA processes of yeast Mss116. (paper)

  8. Identification of unique interactions between the flexible linker and the RecA-like domains of DEAD-box helicase Mss116

    Science.gov (United States)

    Zhang, Yuan; Palla, Mirkó; Sun, Andrew; Liao, Jung-Chi

    2013-09-01

    DEAD-box RNA helicases are ATP-dependent proteins implicated in nearly all aspects of RNA metabolism. The yeast DEAD-box helicase Mss116 is unique in its functions of splicing group I and group II introns and activating mRNA translation, but the structural understanding of why it performs these unique functions remains unclear. Here we used sequence analysis and molecular dynamics simulation to identify residues in the flexible linker specific for yeast Mss116, potentially associated with its unique functions. We first identified residues that are 100% conserved in Mss116 of different species of the Saccharomycetaceae family. The amino acids of these conserved residues were then compared with the amino acids of the corresponding residue positions of other RNA helicases to identify residues that have distinct amino acids from other DEAD-box proteins. Four residues in the flexible linker, i.e. N334, E335, P336 and H339, are conserved and Mss116-specific. Molecular dynamics simulation was conducted for the wild-type Mss116 structure and mutant models to examine mutational effects of the linker on the conformational equilibrium. Relatively short MD simulation runs (within 20 ns) were enough for us to observe mutational effects, suggesting serious structural perturbations by these mutations. The mutation of E335 depletes the interactions between E335 and K95 in domain 1. The interactions between N334/P336 and N496/I497 of domain 2 are also abolished by mutation. Our results suggest that tight interactions between the Mss116-specific flexible linker and the two RecA-like domains may be mechanically required to crimp RNA for the unique RNA processes of yeast Mss116.

  9. Targeting helicase-dependent amplification products with an electrochemical genosensor for reliable and sensitive screening of genetically modified organisms.

    Science.gov (United States)

    Moura-Melo, Suely; Miranda-Castro, Rebeca; de-Los-Santos-Álvarez, Noemí; Miranda-Ordieres, Arturo J; Dos Santos Junior, J Ribeiro; da Silva Fonseca, Rosana A; Lobo-Castañón, Maria Jesús

    2015-08-18

    Cultivation of genetically modified organisms (GMOs) and their use in food and feed is constantly expanding; thus, the question of informing consumers about their presence in food has proven of significant interest. The development of sensitive, rapid, robust, and reliable methods for the detection of GMOs is crucial for proper food labeling. In response, we have experimentally characterized the helicase-dependent isothermal amplification (HDA) and sequence-specific detection of a transgene from the Cauliflower Mosaic Virus 35S Promoter (CaMV35S), inserted into most transgenic plants. HDA is one of the simplest approaches for DNA amplification, emulating the bacterial replication machinery, and resembling PCR but under isothermal conditions. However, it usually suffers from a lack of selectivity, which is due to the accumulation of spurious amplification products. To improve the selectivity of HDA, which makes the detection of amplification products more reliable, we have developed an electrochemical platform targeting the central sequence of HDA copies of the transgene. A binary monolayer architecture is built onto a thin gold film where, upon the formation of perfect nucleic acid duplexes with the amplification products, these are enzyme-labeled and electrochemically transduced. The resulting combined system increases genosensor detectability up to 10(6)-fold, allowing Yes/No detection of GMOs with a limit of detection of ∼30 copies of the CaMV35S genomic DNA. A set of general utility rules in the design of genosensors for detection of HDA amplicons, which may assist in the development of point-of-care tests, is also included. The method provides a versatile tool for detecting nucleic acids with extremely low abundance not only for food safety control but also in the diagnostics and environmental control areas.

  10. The DNA repair endonuclease XPG interacts directly and functionally with the WRN helicase defective in Werner syndrome

    Energy Technology Data Exchange (ETDEWEB)

    Trego, Kelly S.; Chernikova, Sophia B.; Davalos, Albert R.; Perry, J. Jefferson P.; Finger, L. David; Ng, Cliff; Tsai, Miaw-Sheue; Yannone, Steven M.; Tainer, John A.; Campisi, Judith; Cooper, Priscilla K.

    2011-04-20

    XPG is a structure-specific endonuclease required for nucleotide excision repair (NER). XPG incision defects result in the cancer-prone syndrome xeroderma pigmentosum, whereas truncating mutations of XPG cause the severe postnatal progeroid developmental disorder Cockayne syndrome. We show that XPG interacts directly with WRN protein, which is defective in the premature aging disorder Werner syndrome, and that the two proteins undergo similar sub-nuclear redistribution in S-phase and co-localize in nuclear foci. The co-localization was observed in mid- to late-S-phase, when WRN moves from nucleoli to nuclear foci that have been shown to contain protein markers of both stalled replication forks and telomeric proteins. We mapped the interaction between XPG and WRN to the C-terminal domains of each and show that interaction with the C-terminal domain of XPG strongly stimulates WRN helicase activity. WRN also possesses a competing DNA single-strand annealing activity that, combined with unwinding, has been shown to coordinate regression of model replication forks to form Holliday junction/chicken foot intermediate structures. We tested whether XPG stimulated WRN annealing activity and found that XPG itself has intrinsic strand annealing activity that requires the unstructured R- and C-terminal domains, but not the conserved catalytic core or endonuclease activity. Annealing by XPG is cooperative, rather than additive, with WRN annealing. Taken together, our results suggest a novel function for XPG in S-phase that is at least in part carried out coordinately with WRN, and which may contribute to the severity of the phenotypes that occur upon loss of XPG.

  11. DNA2 cooperates with the WRN and BLM RecQ helicases to mediate long-range DNA end resection in human cells

    Czech Academy of Sciences Publication Activity Database

    Sturzenegger, A.; Burdová, Kamila; Kanagaraj, R.; Levikova, M.; Pinto, C.; Cejka, P.; Janščák, Pavel

    2014-01-01

    Roč. 289, č. 39 (2014), s. 27314-27326 ISSN 0021-9258 R&D Projects: GA ČR GAP305/10/0281 Grant - others:Swiss National Science Foundation(CH) 31003A-129747; Swiss National Science Foundation(CH) 31003A_146206; Swiss National Science Foundation(CH) PP00P3 133636; University of Zurich(CH) FK-13-098 Institutional support: RVO:68378050 Keywords : DNA Damage * DNA Helicase * DNA Recombination * DNA Repair * Genomic Instability * RecQ Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 4.573, year: 2014

  12. Regulated eukaryotic DNA replication origin firing with purified proteins.

    Science.gov (United States)

    Yeeles, Joseph T P; Deegan, Tom D; Janska, Agnieszka; Early, Anne; Diffley, John F X

    2015-03-26

    Eukaryotic cells initiate DNA replication from multiple origins, which must be tightly regulated to promote precise genome duplication in every cell cycle. To accomplish this, initiation is partitioned into two temporally discrete steps: a double hexameric minichromosome maintenance (MCM) complex is first loaded at replication origins during G1 phase, and then converted to the active CMG (Cdc45-MCM-GINS) helicase during S phase. Here we describe the reconstitution of budding yeast DNA replication initiation with 16 purified replication factors, made from 42 polypeptides. Origin-dependent initiation recapitulates regulation seen in vivo. Cyclin-dependent kinase (CDK) inhibits MCM loading by phosphorylating the origin recognition complex (ORC) and promotes CMG formation by phosphorylating Sld2 and Sld3. Dbf4-dependent kinase (DDK) promotes replication by phosphorylating MCM, and can act either before or after CDK. These experiments define the minimum complement of proteins, protein kinase substrates and co-factors required for regulated eukaryotic DNA replication.

  13. 7 CFR 29.75 - Accessibility of tobacco.

    Science.gov (United States)

    2010-01-01

    ... 7 Agriculture 2 2010-01-01 2010-01-01 false Accessibility of tobacco. 29.75 Section 29.75... REGULATIONS TOBACCO INSPECTION Regulations Mandatory Inspection § 29.75 Accessibility of tobacco. (a) All tobacco subject to mandatory inspection shall be made readily accessible for inspection. (b)(1) Each...

  14. Mycobacterial UvrD1 is a Ku-dependent DNA helicase that plays a role in multiple DNA repair events, including double-strand break repair.

    Science.gov (United States)

    Sinha, Krishna Murari; Stephanou, Nicolas C; Gao, Feng; Glickman, Michael S; Shuman, Stewart

    2007-05-18

    Mycobacterium tuberculosis and other bacterial pathogens have a Ku-dependent nonhomologous end joining pathway of DNA double-strand break repair. Here we identify mycobacterial UvrD1 as a novel interaction partner for Ku in a genome-wide yeast two-hybrid screen. UvrD1 per se is a vigorous DNA-dependent ATPase but a feeble DNA helicase. Ku stimulates UvrD1 to catalyze ATP-dependent unwinding of 3'-tailed DNAs. UvrD1, Ku, and DNA form a stable ternary complex in the absence of ATP. The Ku binding determinants are located in the distinctive C-terminal segment of UvrD1. A second mycobacterial paralog, UvrD2, is a vigorous Ku-independent DNA helicase. Ablation of UvrD1 sensitizes Mycobacterium smegmatis to killing by ultraviolet and ionizing radiation and to a single chromosomal break generated by I-SceI endonuclease. The physical and functional interactions of bacterial Ku and UvrD1 highlight the potential for cross-talk between components of nonhomologous end joining and nucleotide excision repair pathways.

  15. DndEi Exhibits Helicase Activity Essential for DNA Phosphorothioate Modification and ATPase Activity Strongly Stimulated by DNA Substrate with a GAAC/GTTC Motif.

    Science.gov (United States)

    Zheng, Tao; Jiang, Pan; Cao, Bo; Cheng, Qiuxiang; Kong, Lingxin; Zheng, Xiaoqing; Hu, Qinghai; You, Delin

    2016-01-15

    Phosphorothioate (PT) modification of DNA, in which the non-bridging oxygen of the backbone phosphate group is replaced by sulfur, is governed by the DndA-E proteins in prokaryotes. To better understand the biochemical mechanism of PT modification, functional analysis of the recently found PT-modifying enzyme DndEi, which has an additional domain compared with canonical DndE, from Riemerella anatipestifer is performed in this study. The additional domain is identified as a DNA helicase, and functional deletion of this domain in vivo leads to PT modification deficiency, indicating an essential role of helicase activity in PT modification. Subsequent analysis reveals that the additional domain has an ATPase activity. Intriguingly, the ATPase activity is strongly stimulated by DNA substrate containing a GAAC/GTTC motif (i.e. the motif at which PT modifications occur in R. anatipestifer) when the additional domain and the other domain (homologous to canonical DndE) are co-expressed as a full-length DndEi. These results reveal that PT modification is a biochemical process with DNA strand separation and intense ATP hydrolysis. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  16. Helicase-dependent isothermal amplification: a novel tool in the development of molecular-based analytical systems for rapid pathogen detection.

    Science.gov (United States)

    Barreda-García, Susana; Miranda-Castro, Rebeca; de-Los-Santos-Álvarez, Noemí; Miranda-Ordieres, Arturo J; Lobo-Castañón, María Jesús

    2018-01-01

    Highly sensitive testing of nucleic acids is essential to improve the detection of pathogens, which pose a major threat for public health worldwide. Currently available molecular assays, mainly based on PCR, have a limited utility in point-of-need control or resource-limited settings. Consequently, there is a strong interest in developing cost-effective, robust, and portable platforms for early detection of these harmful microorganisms. Since its description in 2004, isothermal helicase-dependent amplification (HDA) has been successfully applied in the development of novel molecular-based technologies for rapid, sensitive, and selective detection of viruses and bacteria. In this review, we highlight relevant analytical systems using this simple nucleic acid amplification methodology that takes place at a constant temperature and that is readily compatible with microfluidic technologies. Different strategies for monitoring HDA amplification products are described. In addition, we present technological advances for integrating sample preparation, HDA amplification, and detection. Future perspectives and challenges toward point-of-need use not only for clinical diagnosis but also in food safety testing and environmental monitoring are also discussed. Graphical Abstract Expanding the analytical toolbox for the detection of DNA sequences specific of pathogens with isothermal helicase dependent amplification (HDA).

  17. Formation of a Trimeric Xpo1-Ran[GTP]-Ded1 Exportin Complex Modulates ATPase and Helicase Activities of Ded1.

    Directory of Open Access Journals (Sweden)

    Glenn Hauk

    Full Text Available The DEAD-box RNA helicase Ded1, which is essential in yeast and known as DDX3 in humans, shuttles between the nucleus and cytoplasm and takes part in several basic processes including RNA processing and translation. A key interacting partner of Ded1 is the exportin Xpo1, which together with the GTP-bound state of the small GTPase Ran, facilitates unidirectional transport of Ded1 out of the nucleus. Here we demonstrate that Xpo1 and Ran[GTP] together reduce the RNA-stimulated ATPase and helicase activities of Ded1. Binding and inhibition of Ded1 by Xpo1 depend on the affinity of the Ded1 nuclear export sequence (NES for Xpo1 and the presence of Ran[GTP]. Association with Xpo1/Ran[GTP] reduces RNA-stimulated ATPase activity of Ded1 by increasing the apparent KM for the RNA substrate. Despite the increased KM, the Ded1:Xpo1:Ran[GTP] ternary complex retains the ability to bind single stranded RNA, suggesting that Xpo1/Ran[GTP] may modulate the substrate specificity of Ded1. These results demonstrate that, in addition to transport, exportins such as Xpo1 also have the capability to alter enzymatic activities of their cargo.

  18. The helicase and RNaseIIIa domains of Arabidopsis Dicer-Like1 modulate catalytic parameters during MicroRNA biogenesis

    KAUST Repository

    Liu, Chenggang

    2012-04-03

    Dicer-Like1 (DCL1), an RNaseIII endonuclease, and Hyponastic Leaves1 (HYL1), a double-stranded RNA-binding protein, are core components of the plant microRNA (miRNA) biogenesis machinery. hyl1 mutants accumulate low levels of miRNAs and display pleiotropic developmental phenotypes. We report the identification of five new hyl1 suppressor mutants, all of which are alleles of DCL1. These new alleles affect either the helicase or the RNaseIIIa domains of DCL1, highlighting the critical functions of these domains. Biochemical analysis of the DCL1 suppressor variants reveals that they process the primary transcript (pri-miRNA) more efficiently than wild-type DCL1, with both higher Kcat and lower Km values. The DCL1 variants largely rescue wild-type miRNA accumulation levels in vivo, but do not rescue the MIRNA processing precision defects of the hyl1 mutant. In vitro, the helicase domain confers ATP dependence on DCL1-catalyzed MIRNA processing, attenuates DCL1 cleavage activity, and is required for precise MIRNA processing of some substrates. © 2012 American Society of Plant Biologists.

  19. Cas3 is a single-stranded DNA nuclease and ATP-dependent helicase in the CRISPR/Cas immune system.

    Science.gov (United States)

    Sinkunas, Tomas; Gasiunas, Giedrius; Fremaux, Christophe; Barrangou, Rodolphe; Horvath, Philippe; Siksnys, Virginijus

    2011-04-06

    Clustered regularly interspaced short palindromic repeat (CRISPR) is a recently discovered adaptive prokaryotic immune system that provides acquired immunity against foreign nucleic acids by utilizing small guide crRNAs (CRISPR RNAs) to interfere with invading viruses and plasmids. In Escherichia coli, Cas3 is essential for crRNA-guided interference with virus proliferation. Cas3 contains N-terminal HD phosphohydrolase and C-terminal Superfamily 2 (SF2) helicase domains. Here, we provide the first report of the cloning, expression, purification and in vitro functional analysis of the Cas3 protein of the Streptococcus thermophilus CRISPR4 (Ecoli subtype) system. Cas3 possesses a single-stranded DNA (ssDNA)-stimulated ATPase activity, which is coupled to unwinding of DNA/DNA and RNA/DNA duplexes. Cas3 also shows ATP-independent nuclease activity located in the HD domain with a preference for ssDNA substrates. To dissect the contribution of individual domains, Cas3 separation-of-function mutants (ATPase(+)/nuclease(-) and ATPase(-)/nuclease(+)) were obtained by site-directed mutagenesis. We propose that the Cas3 ATPase/helicase domain acts as a motor protein, which assists delivery of the nuclease activity to Cascade-crRNA complex targeting foreign DNA.

  20. The rem mutations in the ATP-binding groove of the Rad3/XPD helicase lead to Xeroderma pigmentosum-Cockayne syndrome-like phenotypes.

    Science.gov (United States)

    Herrera-Moyano, Emilia; Moriel-Carretero, María; Montelone, Beth A; Aguilera, Andrés

    2014-12-01

    The eukaryotic TFIIH complex is involved in Nucleotide Excision Repair and transcription initiation. We analyzed three yeast mutations of the Rad3/XPD helicase of TFIIH known as rem (recombination and mutation phenotypes). We found that, in these mutants, incomplete NER reactions lead to replication fork breaking and the subsequent engagement of the homologous recombination machinery to restore them. Nevertheless, the penetrance varies among mutants, giving rise to a phenotype gradient. Interestingly, the mutations analyzed reside at the ATP-binding groove of Rad3 and in vivo experiments reveal a gain of DNA affinity upon damage of the mutant Rad3 proteins. Since mutations at the ATP-binding groove of XPD in humans are present in the Xeroderma pigmentosum-Cockayne Syndrome (XP-CS), we recreated rem mutations in human cells, and found that these are XP-CS-like. We propose that the balance between the loss of helicase activity and the gain of DNA affinity controls the capacity of TFIIH to open DNA during NER, and its persistence at both DNA lesions and promoters. This conditions NER efficiency and transcription resumption after damage, which in human cells would explain the XP-CS phenotype, opening new perspectives to understand the molecular basis of the role of XPD in human disease.

  1. NS3 from Hepatitis C Virus Strain JFH-1 Is an Unusually Robust Helicase That Is Primed To Bind and Unwind Viral RNA

    Energy Technology Data Exchange (ETDEWEB)

    Zhou, Ting; Ren, Xiaoming; Adams, Rebecca L.; Pyle, Anna Marie; Ou, J. -H. James

    2017-10-25

    Hepatitis C viruses (HCV) encode a helicase enzyme that is essential for viral replication and assembly (nonstructural protein 3 [NS3]). This helicase has become the focus of extensive basic research on the general helicase mechanism, and it is also of interest as a novel drug target. Despite the importance of this protein, mechanistic work on NS3 has been conducted almost exclusively on variants from HCV genotype 1. Our understanding of NS3 from the highly active HCV strains that are used to study HCV genetics and mechanism in cell culture (such as JFH-1) is lacking. We therefore set out to determine whether NS3 from the replicatively efficient genotype 2a strain JFH-1 displays novel functional or structural properties. Using biochemical assays for RNA binding and duplex unwinding, we show that JFH-1 NS3 binds RNA much more rapidly than the previously studied NS3 variants from genotype 1b. Unlike NS3 variants from other genotypes, JFH-1 NS3 binds RNA with high affinity in a functionally active form that is capable of immediately unwinding RNA duplexes without undergoing rate-limiting conformational changes that precede activation. Unlike other superfamily 2 (SF2) helicases, JFH-1 NS3 does not require long 3' overhangs, and it unwinds duplexes that are flanked by only a few nucleotides, as in the folded HCV genome. To understand the physical basis for this, we solved the crystal structure of JFH-1 NS3, revealing a novel conformation that contains an open, positively charged RNA binding cleft that is primed for productive interaction with RNA targets, potentially explaining robust replication by HCV JFH-1.

    IMPORTANCEGenotypes of HCV are as divergent as different types of flavivirus, and yet mechanistic features of HCV variants are presumed to be held in common. One of the most well-studied components of the HCV replication complex is a helicase known as nonstructural protein 3 (NS3). We set out to determine whether this important

  2. 7 CFR 29.403 - Accessibility of tobacco.

    Science.gov (United States)

    2010-01-01

    ... 7 Agriculture 2 2010-01-01 2010-01-01 false Accessibility of tobacco. 29.403 Section 29.403 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards... REGULATIONS TOBACCO INSPECTION Regulations Miscellaneous § 29.403 Accessibility of tobacco. All tobacco...

  3. IMPROVING ACCESS TO DRUGS

    Directory of Open Access Journals (Sweden)

    Max Joseph Herman

    2012-11-01

    Full Text Available Although essentially not all therapies need drug intervention, drugs is still an important components in health sector, either in preventive, curative, rehabilitative or promotion efforts. Hence the access to drugs is a main problem, either in international or national scale even to the smallest unit. The problem on access to drugs is very complicated and cannot be separated especially from pharmacy management problems; moreover in general from the overall lack of policy development and effective of health policy, and also the implementation process. With the policy development and effective health policy, rational drug uses, sufficient health service budget so a country can overcome the health problems. Besides infrastructures, regulations, distribution and cultural influences; the main obstacles for drug access is drugs affordability if the price of drugs is an important part and determined by many factors, especially the drug status whether is still patent orgenerics that significantly decrease cost of health cares and enhance the drugs affordability. The determination of essential drug prices in developing countries should based on equity principal so that poor people pay cheaper and could afford the essential drugs. WHO predicts two third of world population can not afford the essential drugs in which in developing countries, some are because of in efficient budget allocation in consequence of drug distribution management, including incorrect selection and allocation and also irrational uses. In part these could be overcome by enhancing performances on the allocation pharmacy needs, including the management of information system, inventory management, stock management and the distribution. Key words: access, drugs, essential drugs, generic drugs

  4. 78 FR 10166 - Access Interpreting; Transfer of Data

    Science.gov (United States)

    2013-02-13

    ... regulations. Access Interpreting has been awarded a contract to perform work for OPP, and access to this information will enable Access Interpreting to fulfill the obligations of the contract. DATES: Access.... Contractor Requirements Under Contract No. EP10H000109, this contract is to provide the Environmental...

  5. A Novel Rrm3 Function in Restricting DNA Replication via an Orc5-Binding Domain Is Genetically Separable from Rrm3 Function as an ATPase/Helicase in Facilitating Fork Progression

    DEFF Research Database (Denmark)

    Syed, Salahuddin; Madsen, Claus Desler; Rasmussen, Lene J.

    2016-01-01

    hydroxyurea. This novel Rrm3 function is independent of its established role as an ATPase/helicase in facilitating replication fork progression through polymerase blocking obstacles. Using quantitative mass spectrometry and genetic analyses, we find that the homologous recombination factor Rdh54 and Rad5...

  6. Enterovirus Exposure Uniquely Discriminates Type 1 Diabetes Patients with a Homozygous from a Heterozygous Melanoma Differentiation-Associated Protein 5/Interferon Induced with Helicase C Domain 1 A946T Genotype

    NARCIS (Netherlands)

    Schulte, B.M.; Gielen, P.R.; Kers-Rebel, E.D.; Prosser, A.C.; Lind, K.; Flodstrom-Tullberg, M.; Tack, C.J.J.; Elving, L.D.; Adema, G.J.

    2016-01-01

    In children at risk for type 1 diabetes, innate immune activity is detected before seroconversion. Enterovirus infections have been linked to diabetes development, and a polymorphism (A946T) in the innate immune sensor recognizing enterovirus RNA, interferon-induced with helicase C domain 1/melanoma

  7. Decision Analysis of Dynamic Spectrum Access Rules

    Energy Technology Data Exchange (ETDEWEB)

    Juan D. Deaton; Luiz A. DaSilva; Christian Wernz

    2011-12-01

    A current trend in spectrum regulation is to incorporate spectrum sharing through the design of spectrum access rules that support Dynamic Spectrum Access (DSA). This paper develops a decision-theoretic framework for regulators to assess the impacts of different decision rules on both primary and secondary operators. We analyze access rules based on sensing and exclusion areas, which in practice can be enforced through geolocation databases. Our results show that receiver-only sensing provides insufficient protection for primary and co-existing secondary users and overall low social welfare. On the other hand, using sensing information between the transmitter and receiver of a communication link, provides dramatic increases in system performance. The performance of using these link end points is relatively close to that of using many cooperative sensing nodes associated to the same access point and large link exclusion areas. These results are useful to regulators and network developers in understanding in developing rules for future DSA regulation.

  8. Farmers Perception of the Quality and Accessibility of ...

    African Journals Online (AJOL)

    User

    enforcement of pesticide regulations, high cost, and porous borders. This study therefore .... personal savings were very accessible, while accessibility of agrochemicals from ..... Valuing the Benefits and Costs of Improved Food Safety and.

  9. A new MCM modification cycle regulates DNA replication initiation.

    Science.gov (United States)

    Wei, Lei; Zhao, Xiaolan

    2016-03-01

    The MCM DNA helicase is a central regulatory target during genome replication. MCM is kept inactive during G1, and it initiates replication after being activated in S phase. During this transition, the only known chemical change to MCM is the gain of multisite phosphorylation that promotes cofactor recruitment. Because replication initiation is intimately linked to multiple biological cues, additional changes to MCM can provide further regulatory points. Here, we describe a yeast MCM SUMOylation cycle that regulates replication. MCM subunits undergo SUMOylation upon loading at origins in G1 before MCM phosphorylation. MCM SUMOylation levels then decline as MCM phosphorylation levels rise, thus suggesting an inhibitory role of MCM SUMOylation during replication. Indeed, increasing MCM SUMOylation impairs replication initiation, partly through promoting the recruitment of a phosphatase that decreases MCM phosphorylation and activation. We propose that MCM SUMOylation counterbalances kinase-based regulation, thus ensuring accurate control of replication initiation.

  10. Conviviality-driven access control policy

    NARCIS (Netherlands)

    El Kateb, Donia; Zannone, N.; Moawad, Assaad; Caire, Patrice; Nain, Grégory; Mouelhi, Tejeddine; Le Traon, Yves

    2015-01-01

    Nowadays many organizations experience security incidents due to unauthorized access to information. To reduce the risk of such incidents, security policies are often employed to regulate access to information. Such policies, however, are often too restrictive, and users do not have the rights

  11. Mycobacterium smegmatis RqlH defines a novel clade of bacterial RecQ-like DNA helicases with ATP-dependent 3'-5' translocase and duplex unwinding activities.

    Science.gov (United States)

    Ordonez, Heather; Unciuleac, Mihaela; Shuman, Stewart

    2012-05-01

    The Escherichia coli RecQ DNA helicase participates in a pathway of DNA repair that operates in parallel to the recombination pathway driven by the multisubunit helicase-nuclease machine RecBCD. The model mycobacterium Mycobacterium smegmatis executes homologous recombination in the absence of its helicase-nuclease machine AdnAB, though it lacks a homolog of E. coli RecQ. Here, we identify and characterize M. smegmatis RqlH, a RecQ-like helicase with a distinctive domain structure. The 691-amino acid RqlH polypeptide consists of a RecQ-like ATPase domain (amino acids 1-346) and tetracysteine zinc-binding domain (amino acids 435-499), separated by an RqlH-specific linker. RqlH lacks the C-terminal HRDC domain found in E. coli RecQ. Rather, the RqlH C-domain resembles bacterial ComF proteins and includes a phosphoribosyltransferase-like module. We show that RqlH is a DNA-dependent ATPase/dATPase that translocates 3'-5' on single-stranded DNA and has 3'-5' helicase activity. These functions inhere to RqlH-(1-505), a monomeric motor unit comprising the ATPase, linker and zinc-binding domains. RqlH homologs are distributed widely among bacterial taxa. The mycobacteria that encode RqlH lack a classical RecQ, though many other Actinobacteria have both RqlH and RecQ. Whereas E. coli K12 encodes RecQ but lacks a homolog of RqlH, other strains of E. coli have both RqlH and RecQ.

  12. The UL5 and UL52 subunits of the herpes simplex virus type 1 helicase-primase subcomplex exhibit a complex interdependence for DNA binding.

    Science.gov (United States)

    Biswas, N; Weller, S K

    2001-05-18

    Herpes simplex virus type 1 encodes a heterotrimeric helicase-primase complex composed of the products of the UL5, UL52, and UL8 genes. The UL5 protein contains seven motifs found in all members of helicase Superfamily 1 (SF1), and the UL52 protein contains several conserved motifs found in primases; however, the contributions of each subunit to the biochemical activities of the subcomplex are not clear. In this work, the DNA binding properties of wild type and mutant subcomplexes were examined using single-stranded, duplex, and forked substrates. A gel mobility shift assay indicated that the UL5-UL52 subcomplex binds more efficiently to the forked substrate than to either single strand or duplex DNA. Although nucleotides are not absolutely required for DNA binding, ADP stimulated the binding of UL5-UL52 to single strand DNA whereas ATP, ADP, and adenosine 5'-O-(thiotriphosphate) stimulated the binding to a forked substrate. We have previously shown that both subunits contact single-stranded DNA in a photocross-linking assay (Biswas, N., and Weller, S. K. (1999) J. Biol. Chem. 274, 8068-8076). In this study, photocross-linking assays with forked substrates indicate that the UL5 and UL52 subunits contact the forked substrates at different positions, UL52 at the single-stranded DNA tail and UL5 near the junction between single-stranded and double-stranded DNA. Neither subunit was able to cross-link a forked substrate when 5-iododeoxyuridine was located within the duplex portion. Photocross-linking experiments with subcomplexes containing mutant versions of UL5 and wild type UL52 indicated that the integrity of the ATP binding region is important for DNA binding of both subunits. These results support our previous proposal that UL5 and UL52 exhibit a complex interdependence for DNA binding (Biswas, N., and Weller, S. K. (1999) J. Biol. Chem. 274, 8068-8076) and indicate that the UL52 subunit may play a more active role in helicase activity than had previously been

  13. The dsRNA binding protein RDE-4 interacts with RDE-1, DCR-1, and a DExH-box helicase to direct RNAi in C. elegans.

    Science.gov (United States)

    Tabara, Hiroaki; Yigit, Erbay; Siomi, Haruhiko; Mello, Craig C

    2002-06-28

    Double-stranded (ds) RNA induces potent gene silencing, termed RNA interference (RNAi). At an early step in RNAi, an RNaseIII-related enzyme, Dicer (DCR-1), processes long-trigger dsRNA into small interfering RNAs (siRNAs). DCR-1 is also required for processing endogenous regulatory RNAs called miRNAs, but how DCR-1 recognizes its endogenous and foreign substrates is not yet understood. Here we show that the C. elegans RNAi pathway gene, rde-4, encodes a dsRNA binding protein that interacts during RNAi with RNA identical to the trigger dsRNA. RDE-4 protein also interacts in vivo with DCR-1, RDE-1, and a conserved DExH-box helicase. Our findings suggest a model in which RDE-4 and RDE-1 function together to detect and retain foreign dsRNA and to present this dsRNA to DCR-1 for processing.

  14. The Crystal Structure of the Drosophila Germline Inducer Oskar Identifies Two Domains with Distinct Vasa Helicase- and RNA-Binding Activities

    Directory of Open Access Journals (Sweden)

    Mandy Jeske

    2015-07-01

    Full Text Available In many animals, the germ plasm segregates germline from soma during early development. Oskar protein is known for its ability to induce germ plasm formation and germ cells in Drosophila. However, the molecular basis of germ plasm formation remains unclear. Here, we show that Oskar is an RNA-binding protein in vivo, crosslinking to nanos, polar granule component, and germ cell-less mRNAs, each of which has a role in germline formation. Furthermore, we present high-resolution crystal structures of the two Oskar domains. RNA-binding maps in vitro to the C-terminal domain, which shows structural similarity to SGNH hydrolases. The highly conserved N-terminal LOTUS domain forms dimers and mediates Oskar interaction with the germline-specific RNA helicase Vasa in vitro. Our findings suggest a dual function of Oskar in RNA and Vasa binding, providing molecular clues to its germ plasm function.

  15. Mycobacterium tuberculosis UvrB Is a Robust DNA-Stimulated ATPase That Also Possesses Structure-Specific ATP-Dependent DNA Helicase Activity.

    Science.gov (United States)

    Thakur, Manoj; Kumar, Mohan B J; Muniyappa, K

    2016-10-18

    Much is known about the Escherichia coli nucleotide excision repair (NER) pathway; however, very little is understood about the proteins involved and the molecular mechanism of NER in mycobacteria. In this study, we show that Mycobacterium tuberculosis UvrB (MtUvrB), which exists in solution as a monomer, binds to DNA in a structure-dependent manner. A systematic examination of MtUvrB substrate specificity reveals that it associates preferentially with single-stranded DNA, duplexes with 3' or 5' overhangs, and linear duplex DNA with splayed arms. Whereas E. coli UvrB (EcUvrB) binds weakly to undamaged DNA and has no ATPase activity, MtUvrB possesses intrinsic ATPase activity that is greatly stimulated by both single- and double-stranded DNA. Strikingly, we found that MtUvrB, but not EcUvrB, possesses the DNA unwinding activity characteristic of an ATP-dependent DNA helicase. The helicase activity of MtUvrB proceeds in the 3' to 5' direction and is strongly modulated by a nontranslocating 5' single-stranded tail, indicating that in addition to the translocating strand it also interacts with the 5' end of the substrate. The fraction of DNA unwound by MtUvrB decreases significantly as the length of the duplex increases: it fails to unwind duplexes longer than 70 bp. These results, on one hand, reveal significant mechanistic differences between MtUvrB and EcUvrB and, on the other, support an alternative role for UvrB in the processing of key DNA replication intermediates. Altogether, our findings provide insights into the catalytic functions of UvrB and lay the foundation for further understanding of the NER pathway in M. tuberculosis.

  16. A Rad53 independent function of Rad9 becomes crucial for genome maintenance in the absence of the Recq helicase Sgs1.

    Directory of Open Access Journals (Sweden)

    Ida Nielsen

    Full Text Available The conserved family of RecQ DNA helicases consists of caretaker tumour suppressors, that defend genome integrity by acting on several pathways of DNA repair that maintain genome stability. In budding yeast, Sgs1 is the sole RecQ helicase and it has been implicated in checkpoint responses, replisome stability and dissolution of double Holliday junctions during homologous recombination. In this study we investigate a possible genetic interaction between SGS1 and RAD9 in the cellular response to methyl methane sulphonate (MMS induced damage and compare this with the genetic interaction between SGS1 and RAD24. The Rad9 protein, an adaptor for effector kinase activation, plays well-characterized roles in the DNA damage checkpoint response, whereas Rad24 is characterized as a sensor protein also in the DNA damage checkpoint response. Here we unveil novel insights into the cellular response to MMS-induced damage. Specifically, we show a strong synergistic functionality between SGS1 and RAD9 for recovery from MMS induced damage and for suppression of gross chromosomal rearrangements, which is not the case for SGS1 and RAD24. Intriguingly, it is a Rad53 independent function of Rad9, which becomes crucial for genome maintenance in the absence of Sgs1. Despite this, our dissection of the MMS checkpoint response reveals parallel, but unequal pathways for Rad53 activation and highlights significant differences between MMS- and hydroxyurea (HU-induced checkpoint responses with relation to the requirement of the Sgs1 interacting partner Topoisomerase III (Top3. Thus, whereas earlier studies have documented a Top3-independent role of Sgs1 for an HU-induced checkpoint response, we show here that upon MMS treatment, Sgs1 and Top3 together define a minor but parallel pathway to that of Rad9.

  17. Structure of the SPRY domain of the human RNA helicase DDX1, a putative interaction platform within a DEAD-box protein

    Energy Technology Data Exchange (ETDEWEB)

    Kellner, Julian N.; Meinhart, Anton, E-mail: anton.meinhart@mpimf-heidelberg.mpg.de [Max Planck Institute for Medical Research, Jahnstrasse 29, 69120 Heidelberg (Germany)

    2015-08-25

    The structure of the SPRY domain of the human RNA helicase DDX1 was determined at 2.0 Å resolution. The SPRY domain provides a putative protein–protein interaction platform within DDX1 that differs from other SPRY domains in its structure and conserved regions. The human RNA helicase DDX1 in the DEAD-box family plays an important role in RNA processing and has been associated with HIV-1 replication and tumour progression. Whereas previously described DEAD-box proteins have a structurally conserved core, DDX1 shows a unique structural feature: a large SPRY-domain insertion in its RecA-like consensus fold. SPRY domains are known to function as protein–protein interaction platforms. Here, the crystal structure of the SPRY domain of human DDX1 (hDSPRY) is reported at 2.0 Å resolution. The structure reveals two layers of concave, antiparallel β-sheets that stack onto each other and a third β-sheet beneath the β-sandwich. A comparison with SPRY-domain structures from other eukaryotic proteins showed that the general β-sandwich fold is conserved; however, differences were detected in the loop regions, which were identified in other SPRY domains to be essential for interaction with cognate partners. In contrast, in hDSPRY these loop regions are not strictly conserved across species. Interestingly, though, a conserved patch of positive surface charge is found that may replace the connecting loops as a protein–protein interaction surface. The data presented here comprise the first structural information on DDX1 and provide insights into the unique domain architecture of this DEAD-box protein. By providing the structure of a putative interaction domain of DDX1, this work will serve as a basis for further studies of the interaction network within the hetero-oligomeric complexes of DDX1 and of its recruitment to the HIV-1 Rev protein as a viral replication factor.

  18. Structure of a Novel DNA-binding Domain of Helicase-like Transcription Factor (HLTF) and Its Functional Implication in DNA Damage Tolerance.

    Science.gov (United States)

    Hishiki, Asami; Hara, Kodai; Ikegaya, Yuzu; Yokoyama, Hideshi; Shimizu, Toshiyuki; Sato, Mamoru; Hashimoto, Hiroshi

    2015-05-22

    HLTF (helicase-like transcription factor) is a yeast RAD5 homolog found in mammals. HLTF has E3 ubiquitin ligase and DNA helicase activities, and plays a pivotal role in the template-switching pathway of DNA damage tolerance. HLTF has an N-terminal domain that has been designated the HIRAN (HIP116 and RAD5 N-terminal) domain. The HIRAN domain has been hypothesized to play a role in DNA binding; however, the structural basis of, and functional evidence for, the HIRAN domain in DNA binding has remained unclear. Here we show for the first time the crystal structure of the HIRAN domain of human HLTF in complex with DNA. The HIRAN domain is composed of six β-strands and two α-helices, forming an OB-fold structure frequently found in ssDNA-binding proteins, including in replication factor A (RPA). Interestingly, this study reveals that the HIRAN domain interacts with not only with a single-stranded DNA but also with a duplex DNA. Furthermore, the structure unexpectedly clarifies that the HIRAN domain specifically recognizes the 3'-end of DNA. These results suggest that the HIRAN domain functions as a sensor to the 3'-end of the primer strand at the stalled replication fork and that the domain facilitates fork regression. HLTF is recruited to a damaged site through the HIRAN domain at the stalled replication fork. Furthermore, our results have implications for the mechanism of template switching. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  19. Fishing Access Areas

    Data.gov (United States)

    Vermont Center for Geographic Information — The Vermont Fish & Wildlife Department maintains developed fishing access areas. These sites provide public access to waters in Vermont for shore fishing...

  20. Accessibility and sensory experiences

    DEFF Research Database (Denmark)

    Ryhl, Camilla

    2010-01-01

    and accessibility. Sensory accessibility accommodates aspects of a sensory disability and describes architectural design requirements needed to ensure access to architectural experiences. In the context of architecture accessibility has become a design concept of its own. It is generally described as ensuring...... physical access to the built environment by accommodating physical disabilities. While the existing concept of accessibility ensures the physical access of everyone to a given space, sensory accessibility ensures the choice of everyone to stay and be able to participate and experience....

  1. JISC Open Access Briefing Paper

    OpenAIRE

    Swan, Alma

    2005-01-01

    What Open Access is. What Open Access is not. How is Open Access provided? Open Access archives or repositories. Open Access journals. Why should authors provide Open Access to their work? Further information and resources

  2. The PCNA-associated protein PARI negatively regulates homologous recombination via the inhibition of DNA repair synthesis

    DEFF Research Database (Denmark)

    Burkovics, Peter; Dome, Lili; Juhasz, Szilvia

    2016-01-01

    to inhibit homologous recombination (HR) events. Here, we describe a biochemical mechanism in which PARI functions as an HR regulator after replication fork stalling and during double-strand break repair. In our reconstituted biochemical system, we show that PARI inhibits DNA repair synthesis during...... recombination events in a PCNA interaction-dependent way but independently of its UvrD-like helicase domain. In accordance, we demonstrate that PARI inhibits HR in vivo, and its knockdown suppresses the UV sensitivity of RAD18-depleted cells. Our data reveal a novel human regulatory mechanism that limits...

  3. 7 CFR 29.9241 - Accessibility of tobacco.

    Science.gov (United States)

    2010-01-01

    ... 7 Agriculture 2 2010-01-01 2010-01-01 false Accessibility of tobacco. 29.9241 Section 29.9241... REGULATIONS TOBACCO INSPECTION Policy Statement and Regulations Governing the Identification and Certification of Nonquota Tobacco Produced and Marketed in a Quota Area Administration § 29.9241 Accessibility of...

  4. 7 CFR 29.65 - Accessibility of tobacco.

    Science.gov (United States)

    2010-01-01

    ... 7 Agriculture 2 2010-01-01 2010-01-01 false Accessibility of tobacco. 29.65 Section 29.65... REGULATIONS TOBACCO INSPECTION Regulations Permissive Inspection § 29.65 Accessibility of tobacco. All tobacco... characteristics or for drawing of samples. In the case of tobacco in packages, the coverings shall be removed by...

  5. 48 CFR 3052.204-71 - Contractor employee access.

    Science.gov (United States)

    2010-10-01

    ... 48 Federal Acquisition Regulations System 7 2010-10-01 2010-10-01 false Contractor employee access... CLAUSES Text of Provisions and Clauses 3052.204-71 Contractor employee access. As prescribed in (HSAR) 48...: Contractor Employee Access (JUN 2006) (a) “Sensitive Information,” as used in this Chapter, means any...

  6. Professional Access 2013 programming

    CERN Document Server

    Hennig, Teresa; Hepworth, George; Yudovich, Dagi (Doug)

    2013-01-01

    Authoritative and comprehensive coverage for building Access 2013 Solutions Access, the most popular database system in the world, just opened a new frontier in the Cloud. Access 2013 provides significant new features for building robust line-of-business solutions for web, client and integrated environments.  This book was written by a team of Microsoft Access MVPs, with consulting and editing by Access experts, MVPs and members of the Microsoft Access team. It gives you the information and examples to expand your areas of expertise and immediately start to develop and upgrade projects. Exp

  7. Access 2010 Programmer's Reference

    CERN Document Server

    Hennig, Teresa; Griffith, Geoffrey L

    2010-01-01

    A comprehensive guide to programming for Access 2010 and 2007. Millions of people use the Access database applications, and hundreds of thousands of developers work with Access daily. Access 2010 brings better integration with SQL Server and enhanced XML support; this Wrox guide shows developers how to take advantage of these and other improvements. With in-depth coverage of VBA, macros, and other programming methods for building Access applications, this book also provides real-world code examples to demonstrate each topic.: Access is the leading database that is used worldwide; While VBA rem

  8. Computational study on the inhibitor binding mode and allosteric regulation mechanism in hepatitis C virus NS3/4A protein.

    Directory of Open Access Journals (Sweden)

    Weiwei Xue

    Full Text Available HCV NS3/4A protein is an attractive therapeutic target responsible for harboring serine protease and RNA helicase activities during the viral replication. Small molecules binding at the interface between the protease and helicase domains can stabilize the closed conformation of the protein and thus block the catalytic function of HCV NS3/4A protein via an allosteric regulation mechanism. But the detailed mechanism remains elusive. Here, we aimed to provide some insight into the inhibitor binding mode and allosteric regulation mechanism of HCV NS3/4A protein by using computational methods. Four simulation systems were investigated. They include: apo state of HCV NS3/4A protein, HCV NS3/4A protein in complex with an allosteric inhibitor and the truncated form of the above two systems. The molecular dynamics simulation results indicate HCV NS3/4A protein in complex with the allosteric inhibitor 4VA adopts a closed conformation (inactive state, while the truncated apo protein adopts an open conformation (active state. Further residue interaction network analysis suggests the communication of the domain-domain interface play an important role in the transition from closed to open conformation of HCV NS3/4A protein. However, the inhibitor stabilizes the closed conformation through interaction with several key residues from both the protease and helicase domains, including His57, Asp79, Asp81, Asp168, Met485, Cys525 and Asp527, which blocks the information communication between the functional domains interface. Finally, a dynamic model about the allosteric regulation and conformational changes of HCV NS3/4A protein was proposed and could provide fundamental insights into the allosteric mechanism of HCV NS3/4A protein function regulation and design of new potent inhibitors.

  9. Overview: Routes to Open Access

    OpenAIRE

    Tullney, Marco; van Wezenbeek, Wilma

    2017-01-01

    Slides of an overview presentation given at a CESAER workshop on Open Access, February 2nd, 2017, in Brussels Cover major routes to more open access as discussed in the Task Force Open Science of CESAER: (national) open access strategies open access mandates open access incentives open access awareness open access publishing open access infrastructure

  10. Physical Access Control Database -

    Data.gov (United States)

    Department of Transportation — This data set contains the personnel access card data (photo, name, activation/expiration dates, card number, and access level) as well as data about turnstiles and...

  11. Design for Accessibility

    DEFF Research Database (Denmark)

    Herriott, Richard

    2012-01-01

    A report on how nine rail builder, operators and transport designers deal with design for accessibility......A report on how nine rail builder, operators and transport designers deal with design for accessibility...

  12. Information on Open Access

    African Journals Online (AJOL)

    Open Access (OA), defined most simply, means free full text online. There are over 130 Open Access journals hosted on the AJOL website. You can find a full list of these journals here: OA journals on AJOL ...

  13. Role of the ATPase/helicase maleless (MLE in the assembly, targeting, spreading and function of the male-specific lethal (MSL complex of Drosophila

    Directory of Open Access Journals (Sweden)

    Morra Rosa

    2011-04-01

    Full Text Available Abstract Background The male-specific lethal (MSL complex of Drosophila remodels the chromatin of the X chromosome in males to enhance the level of transcription of most X-linked genes, and thereby achieve dosage compensation. The core complex consists of five proteins and one of two non-coding RNAs. One of the proteins, MOF (males absent on the first, is a histone acetyltransferase that specifically acetylates histone H4 at lysine 16. Another protein, maleless (MLE, is an ATP-dependent helicase with the ability to unwind DNA/RNA or RNA/RNA substrates in vitro. Recently, we showed that the ATPase activity of MLE is sufficient for the hypertranscription of genes adjacent to a high-affinity site by MSL complexes located at that site. The helicase activity is required for the spreading of the complex to the hundreds of positions along the X chromosome, where it is normally found. In this study, to further understand the role of MLE in the function of the MSL complex, we analyzed its relationship to the other complex components by creating a series of deletions or mutations in its putative functional domains, and testing their effect on the distribution and function of the complex in vivo. Results The presence of the RB2 RNA-binding domain is necessary for the association of the MSL3 protein with the other complex subunits. In its absence, the activity of the MOF subunit was compromised, and the complex failed to acetylate histone H4 at lysine 16. Deletion of the RB1 RNA-binding domain resulted in complexes that maintained substantial acetylation activity but failed to spread beyond the high-affinity sites. Flies bearing this mutation exhibited low levels of roX RNAs, indicating that these RNAs failed to associate with the proteins of the complex and were degraded, or that MLE contributes to their synthesis. Deletion of the glycine-rich C-terminal region, which contains a nuclear localization sequence, caused a substantial level of retention of the

  14. Access Customized Forms

    OpenAIRE

    Cosma Emil; Jeflea Victor

    2010-01-01

    By using Word, Excel or PowerPoint one can automate routine operations using the VBA language (Visual Basic for Applications). This language is also used in Access, allowing access to data stored in tables or queries. Thus, Access and VBA resources can be used together. Access is designed for programming forms and reports (among other things), so there won’t be found any of the VBA editor’s specific forms.

  15. Accessing Electronic Journals.

    Science.gov (United States)

    McKay, Sharon Cline

    1999-01-01

    Discusses issues librarians need to consider when providing access to electronic journals. Topics include gateways; index and abstract services; validation and pay-per-view; title selection; integration with OPACs (online public access catalogs)or Web sites; paper availability; ownership versus access; usage restrictions; and services offered…

  16. Open access and beyond

    Directory of Open Access Journals (Sweden)

    Das Chhaya

    2006-09-01

    Full Text Available Abstract Uncensored exchange of scientific results hastens progress. Open Access does not stop at the removal of price and permission barriers; still, censorship and reading disabilities, to name a few, hamper access to information. Here, we invite the scientific community and the public to discuss new methods to distribute, store and manage literature in order to achieve unfettered access to literature.

  17. The LHC access system LACS and LASS

    CERN Document Server

    Ninin, P

    2005-01-01

    The LHC complex is divided into a number of zones with different levels of access controls.Inside the interlocked areas, the personnel protection is ensured by the LHC Access System.The system is made of two parts:the LHC Access Safety System and the LHC Access Control System. During machine operation,the LHC Access Safety System ensures the collective protection of the personnel against the radiation hazards arising from the operation of the accelerator by interlocking the LHC key safety elements. When the beams are off, the LHC Access Control System regulates the access to the accelerator and its many subsystems.It allows a remote, local or automatic operation of the access control equipment which verifies and identifies all users entering the controlled areas.The global architecture of the LHC Access System is now designed and is being validated to ensure that it meets the safety requirements for operation of the LHC.A pilot installation will be tested in the summer 2005 to validate the concept with the us...

  18. Access 2013 for dummies

    CERN Document Server

    Ulrich Fuller, Laurie

    2013-01-01

    The easy guide to Microsoft Access returns with updates on the latest version! Microsoft Access allows you to store, organize, view, analyze, and share data; the new Access 2013 release enables you to build even more powerful, custom database solutions that integrate with the web and enterprise data sources. Access 2013 For Dummies covers all the new features of the latest version of Accessand serves as an ideal reference, combining the latest Access features with the basics of building usable databases. You'll learn how to create an app from the Welcome screen, get support

  19. Pro Access 2010 Development

    CERN Document Server

    Collins, Mark

    2011-01-01

    Pro Access 2010 Development is a fundamental resource for developing business applications that take advantage of the features of Access 2010 and the many sources of data available to your business. In this book, you'll learn how to build database applications, create Web-based databases, develop macros and Visual Basic for Applications (VBA) tools for Access applications, integrate Access with SharePoint and other business systems, and much more. Using a practical, hands-on approach, this book will take you through all the facets of developing Access-based solutions, such as data modeling, co

  20. Android Access Control Extension

    Directory of Open Access Journals (Sweden)

    Anton Baláž

    2015-12-01

    Full Text Available The main objective of this work is to analyze and extend security model of mobile devices running on Android OS. Provided security extension is a Linux kernel security module that allows the system administrator to restrict program's capabilities with per-program profiles. Profiles can allow capabilities like network access, raw socket access, and the permission to read, write, or execute files on matching paths. Module supplements the traditional Android capability access control model by providing mandatory access control (MAC based on path. This extension increases security of access to system objects in a device and allows creating security sandboxes per application.

  1. Open Access @ DTU

    DEFF Research Database (Denmark)

    Ekstrøm, Jeannette

    Open Access is high on the agenda in Denmark and internationally. Denmark has announced a national strategy for Open Access that aims to achieve Open Access to 80% in 2017 and 100% in 2022 to peer review research articles. All public Danish funders as well as H2020 requires that all peer review...... articles that is an outcome of their funding will be Open Access. Uploading your full texts (your final author manuscript after review ) to DTU Orbit is a fundamental part of providing Open Access to your research. We are here to answer all your questions with regards to Open Access and related topics...... such as copyright, DTU Orbit, Open Access journals, APCs, Vouchers etc....

  2. Structure and reconstitution of yeast Mpp6-nuclear exosome complexes reveals that Mpp6 stimulates RNA decay and recruits the Mtr4 helicase

    Energy Technology Data Exchange (ETDEWEB)

    Wasmuth, Elizabeth V. [Structural Biology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, United States; Zinder, John C. [Structural Biology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, United States; Tri-Institutional Training Program in Chemical Biology, Memorial Sloan Kettering Cancer Center, New York, United States; Zattas, Dimitrios [Structural Biology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, United States; Das, Mom [Structural Biology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, United States; Lima, Christopher D. [Structural Biology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, United States; Howard Hughes Medical Institute, Memorial Sloan Kettering Cancer Center, New York, United States

    2017-07-25

    Nuclear RNA exosomes catalyze a range of RNA processing and decay activities that are coordinated in part by cofactors, including Mpp6, Rrp47, and the Mtr4 RNA helicase. Mpp6 interacts with the nine-subunit exosome core, while Rrp47 stabilizes the exoribonuclease Rrp6 and recruits Mtr4, but it is less clear if these cofactors work together. Using biochemistry with Saccharomyces cerevisiae proteins, we show that Rrp47 and Mpp6 stimulate exosome-mediated RNA decay, albeit with unique dependencies on elements within the nuclear exosome. Mpp6-exosomes can recruit Mtr4, while Mpp6 and Rrp47 each contribute to Mtr4-dependent RNA decay, with maximal Mtr4-dependent decay observed with both cofactors. The 3.3 Å structure of a twelve-subunit nuclear Mpp6 exosome bound to RNA shows the central region of Mpp6 bound to the exosome core, positioning its Mtr4 recruitment domain next to Rrp6 and the exosome central channel. Genetic analysis reveals interactions that are largely consistent with our model.

  3. Redistribution of demethylated RNA helicase A during foot-and-mouth disease virus infection: Role of Jumonji C-domain containing protein 6 in RHA demethylation

    International Nuclear Information System (INIS)

    Lawrence, Paul; Conderino, Joseph S.; Rieder, Elizabeth

    2014-01-01

    Previously, RNA helicase A (RHA) re-localization from the nucleus to the cytoplasm in foot-and-mouth disease virus (FMDV) infected cells was shown to coincide with loss of RHA methylated arginine residues at its C-terminus. The potential interaction between RHA and Jumonji C-domain (JmjC) protein 6 (JMJD6) arginine demethylase in infected cells was investigated. Treatment with N-oxalylglycine (NOG) inhibitor of JmjC demethylases prevented FMDV-induced RHA demethylation and re-localization, and also decreased viral protein synthesis and virus titers. Physical interaction between JMJD6 and RHA was demonstrated via reciprocal co-immunoprecipitation, where RHA preferentially bound JMJD6 monomers. Nuclear efflux of demethylated RHA (DM-RHA) coincided with nuclear influx of JMJD6, which was not observed using another picornavirus. A modified biochemical assay demonstrated JMJD6 induced dose-dependent demethylation of RHA and two RHA-derived isoforms, which could be inhibited by NOG. We propose a role for JMJD6 in RHA demethylation stimulated by FMDV, that appears to facilitate virus replication. - Highlights: • We examined the role of JMJD6 in FMDV-induced RHA demethylation process. • Using an arginine demethylation assay showed that JMJD6 is involved in RHA demethylation. • A demethylases inhibitor reduced cytoplasmic accumulation of RHA and FMDV titers

  4. Redistribution of demethylated RNA helicase A during foot-and-mouth disease virus infection: Role of Jumonji C-domain containing protein 6 in RHA demethylation

    Energy Technology Data Exchange (ETDEWEB)

    Lawrence, Paul; Conderino, Joseph S.; Rieder, Elizabeth, E-mail: elizabeth.rieder@ars.usda.gov

    2014-03-15

    Previously, RNA helicase A (RHA) re-localization from the nucleus to the cytoplasm in foot-and-mouth disease virus (FMDV) infected cells was shown to coincide with loss of RHA methylated arginine residues at its C-terminus. The potential interaction between RHA and Jumonji C-domain (JmjC) protein 6 (JMJD6) arginine demethylase in infected cells was investigated. Treatment with N-oxalylglycine (NOG) inhibitor of JmjC demethylases prevented FMDV-induced RHA demethylation and re-localization, and also decreased viral protein synthesis and virus titers. Physical interaction between JMJD6 and RHA was demonstrated via reciprocal co-immunoprecipitation, where RHA preferentially bound JMJD6 monomers. Nuclear efflux of demethylated RHA (DM-RHA) coincided with nuclear influx of JMJD6, which was not observed using another picornavirus. A modified biochemical assay demonstrated JMJD6 induced dose-dependent demethylation of RHA and two RHA-derived isoforms, which could be inhibited by NOG. We propose a role for JMJD6 in RHA demethylation stimulated by FMDV, that appears to facilitate virus replication. - Highlights: • We examined the role of JMJD6 in FMDV-induced RHA demethylation process. • Using an arginine demethylation assay showed that JMJD6 is involved in RHA demethylation. • A demethylases inhibitor reduced cytoplasmic accumulation of RHA and FMDV titers.

  5. General Methods for Analysis of Sequential “n-step” Kinetic Mechanisms: Application to Single Turnover Kinetics of Helicase-Catalyzed DNA Unwinding

    Science.gov (United States)

    Lucius, Aaron L.; Maluf, Nasib K.; Fischer, Christopher J.; Lohman, Timothy M.

    2003-01-01

    Helicase-catalyzed DNA unwinding is often studied using “all or none” assays that detect only the final product of fully unwound DNA. Even using these assays, quantitative analysis of DNA unwinding time courses for DNA duplexes of different lengths, L, using “n-step” sequential mechanisms, can reveal information about the number of intermediates in the unwinding reaction and the “kinetic step size”, m, defined as the average number of basepairs unwound between two successive rate limiting steps in the unwinding cycle. Simultaneous nonlinear least-squares analysis using “n-step” sequential mechanisms has previously been limited by an inability to float the number of “unwinding steps”, n, and m, in the fitting algorithm. Here we discuss the behavior of single turnover DNA unwinding time courses and describe novel methods for nonlinear least-squares analysis that overcome these problems. Analytic expressions for the time courses, fss(t), when obtainable, can be written using gamma and incomplete gamma functions. When analytic expressions are not obtainable, the numerical solution of the inverse Laplace transform can be used to obtain fss(t). Both methods allow n and m to be continuous fitting parameters. These approaches are generally applicable to enzymes that translocate along a lattice or require repetition of a series of steps before product formation. PMID:14507688

  6. General methods for analysis of sequential "n-step" kinetic mechanisms: application to single turnover kinetics of helicase-catalyzed DNA unwinding.

    Science.gov (United States)

    Lucius, Aaron L; Maluf, Nasib K; Fischer, Christopher J; Lohman, Timothy M

    2003-10-01

    Helicase-catalyzed DNA unwinding is often studied using "all or none" assays that detect only the final product of fully unwound DNA. Even using these assays, quantitative analysis of DNA unwinding time courses for DNA duplexes of different lengths, L, using "n-step" sequential mechanisms, can reveal information about the number of intermediates in the unwinding reaction and the "kinetic step size", m, defined as the average number of basepairs unwound between two successive rate limiting steps in the unwinding cycle. Simultaneous nonlinear least-squares analysis using "n-step" sequential mechanisms has previously been limited by an inability to float the number of "unwinding steps", n, and m, in the fitting algorithm. Here we discuss the behavior of single turnover DNA unwinding time courses and describe novel methods for nonlinear least-squares analysis that overcome these problems. Analytic expressions for the time courses, f(ss)(t), when obtainable, can be written using gamma and incomplete gamma functions. When analytic expressions are not obtainable, the numerical solution of the inverse Laplace transform can be used to obtain f(ss)(t). Both methods allow n and m to be continuous fitting parameters. These approaches are generally applicable to enzymes that translocate along a lattice or require repetition of a series of steps before product formation.

  7. Minichromosome maintenance helicase paralog MCM9 is dispensible for DNA replication but functions in germ-line stem cells and tumor suppression.

    Science.gov (United States)

    Hartford, Suzanne A; Luo, Yunhai; Southard, Teresa L; Min, Irene M; Lis, John T; Schimenti, John C

    2011-10-25

    Effective DNA replication is critical to the health and reproductive success of organisms. The six MCM2-7 proteins, which form the replicative helicase, are essential for high-fidelity replication of the genome. Many eukaryotes have a divergent paralog, MCM9, that was reported to be essential for loading MCM2-7 onto replication origins in the Xenopus oocyte extract system. To address the in vivo role of mammalian MCM9, we created and analyzed the phenotypes of mice with various mutations in Mcm9 and an intronic DNA replication-related gene Asf1a. Ablation of Mcm9 was compatible with cell proliferation and mouse viability, showing that it is nonessential for MCM2-7 loading or DNA replication. Mcm9 mutants underwent p53-independent embryonic germ-cell depletion in both sexes, with males also exhibiting defective spermatogonial stem-cell renewal. MCM9-deficient cells had elevated genomic instability and defective cell cycle reentry following replication stress, and mutant animals were prone to sex-specific cancers, most notably hepatocellular carcinoma in males. The phenotypes of mutant mice and cells suggest that MCM9 evolved a specialized but nonessential role in DNA replication or replication-linked quality-control mechanisms that are especially important for germ-line stem cells, and also for tumor suppression and genome maintenance in the soma.

  8. Crystal Structure of the Phage T4 Recombinase UvsX and Its Functional Interaction with the T4 SF2 Helicase UvsW

    Energy Technology Data Exchange (ETDEWEB)

    Gajewski, Stefan; Webb, Michael R.; Galkin, Vitold; Egelman, Edward H.; Kreuzer, Kenneth N.; White, Stephen W. (Duke); (UV); (SJCH)

    2012-07-11

    Bacteriophage T4 provides an important model system for studying the mechanism of homologous recombination. We have determined the crystal structure of the T4 UvsX recombinase, and the overall architecture and fold closely resemble those of RecA, including a highly conserved ATP binding site. Based on this new structure, we reanalyzed electron microscopy reconstructions of UvsX-DNA filaments and docked the UvsX crystal structure into two different filament forms: a compressed filament generated in the presence of ADP and an elongated filament generated in the presence of ATP and aluminum fluoride. In these reconstructions, the ATP binding site sits at the protomer interface, as in the RecA filament crystal structure. However, the environment of the ATP binding site is altered in the two filament reconstructions, suggesting that nucleotide cannot be as easily accommodated at the protomer interface of the compressed filament. Finally, we show that the phage helicase UvsW completes the UvsX-promoted strand-exchange reaction, allowing the generation of a simple nicked circular product rather than complex networks of partially exchanged substrates.

  9. Regulating the Regulator

    Energy Technology Data Exchange (ETDEWEB)

    1992-08-26

    The article reports on a challenge to the UK electricity regulator to defend his record by the Coalition for Fair Electricity Regulation (COFFER). The challenge centres on whether the obligation for the regional electric companies (REC) to purchase power from the cheapest source is being enforced. This is related to the wider issue of whether the REC's support of combined-cycle gas turbine (CCGT) is economic. COFFER considers that uneconomic gas-fired power plants are being allowed to displace economic coal-fired stations. Aspects discussed include the background to the dispute and the costs of CCGT and coal fired power generation. 1 fig., 1 tab.

  10. 33 CFR 165.1405 - Regulated Navigation Areas and Security Zones; Designated Escorted Vessels-Philippine Sea and...

    Science.gov (United States)

    2010-07-01

    ...) PORTS AND WATERWAYS SAFETY REGULATED NAVIGATION AREAS AND LIMITED ACCESS AREAS Specific Regulated...). (a) Regulated navigation area. The following areas, designated by coordinates referencing World...

  11. The RecQ helicase-topoisomerase III-Rmi1 complex: a DNA structure-specific 'dissolvasome'?

    DEFF Research Database (Denmark)

    Mankouri, Hocine W; Hickson, Ian D

    2007-01-01

    structures, and we propose here that it functions in a coordinated fashion as a DNA structure-specific 'dissolvasome'. Little is known about how the RTR complex might be regulated or targeted to various DNA structures in vivo. Recent findings indicate that the components of the RTR complex might activate...... the cell cycle checkpoint machinery as well as be a target of checkpoint kinases, suggesting that these events are crucial to ensure faithful DNA replication and chromosome segregation....

  12. Migrants' access to healthcare

    DEFF Research Database (Denmark)

    Norredam, Marie

    2011-01-01

    There are strong pragmatic and moral reasons for receiving societies to address access to healthcare for migrants. Receiving societies have a pragmatic interest in sustaining migrants' health to facilitate integration; they also have a moral obligation to ensure migrants' access to healthcare...... according to international human rights principles. The intention of this thesis is to increase the understanding of migrants' access to healthcare by exploring two study aims: 1) Are there differences in migrants' access to healthcare compared to that of non-migrants? (substudy I and II); and 2) Why...... are there possible differences in migrants' access to healthcare compared to that of non-migrants? (substudy III and IV). The thesis builds on different methodological approaches using both register-based retrospective cohort design, cross-sectional design and survey methods. Two different measures of access were...

  13. Three essays on access pricing

    Science.gov (United States)

    Sydee, Ahmed Nasim

    access pricing with congestion and in which investments in infrastructure are lumpy. To fix ideas, the model is formulated in the context of airport infrastructure investments, which captures both the element of congestion and the lumpiness involved in infrastructure investments. The optimal investment program suggests how many units of capacity should be installed and at which times. Because time is continuous in the model, the discounted cost -- despite the lumpiness of capacity additions -- can be made to vary continuously by varying the time a capacity addition is made. The main results that emerge from the analysis can be described as follows: First, the global demand for air travel rises with time and experiences an upward jump whenever a capacity addition is made. Second, the access price is constant and stays at the basic level when the system is not congested. When the system is congested, a congestion surcharge is imposed on top of the basic level, and the congestion surcharge rises with the level of congestion until the next capacity addition is made at which time the access price takes a downward jump. Third, the individual demand for air travel is constant before congestion sets in and after the last capacity addition takes place. During a time interval in which congestion rises, the individual demand for travel is below the level that prevails when there is no congestion and declines as congestion worsens. The third essay contains a model of access pricing for natural gas transmission pipelines, both when pipeline operators are regulated and when they behave strategically. The high sunk costs involved in building a pipeline network constitute a serious barrier of entry, and competitive behaviour in the transmission pipeline sector cannot be expected. Most of the economic analyses of access pricing for natural gas transmission pipelines are carried out from the regulatory perspective, and the access price paid by shippers are cost-based. The model formalized

  14. Access Data Analysis Cookbook

    CERN Document Server

    Bluttman, Ken

    2008-01-01

    This book offers practical recipes to solve a variety of common problems that users have with extracting Access data and performing calculations on it. Whether you use Access 2007 or an earlier version, this book will teach you new methods to query data, different ways to move data in and out of Access, how to calculate answers to financial and investment issues, how to jump beyond SQL by manipulating data with VBA, and more.

  15. Strategic Accessibility Competition

    OpenAIRE

    Bacchiega, Emanuele; Randon, Emanuela; Zirulia, Lorenzo

    2010-01-01

    We analyze the effect of competition in market-accessibility enhancement among quality-differentiated firms. Firms are located in regions with different ex-ante transport costs to reach the final market. We characterize the equilibrium of the two-stage game in which firms first invest to improve market accessibility and then compete in prices. Efforts in accessibility improvement crucially depend on the interplay between the willingness to pay for the quality premium of the median consumer an...

  16. Are PDF Documents Accessible?

    Directory of Open Access Journals (Sweden)

    Mireia Ribera Turró

    2008-09-01

    Full Text Available Adobe PDF is one of the most widely used formats in scientific communications and in administrative documents. In its latest versions it has incorporated structural tags and improvements that increase its level of accessibility. This article reviews the concept of accessibility in the reading of digital documents and evaluates the accessibility of PDF according to the most widely established standards.

  17. Open Access Monitor - DK

    DEFF Research Database (Denmark)

    Svendsen, Michael; Hansen, Lars Asger Juel; Andersen, Dorte

    2017-01-01

    Open Access Monitor - DK (OAM-DK) is a 2-year DEFF funded [DEFF.2016-0018] national project running in 2017-2018 with the aim of collecting, documenting and administrating Open Access publishing costs. OAM-DK is lead by Copenhagen University Library under the Royal Danish Library with participation...... of all Danish University Libraries. This poster presents the first results of Open Access costs related to 2015 publications at the The University of Copenhagen....

  18. 78 FR 45454 - Patient Access to Records

    Science.gov (United States)

    2013-07-29

    ... regarding current and future health care. Removing barriers to a veteran's access to VA records will support... patient autonomy and shared decision making. Removing this regulation will directly benefit veterans by... property, Infants and children, Inventions and patents, Parking, Penalties, Privacy, Reporting and...

  19. 34 CFR 108.6 - Equal access.

    Science.gov (United States)

    2010-07-01

    ... 34 Education 1 2010-07-01 2010-07-01 false Equal access. 108.6 Section 108.6 Education Regulations of the Offices of the Department of Education OFFICE FOR CIVIL RIGHTS, DEPARTMENT OF EDUCATION EQUAL... limited to, school-related means of communication, such as bulletin board notices and literature...

  20. Demystifying Open Access

    International Nuclear Information System (INIS)

    Mele, Salvatore

    2007-01-01

    The tenets of Open Access are to grant anyone, anywhere and anytime free access to the results of scientific research. HEP spearheaded the Open Access dissemination of scientific results with the mass mailing of preprints in the pre-WWW era and with the launch of the arXiv preprint system at the dawn of the '90s. The HEP community is now ready for a further push to Open Access while retaining all the advantages of the peer-review system and, at the same time, bring the spiralling cost of journal subscriptions under control. I will present a possible plan for the conversion to Open Access of HEP peer-reviewed journals, through a consortium of HEP funding agencies, laboratories and libraries: SCOAP3 (Sponsoring Consortium for Open Access Publishing in Particle Physics). SCOAP3 will engage with scientific publishers towards building a sustainable model for Open Access publishing, which is as transparent as possible for HEP authors. The current system in which journals income comes from subscription fees is replaced with a scheme where SCOAP3 compensates publishers for the costs incurred to organise the peer-review service and give Open Access to the final version of articles. SCOAP3 will be funded by all countries active in HEP under a 'fair share' scenario, according to their production of HEP articles. In this talk I will present a short overview of the history of Open Access in HEP, the details of the SCOAP3 model and the outlook for its implementation.

  1. OGIS Access System

    Data.gov (United States)

    National Archives and Records Administration — The OGIS Access System (OAS) provides case management, stakeholder collaboration, and public communications activities including a web presence via a web portal.

  2. Access 2013 bible

    CERN Document Server

    Alexander, Michael

    2013-01-01

    A comprehensive reference to the updated and new features of Access 2013 As the world's most popular database management tool, Access enables you to organize, present, analyze, and share data as well as build powerful database solutions. However, databases can be complex. That's why you need the expert guidance in this comprehensive reference. Access 2013 Bible helps you gain a solid understanding of database purpose, construction, and application so that whether you're new to Access or looking to upgrade to the 2013 version, this well-rounded resource provides you with a th

  3. Web Accessibility and Guidelines

    Science.gov (United States)

    Harper, Simon; Yesilada, Yeliz

    Access to, and movement around, complex online environments, of which the World Wide Web (Web) is the most popular example, has long been considered an important and major issue in the Web design and usability field. The commonly used slang phrase ‘surfing the Web’ implies rapid and free access, pointing to its importance among designers and users alike. It has also been long established that this potentially complex and difficult access is further complicated, and becomes neither rapid nor free, if the user is disabled. There are millions of people who have disabilities that affect their use of the Web. Web accessibility aims to help these people to perceive, understand, navigate, and interact with, as well as contribute to, the Web, and thereby the society in general. This accessibility is, in part, facilitated by the Web Content Accessibility Guidelines (WCAG) currently moving from version one to two. These guidelines are intended to encourage designers to make sure their sites conform to specifications, and in that conformance enable the assistive technologies of disabled users to better interact with the page content. In this way, it was hoped that accessibility could be supported. While this is in part true, guidelines do not solve all problems and the new WCAG version two guidelines are surrounded by controversy and intrigue. This chapter aims to establish the published literature related to Web accessibility and Web accessibility guidelines, and discuss limitations of the current guidelines and future directions.

  4. Market, Regulation, Market, Regulation

    DEFF Research Database (Denmark)

    Frankel, Christian; Galland, Jean-Pierre

    2015-01-01

    barriers to trade in Europe, realized the free movement of products by organizing progressively several orders of markets and regulation. Based on historical and institutional documents, on technical publications, and on interviews, this article relates how the European Commission and the Member States had......This paper focuses on the European Regulatory system which was settled both for opening the Single Market for products and ensuring the consumers' safety. It claims that the New Approach and Standardization, and the Global Approach to conformity assessment, which suppressed the last technical...... alternatively recourse to markets and to regulations, at the three main levels of the New Approach Directives implementation. The article focuses also more specifically on the Medical Devices sector, not only because this New Approach sector has long been controversial in Europe, and has recently been concerned...

  5. Optical Access Networks

    Science.gov (United States)

    Zheng, Jun; Ansari, Nirwan

    2005-06-01

    Call for Papers: Optical Access Networks With the wide deployment of fiber-optic technology over the past two decades, we have witnessed a tremendous growth of bandwidth capacity in the backbone networks of today's telecommunications infrastructure. However, access networks, which cover the "last-mile" areas and serve numerous residential and small business users, have not been scaled up commensurately. The local subscriber lines for telephone and cable television are still using twisted pairs and coaxial cables. Most residential connections to the Internet are still through dial-up modems operating at a low speed on twisted pairs. As the demand for access bandwidth increases with emerging high-bandwidth applications, such as distance learning, high-definition television (HDTV), and video on demand (VoD), the last-mile access networks have become a bandwidth bottleneck in today's telecommunications infrastructure. To ease this bottleneck, it is imperative to provide sufficient bandwidth capacity in the access networks to open the bottleneck and thus present more opportunities for the provisioning of multiservices. Optical access solutions promise huge bandwidth to service providers and low-cost high-bandwidth services to end users and are therefore widely considered the technology of choice for next-generation access networks. To realize the vision of optical access networks, however, many key issues still need to be addressed, such as network architectures, signaling protocols, and implementation standards. The major challenges lie in the fact that an optical solution must be not only robust, scalable, and flexible, but also implemented at a low cost comparable to that of existing access solutions in order to increase the economic viability of many potential high-bandwidth applications. In recent years, optical access networks have been receiving tremendous attention from both academia and industry. A large number of research activities have been carried out or

  6. Accessibility of public services

    NARCIS (Netherlands)

    Poort, J.P.; Groot, I.; Kok, L.; de Graaf, D.; Hof, B.J.F.

    2005-01-01

    The accessibility of certain products and services to all people, irrespective of their income, age, health and geographical location is considered to be of great social importance. Think for instance of health care, education, electricity, and sanitation. Accessibility can be secured in a variety

  7. Market Access and Welfare

    DEFF Research Database (Denmark)

    Raimondos-Møller, Pascalis; Woodland, Alan D.

    2015-01-01

    Well known tariff reform rules that are guaranteed to increase welfare will not necessarily increase market access, while rules that are guaranteed to increase market access will not necessarily increase welfare. The present paper proposes a new set of tariff reforms that can achieve both...

  8. Standards and Access.

    Science.gov (United States)

    Fox, Tom

    1993-01-01

    Argues that easy claims about the relationship between language mastery and academic or economic access (made by both conservative commentators on education and mainstream writing teachers) are false and obscure real social and political boundaries, such as racism, sexism, elitism, and homophobia, that really do prevent access. (SR)

  9. Albania’s challenge for access and competitiveness in EU Market: When good will requires more than legal regulation. An inside of Albania’s efforts to increase the presence of its domestic product in EU markets.

    Directory of Open Access Journals (Sweden)

    Edi Spaho

    2012-06-01

    Full Text Available Through the presentation of two case studies this paper aims to engage the theoretical debate on the persistence of public space in the contemporary city, and focuses the attention on the way people practice these spaces and on the policies which are regulating their uses. Starting from the description of different ways in which some urban spaces in Rome (Italy are used by two different communities of people, one mainly composed by immigrants and the other by young city users, and the diverse ways in which their different practices are seen and tolerated, the aim of this paper is to reflect on public spaces. Observing how city users practice public spaces, and analysing the way in which these practices are considered, are particularly exciting perspectives that can offer an interesting vision of the spatial and social reality of the city and of hegemonic relations which govern it.

  10. Transmission access issues: Present and future

    International Nuclear Information System (INIS)

    Bahl, P.K.; Gray, R.G.

    1992-01-01

    In recent years, the electric industry has undergone dramatic changes as the federal and state governments have encouraged bulk power production by Independent Power Producers (IPPS) and Qualified Facilities (QFs). With decentralization and the consequent competition in the field of bulk power production, there has emerged the problem of non-utility generators (NUGS) and of transmission dependent utilities accessing the existing transmission systems owned by electric utilities. This paper presents current and future issues related to transmission access by IPPS, QFs and transmission dependent utilities. These issues include: (a) impact on system reliability, (b) impact of pricing strategies, (c) changes in state and federal regulations, (d) transmission proposals by various entities, (e) present access arrangements, (f) formation of the Western Systems Power Pool (WSPP), and (g) siting difficulties. The transmission access problem warrants consideration of transmission services, coordination among electric utilities, joint planning on a regional basis, and accommodation of IPPs and NUGs

  11. A remark on accessibility

    International Nuclear Information System (INIS)

    Wu, Xinxing; Wang, Jianjun

    2016-01-01

    Highlights: • Obtain some characteristics of accessibility and Kato’s chaos. • Answer negatively a question in [Li R, Wang H, Zhao Y. Kato’s chaos in duopoly games. Chaos Solit Fract 2016;84:69–72]. • A dynamical system is indecomposable if and only if it is weakly transitive. - Abstract: This note obtains some characteristics of accessibility and Kato’s chaos. Applying these results, an accessible dynamical system whose product system is not accessible is constructed, giving a negative answer to a question in [Li R, Wang H, Zhao Y. Kato’s chaos in duopoly games. Chaos Solit Fract 2016;84:69–72]. Besides, it is proved that every transitive interval self-map is accessible.

  12. 5 CFR 2504.11 - Access to the accounting of disclosures from records.

    Science.gov (United States)

    2010-01-01

    ... 5 Administrative Personnel 3 2010-01-01 2010-01-01 false Access to the accounting of disclosures... OFFICE OF THE PRESIDENT PRIVACY ACT REGULATIONS § 2504.11 Access to the accounting of disclosures from records. Rules governing access to the accounting of disclosures are the same as those granting access to...

  13. 5 CFR 1630.9 - Access to the history (accounting) of disclosures from records.

    Science.gov (United States)

    2010-01-01

    ... BOARD PRIVACY ACT REGULATIONS § 1630.9 Access to the history (accounting) of disclosures from records. Rules governing access to the accounting of disclosures are the same as those for granting access to the... 5 Administrative Personnel 3 2010-01-01 2010-01-01 false Access to the history (accounting) of...

  14. Molecular and Functional Characterization of RecD, a Novel Member of the SF1 Family of Helicases, from Mycobacterium tuberculosis.

    Science.gov (United States)

    Dewhare, Shivendra Singh; Umesh, T G; Muniyappa, K

    2015-05-08

    The annotated whole-genome sequence of Mycobacterium tuberculosis revealed the presence of a putative recD gene; however, the biochemical characteristics of its encoded protein product (MtRecD) remain largely unknown. Here, we show that MtRecD exists in solution as a stable homodimer. Protein-DNA binding assays revealed that MtRecD binds efficiently to single-stranded DNA and linear duplexes containing 5' overhangs relative to the 3' overhangs but not to blunt-ended duplex. Furthermore, MtRecD bound more robustly to a variety of Y-shaped DNA structures having ≥18-nucleotide overhangs but not to a similar substrate containing 5-nucleotide overhangs. MtRecD formed more salt-tolerant complexes with Y-shaped structures compared with linear duplex having 3' overhangs. The intrinsic ATPase activity of MtRecD was stimulated by single-stranded DNA. Site-specific mutagenesis of Lys-179 in motif I abolished the ATPase activity of MtRecD. Interestingly, although MtRecD-catalyzed unwinding showed a markedly higher preference for duplex substrates with 5' overhangs, it could also catalyze significant unwinding of substrates containing 3' overhangs. These results support the notion that MtRecD is a bipolar helicase with strong 5' → 3' and weak 3' → 5' unwinding activities. The extent of unwinding of Y-shaped DNA structures was ∼3-fold lower compared with duplexes with 5' overhangs. Notably, direct interaction between MtRecD and its cognate RecA led to inhibition of DNA strand exchange promoted by RecA. Altogether, these studies provide the first detailed characterization of MtRecD and present important insights into the type of DNA structure the enzyme is likely to act upon during the processes of DNA repair or homologous recombination. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  15. RIG-I Helicase-Independent Pathway in Sendai Virus-Activated Dendritic Cells Is Critical for Preventing Lung Metastasis of AT6.3 Prostate Cancer

    Directory of Open Access Journals (Sweden)

    Tomonori Kato

    2010-11-01

    Full Text Available We recently demonstrated highly efficient antitumor immunity against dermal tumors of B16F10 murine melanoma with the use of dendritic cells (DCs activated by replication-competent, as well as nontransmissible-type, recombinant Sendai viruses (rSeV, and proposed a new concept, “immunostimulatory virotherapy,” for cancer immunotherapy. However, there has been little information on the efficacies of thismethod: 1 inmore clinically relevant situations including metastatic diseases, 2 on other tumor types and other animal species, and 3 on the related molecular/cellular mechanisms. In this study, therefore, we investigated the efficacy of vaccinating DCs activated by fusion gene-deleted nontransmissible rSeV on a rat model of lung metastasis using a highly malignant subline of Dunning R-3327 prostate cancer, AT6.3. rSeV/dF-green fluorescent protein (GFP-activated bone marrow-derived DCs (rSeV/dF-GFP-DC, consistent with results previously observed in murine DCs. Vaccination of rSeV/dF-GFP-DC was highly effective at preventing lung metastasis after intravenous loading of R-3327 tumor cells, compared with the effects observed with immature DCs or lipopolysaccharide-activated DCs. Interestingly, neither CTL activity nor DC trafficking showed any apparent difference among groups. Notably, rSeV/dF-DCs expressing a dominant-negative mutant of retinoic acid-inducible gene I (RIG-I (rSeV/dF-RIGIC-DC, an RNA helicase that recognizes the rSeV genome for inducing type I interferons, largely lost the expression of proinflammatory cytokines without any impairment of antitumor activity. These results indicate the essential role of RIG-I-independent signaling on antimetastatic effect induced by rSeV-activated DCs and may provide important insights to DC-based immunotherapy for advanced malignancies.

  16. Nuclear Export of Pre-Ribosomal Subunits Requires Dbp5, but Not as an RNA-Helicase as for mRNA Export.

    Science.gov (United States)

    Neumann, Bettina; Wu, Haijia; Hackmann, Alexandra; Krebber, Heike

    2016-01-01

    The DEAD-box RNA-helicase Dbp5/Rat8 is known for its function in nuclear mRNA export, where it displaces the export receptor Mex67 from the mRNA at the cytoplasmic side of the nuclear pore complex (NPC). Here we show that Dbp5 is also required for the nuclear export of both pre-ribosomal subunits. Yeast temperature-sensitive dbp5 mutants accumulate both ribosomal particles in their nuclei. Furthermore, Dbp5 genetically and physically interacts with known ribosomal transport factors such as Nmd3. Similar to mRNA export we show that also for ribosomal transport Dbp5 is required at the cytoplasmic side of the NPC. However, unlike its role in mRNA export, Dbp5 does not seem to undergo its ATPase cycle for this function, as ATPase-deficient dbp5 mutants that selectively inhibit mRNA export do not affect ribosomal transport. Furthermore, mutants of GLE1, the ATPase stimulating factor of Dbp5, show no major ribosomal export defects. Consequently, while Dbp5 uses its ATPase cycle to displace the export receptor Mex67 from the translocated mRNAs, Mex67 remains bound to ribosomal subunits upon transit to the cytoplasm, where it is detectable on translating ribosomes. Therefore, we propose a model, in which Dbp5 supports ribosomal transport by capturing ribosomal subunits upon their cytoplasmic appearance at the NPC, possibly by binding export factors such as Mex67. Thus, our findings reveal that although different ribonucleoparticles, mRNAs and pre-ribosomal subunits, use shared export factors, they utilize different transport mechanisms.

  17. Wheelchair accessibility to public buildings in Istanbul.

    Science.gov (United States)

    Evcil, A Nilay

    2009-03-01

    Accessibility to public environment is the human right and basic need of each citizen and is one of the fundamental considerations for urban planning. The aim of this study is to determine the compliance of public buildings in central business districts (CBD) of Istanbul, Turkey, to wheelchair accessibility to the guidelines of the instrument and identify architectural barriers faced by wheelchair users. This is a descriptive study of 26 public buildings in CBD of Istanbul. The instrument used is the adapted Useh, Moyo and Munyonga questionnaire to collect the data from direct observation and measurement. Descriptive statistics of simple percentages and means are used to explain the compliance to the guidelines of the instrument and wheelchair accessibility. The descriptive survey results indicate that wheelchair users experience many accessibility problems in public environment of the most urbanised city (cultural capital of Europe in 2010) in a developing country. It is found that the major architectural barrier is the public transportation items with the lowest mean compliance (25%). Beside this, the most compliant to the instrument is entrance to building items with 79% as mean percentage. It is also found that there is an intention to improve accessibility when building construction period is investigated. This article describes the example of the compliance of public buildings accessibility when the country has legislation, but lacking regulations about accessibility for the wheelchair users.

  18. 48 CFR 1509.505-4 - Obtaining access to proprietary information.

    Science.gov (United States)

    2010-10-01

    ... proprietary information. 1509.505-4 Section 1509.505-4 Federal Acquisition Regulations System ENVIRONMENTAL....505-4 Obtaining access to proprietary information. Contractors gaining access to confidential business... business information. ...

  19. Pediatric vascular access

    International Nuclear Information System (INIS)

    Donaldson, James S.

    2006-01-01

    Pediatric interventional radiologists are ideally suited to provide vascular access services to children because of inherent safety advantages and higher success from using image-guided techniques. The performance of vascular access procedures has become routine at many adult interventional radiology practices, but this service is not as widely developed at pediatric institutions. Although interventional radiologists at some children's hospitals offer full-service vascular access, there is little or none at others. Developing and maintaining a pediatric vascular access service is a challenge. Interventionalists skilled in performing such procedures are limited at pediatric institutions, and institutional support from clerical staff, nursing staff, and technologists might not be sufficiently available to fulfill the needs of such a service. There must also be a strong commitment by all members of the team to support such a demanding service. There is a slippery slope of expected services that becomes steeper and steeper as the vascular access service grows. This review is intended primarily as general education for pediatric radiologists learning vascular access techniques. Additionally, the pediatric or adult interventional radiologist seeking to expand services might find helpful tips. The article also provides education for the diagnostic radiologist who routinely interprets radiographs containing vascular access devices. (orig.)

  20. Regulating deregulated energy markets

    International Nuclear Information System (INIS)

    Jackson, M.

    2002-01-01

    The North American gas and electricity markets are fast evolving, and regulators are currently faced with a host of issues such as market-based rates, unbundling, stranded costs, open access, and incentive regulation are surfacing as a result of deregulation. The regulatory environment in Ontario was reviewed by the author. Deregulated markets rule, from commodities to gas and electricity. Additionally, there is an evolution of traditional utility regulation. A look at deregulated markets revealed that there are regulations on boundary conditions on the deregulated market. Under the Ontario Energy Board (OEB), all generators, transmitters, distributors, and retailers of electricity must be licensed. The standard supply service (SSS) offered by electricity distributors and system gas which is still being sold by natural gas distributors continues to be regulated by OEB. One issue that was addressed was separation for revenues and costs of the utility's purchase and sale of gas business, at least for accounting purposes. The next issue discussed was cost of system gas and SSS, followed by timely signals and prudent incurred costs. Historical benefits were reviewed, such as historical commitments to low-cost electricity. Pooling transportation costs, transmission pricing continued, market-based rates, unbundling, stranded costs, open access, incentive regulation/ performance based regulation (PBR) were all discussed. Price cap on PBR, both partial and comprehensive were looked at. A requirement to review guidelines on cost of capital and an application to extend blanket approval provisions for gas storage were discussed, as they are amongst some of the challenges of the future. Other challenges include revised rules and practice and procedure; practice directions for cost awards, appeals, and other functions; confidentiality guidelines; and refinements to the role of and approaches to alternative dispute resolution. The future role of regulators was examined in light

  1. Access 2010 for dummies

    CERN Document Server

    Ulrich Fuller, Laurie

    2010-01-01

    A friendly, step-by-step guide to the Microsoft Office database application Access may be the least understood and most challenging application in the Microsoft Office suite. This guide is designed to help anyone who lacks experience in creating and managing a database learn to use Access 2010 quickly and easily. In the classic For Dummies tradition, the book provides an education in Access, the interface, and the architecture of a database. It explains the process of building a database, linking information, sharing data, generating reports, and much more.As the Micr

  2. Optical Access Networks

    Science.gov (United States)

    Zheng, Jun; Ansari, Nirwan

    2005-01-01

    Call for Papers: Optical Access Networks Guest Editors Jun Zheng, University of Ottawa Nirwan Ansari, New Jersey Institute of Technology Submission Deadline: 1 June 2005 Background With the wide deployment of fiber-optic technology over the past two decades, we have witnessed a tremendous growth of bandwidth capacity in the backbone networks of today's telecommunications infrastructure. However, access networks, which cover the "last-mile" areas and serve numerous residential and small business users, have not been scaled up commensurately. The local subscriber lines for telephone and cable television are still using twisted pairs and coaxial cables. Most residential connections to the Internet are still through dial-up modems operating at a low speed on twisted pairs. As the demand for access bandwidth increases with emerging high-bandwidth applications, such as distance learning, high-definition television (HDTV), and video on demand (VoD), the last-mile access networks have become a bandwidth bottleneck in today's telecommunications infrastructure. To ease this bottleneck, it is imperative to provide sufficient bandwidth capacity in the access networks to open the bottleneck and thus present more opportunities for the provisioning of multiservices. Optical access solutions promise huge bandwidth to service providers and low-cost high-bandwidth services to end users and are therefore widely considered the technology of choice for next-generation access networks. To realize the vision of optical access networks, however, many key issues still need to be addressed, such as network architectures, signaling protocols, and implementation standards. The major challenges lie in the fact that an optical solution must be not only robust, scalable, and flexible, but also implemented at a low cost comparable to that of existing access solutions in order to increase the economic viability of many potential high-bandwidth applications. In recent years, optical access networks

  3. 7 CFR 53.15 - Accessibility to livestock.

    Science.gov (United States)

    2010-01-01

    ... 7 Agriculture 3 2010-01-01 2010-01-01 false Accessibility to livestock. 53.15 Section 53.15... AGRICULTURAL MARKETING ACT OF 1946 AND THE EGG PRODUCTS INSPECTION ACT (CONTINUED) LIVESTOCK (GRADING, CERTIFICATION, AND STANDARDS) Regulations Service § 53.15 Accessibility to livestock. (a) The applicant shall...

  4. 24 CFR 200.929a - Fair Housing Accessibility Guidelines.

    Science.gov (United States)

    2010-04-01

    ... Guidelines. 200.929a Section 200.929a Housing and Urban Development Regulations Relating to Housing and Urban... Fair Housing Accessibility Guidelines. Builders and developers may use the Department's Fair Housing Accessibility Guideline when designing or constructing covered multifamily dwelling units in order to comply...

  5. 49 CFR 633.15 - Access to information.

    Science.gov (United States)

    2010-10-01

    ... 49 Transportation 7 2010-10-01 2010-10-01 false Access to information. 633.15 Section 633.15 Transportation Other Regulations Relating to Transportation (Continued) FEDERAL TRANSIT ADMINISTRATION, DEPARTMENT OF TRANSPORTATION PROJECT MANAGEMENT OVERSIGHT Project Management Oversight Services § 633.15 Access to information. A recipient of FTA...

  6. 45 CFR 2490.150 - Program accessibility: Existing facilities.

    Science.gov (United States)

    2010-10-01

    ... 45 Public Welfare 4 2010-10-01 2010-10-01 false Program accessibility: Existing facilities. 2490.150 Section 2490.150 Public Welfare Regulations Relating to Public Welfare (Continued) JAMES MADISON... ACTIVITIES CONDUCTED BY THE JAMES MADISON MEMORIAL FELLOWSHIP FOUNDATION § 2490.150 Program accessibility...

  7. 7 CFR 52.35 - Accessibility for sampling.

    Science.gov (United States)

    2010-01-01

    ... 7 Agriculture 2 2010-01-01 2010-01-01 false Accessibility for sampling. 52.35 Section 52.35... PROCESSED FOOD PRODUCTS 1 Regulations Governing Inspection and Certification Sampling § 52.35 Accessibility for sampling. Each applicant shall cause the processed products for which inspection is requested to...

  8. 17 CFR 242.610 - Access to quotations.

    Science.gov (United States)

    2010-04-01

    ... 17 Commodity and Securities Exchanges 3 2010-04-01 2010-04-01 false Access to quotations. 242.610...-Regulation of the National Market System § 242.610 Access to quotations. (a) Quotations of SRO trading... national securities exchange or national securities association to the quotations in an NMS stock displayed...

  9. Phosphorylation of Large T Antigen Regulates Merkel Cell Polyomavirus Replication

    International Nuclear Information System (INIS)

    Diaz, Jason; Wang, Xin; Tsang, Sabrina H.; Jiao, Jing; You, Jianxin

    2014-01-01

    Merkel Cell Polyomavirus (MCPyV) was recently discovered as a novel human polyomavirus that is associated with ~80% of Merkel Cell Carcinomas. The Large Tumor antigen (LT) is an early viral protein which has a variety of functions, including manipulation of the cell cycle and initiating viral DNA replication. Phosphorylation plays a critical regulatory role for polyomavirus LT proteins, but no investigation of MCPyV LT phosphorylation has been performed to date. In this report mass spectrometry analysis reveals three unique phosphorylation sites: T271, T297 and T299. In vivo replication assays confirm that phosphorylation of T271 does not play a role in viral replication, while modification at T297 and T299 have dramatic and opposing effects on LT’s ability to initiate replication from the viral origin. We test these mutants for their ability to bind, unwind, and act as a functional helicase at the viral origin. These studies provide a framework for understanding how phosphorylation of LT may dynamically regulate viral replication. Although the natural host cell of MCPyV has not yet been established, this work provides a foundation for understanding how LT activity is regulated and provides tools for better exploring this regulation in both natural host cells and Merkel cells

  10. Phosphorylation of Large T Antigen Regulates Merkel Cell Polyomavirus Replication

    Energy Technology Data Exchange (ETDEWEB)

    Diaz, Jason; Wang, Xin; Tsang, Sabrina H. [Department of Microbiology, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104 (United States); Jiao, Jing [Department of Pathology and Laboratory Medicine, Children’s Hospital of Philadelphia, Philadelphia, PA 19104 (United States); You, Jianxin, E-mail: jianyou@mail.med.upenn.edu [Department of Microbiology, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104 (United States)

    2014-07-08

    Merkel Cell Polyomavirus (MCPyV) was recently discovered as a novel human polyomavirus that is associated with ~80% of Merkel Cell Carcinomas. The Large Tumor antigen (LT) is an early viral protein which has a variety of functions, including manipulation of the cell cycle and initiating viral DNA replication. Phosphorylation plays a critical regulatory role for polyomavirus LT proteins, but no investigation of MCPyV LT phosphorylation has been performed to date. In this report mass spectrometry analysis reveals three unique phosphorylation sites: T271, T297 and T299. In vivo replication assays confirm that phosphorylation of T271 does not play a role in viral replication, while modification at T297 and T299 have dramatic and opposing effects on LT’s ability to initiate replication from the viral origin. We test these mutants for their ability to bind, unwind, and act as a functional helicase at the viral origin. These studies provide a framework for understanding how phosphorylation of LT may dynamically regulate viral replication. Although the natural host cell of MCPyV has not yet been established, this work provides a foundation for understanding how LT activity is regulated and provides tools for better exploring this regulation in both natural host cells and Merkel cells.

  11. Roundabouts and access management.

    Science.gov (United States)

    2014-03-01

    Transportation engineers and planners are becoming more interested in using roundabouts to address access : management and safety concerns in the transportation system. While roundabouts are being used increasingly in a : variety of contexts, existin...

  12. Market Access and Welfare

    DEFF Research Database (Denmark)

    Raimondos-Møller, Pascalis; Woodland, Alan D.

    According to the literature, well known tariff reform rules that are guaranteed to increase welfare will not necessarily increase market access, while rules that are guaranteed to increase market access will not necessarily increase welfare. Such conflict between welfare and market access...... objectives of trade policy is problematic and calls for finding alternative tariff reform rules that can achieve both objectives at the same time. The present paper contributes to this aim by using a new set of tariff reforms that are based on local optimality. Using such reforms it is shown that market...... access and consumer welfare will always be weakly compatible, in the sense that reforms based on each objective have the same signed effect on the other objective. For strong compatibility, whereby both objectives increase as a result of a locally optimal tariff reform, we derive both a necessary...

  13. KAUST Open Access policy

    KAUST Repository

    Baessa, Mohamed A.

    2017-01-01

    The library plays a critical role in facilitating open access for their researchers, from managing a repository to providing support and information on the OA publication process to their authors. Janis Tyhurst and Dr Imad

  14. Access to the city

    DEFF Research Database (Denmark)

    Andreasen, Manja Hoppe; Møller-Jensen, Lasse

    2017-01-01

    This paper is concerned with access to the city for urban residents living in the periphery of Dar es Salaam, Tanzania. The paper presents an analysis of the mobility practices of residents and investigates the mobility constraints they experience in relation to the limited accessibility provided...... mobility and access to the city for residents in the periphery. Regular mobility is an ingrained part of residents' livelihood strategies. The majority of households rely on one or more members regularly travelling to central parts of the city in relation to their livelihood activities. The analysis...... by road and traffic conditions and highlights how accessibility problems of peripheral settlements are not easily understood separately from the general dysfunctions of the overall mobility system of city....

  15. Critical Access Hospitals (CAH)

    Science.gov (United States)

    ... for Success Am I Rural? Evidence-based Toolkits Economic Impact Analysis Tool Community Health Gateway Sustainability Planning ... hospitals and improve access to healthcare by keeping essential services in rural communities. To accomplish this goal, ...

  16. Remote Network Access (RNA)

    National Research Council Canada - National Science Library

    2002-01-01

    .... Remote Network Access (RNA) includes or is associated with all communication devices/software, firewalls, intrusion detection systems and virus protection applications to ensure security of the OIG, DoD, Network from remote...

  17. Access/AML -

    Data.gov (United States)

    Department of Transportation — The AccessAML is a web-based internet single application designed to reduce the vulnerability associated with several accounts assinged to a single users. This is a...

  18. Access road reclamation

    International Nuclear Information System (INIS)

    Manson, T.; Blok, M.

    1997-01-01

    A general review of the measures involved in restoring abandoned access road sites in British Columbia was presented. Permits and licences are needed for the use of crown land for roads used by the petroleum and natural gas industry for exploration activities. However, the regulatory framework for road site reclamation is not well developed. The nature of access road reclamation is very site-specific. Some of the issues that are considered for all reclamation projects include slope stability, water control, revegetation, soil rehabilitation, access management and monitoring. The primary objective of reclaiming access road sites is to return the site to conditions that are equal or better than pre-disturbance conditions. Restoration measures must be approved by BC Environment and by the Department of Fisheries and Oceans where federal fisheries responsibilities are involved. 54 refs., 5 tabs., 3 figs

  19. DEAD-box helicase 27 promotes colorectal cancer growth and metastasis and predicts poor survival in CRC patients.

    Science.gov (United States)

    Tang, Jieting; Chen, Huarong; Wong, Chi-Chun; Liu, Dabin; Li, Tong; Wang, Xiaohong; Ji, Jiafu; Sung, Joseph Jy; Fang, Jing-Yuan; Yu, Jun

    2018-03-14

    Copy number alterations (CNAs) are crucial for colorectal cancer (CRC) development. In this study, DEAD box polypeptide 27 (DDX27) was identified to be highly amplified in both TCGA CRC (474/615) and primary CRC (47/103), which was positively correlated with its mRNA overexpression. High DDX27 mRNA (N = 199) and protein expression (N = 260) predicted poor survival in CRC patients. Ectopic expression of DDX27 increased CRC cells proliferation, migration and invasion, but suppressed apoptosis. Conversely, silencing of DDX27 exerted opposite effects in vitro and significantly inhibited murine xenograft tumor growth and lung metastasis in vivo. Up-regulation of DDX27 enhanced and prolonged TNF-α-mediated NF-κB signaling. Nucleophosmin (NPM1) was identified as a binding partner of DDX27. DDX27 increased nuclear NPM1 and NF-κB-p65 interaction to enhance DNA binding activity of NF-κB. Silencing NPM1 abrogated DDX27-activating NF-κB signaling and its tumor-promoting function. Together, DDX27 is overexpressed and plays a pivotal oncogenic role in CRC.

  20. Time dependent accessibility

    OpenAIRE

    Kaza, Nikhil

    2015-01-01

    Many place based accessibility studies ignore the time component. Relying on theoretical frameworks that treat distance between two fixed points as constant, these methods ignore the diurnal and seasonal changes in accessibility. Network distances between two nodes are dependent on the network structure and weight distribution on the edges. These weights can change quite frequently and the network structure itself is subject to modification because of availability and unavailability of links ...

  1. The universal access handbook

    CERN Document Server

    Stephanidis, Constantine

    2009-01-01

    In recent years, the field of Universal Access has made significant progress in consolidating theoretical approaches, scientific methods and technologies, as well as in exploring new application domains. Increasingly, professionals in this rapidly maturing area require a comprehensive and multidisciplinary resource that addresses current principles, methods, and tools. Written by leading international authorities from academic, research, and industrial organizations and nonmarket institutions, The Universal Access Handbook covers the unfolding scientific, methodological, technological, and pol

  2. Accessible Geoscience - Digital Fieldwork

    Science.gov (United States)

    Meara, Rhian

    2017-04-01

    Accessible Geoscience is a developing field of pedagogic research aimed at widening participation in Geography, Earth and Environmental Science (GEES) subjects. These subjects are often less commonly associated with disabilities, ethnic minorities, low income socio-economic groups and females. While advancements and improvements have been made in the inclusivity of these subject areas in recent years, access and participation of disabled students remains low. While universities are legally obligated to provide reasonable adjustments to ensure accessibility, the assumed incompatibility of GEES subjects and disability often deters students from applying to study these courses at a university level. Instead of making reasonable adjustments if and when they are needed, universities should be aiming to develop teaching materials, spaces and opportunities which are accessible to all, which in turn will allow all groups to participate in the GEES subjects. With this in mind, the Swansea Geography Department wish to enhance the accessibility of our undergraduate degree by developing digital field work opportunities. In the first instance, we intend to digitise three afternoon excursions which are run as part of a 1st year undergraduate module. Each of the field trips will be digitized into English- and Welsh-medium formats. In addition, each field trip will be digitized into British Sign Language (BSL) to allow for accessibility for D/deaf and hard of hearing students. Subtitles will also be made available in each version. While the main focus of this work is to provide accessible fieldwork opportunities for students with disabilities, this work also has additional benefits. Students within the Geography Department will be able to revisit the field trips, to revise and complete associated coursework. The use of digitized field work should not replace opportunities for real field work, but its use by the full cohort of students will begin to "normalize" accessible field

  3. CERN access cards

    CERN Multimedia

    HR Department

    2007-01-01

    Holders of CERN access cards are reminded that the card is an official document. It is important to carry it with you at all times when you are on the site. This applies also to those on standby duty who are called out for emergency interventions. As announced in Weekly Bulletin 13/2006, any loss or theft of access cards must be declared to the competent external authorities.

  4. Vascular Access in Children

    International Nuclear Information System (INIS)

    Krishnamurthy, Ganesh; Keller, Marc S.

    2011-01-01

    Establishment of stable vascular access is one of the essential and most challenging procedures in a pediatric hospital. Many clinical specialties provide vascular service in a pediatric hospital. At the top of the “expert procedural pyramid” is the pediatric interventional radiologist, who is best suited and trained to deliver this service. Growing awareness regarding the safety and high success rate of vascular access using image guidance has led to increased demand from clinicians to provide around-the-clock vascular access service by pediatric interventional radiologists. Hence, the success of a vascular access program, with the pediatric interventional radiologist as the key provider, is challenging, and a coordinated multidisciplinary team effort is essential for success. However, there are few dedicated pediatric interventional radiologists across the globe, and also only a couple of training programs exist for pediatric interventions. This article gives an overview of the technical aspects of pediatric vascular access and provides useful tips for obtaining vascular access in children safely and successfully using image guidance.

  5. The RTR Complex Partner RMI2 and the DNA Helicase RTEL1 Are Both Independently Involved in Preserving the Stability of 45S rDNA Repeats in Arabidopsis thaliana.

    Directory of Open Access Journals (Sweden)

    Sarah Röhrig

    2016-10-01

    Full Text Available The stability of repetitive sequences in complex eukaryotic genomes is safeguarded by factors suppressing homologues recombination. Prominent in this is the role of the RTR complex. In plants, it consists of the RecQ helicase RECQ4A, the topoisomerase TOP3α and RMI1. Like mammals, but not yeast, plants harbor an additional complex partner, RMI2. Here, we demonstrate that, in Arabidopsis thaliana, RMI2 is involved in the repair of aberrant replication intermediates in root meristems as well as in intrastrand crosslink repair. In both instances, RMI2 is involved independently of the DNA helicase RTEL1. Surprisingly, simultaneous loss of RMI2 and RTEL1 leads to loss of male fertility. As both the RTR complex and RTEL1 are involved in suppression of homologous recombination (HR, we tested the efficiency of HR in the double mutant rmi2-2 rtel1-1 and found a synergistic enhancement (80-fold. Searching for natural target sequences we found that RTEL1 is required for stabilizing 45S rDNA repeats. In the double mutant with rmi2-2 the number of 45S rDNA repeats is further decreased sustaining independent roles of both factors in this process. Thus, loss of suppression of HR does not only lead to a destabilization of rDNA repeats but might be especially deleterious for tissues undergoing multiple cell divisions such as the male germline.

  6. The RTR Complex Partner RMI2 and the DNA Helicase RTEL1 Are Both Independently Involved in Preserving the Stability of 45S rDNA Repeats in Arabidopsis thaliana.

    Science.gov (United States)

    Röhrig, Sarah; Schröpfer, Susan; Knoll, Alexander; Puchta, Holger

    2016-10-01

    The stability of repetitive sequences in complex eukaryotic genomes is safeguarded by factors suppressing homologues recombination. Prominent in this is the role of the RTR complex. In plants, it consists of the RecQ helicase RECQ4A, the topoisomerase TOP3α and RMI1. Like mammals, but not yeast, plants harbor an additional complex partner, RMI2. Here, we demonstrate that, in Arabidopsis thaliana, RMI2 is involved in the repair of aberrant replication intermediates in root meristems as well as in intrastrand crosslink repair. In both instances, RMI2 is involved independently of the DNA helicase RTEL1. Surprisingly, simultaneous loss of RMI2 and RTEL1 leads to loss of male fertility. As both the RTR complex and RTEL1 are involved in suppression of homologous recombination (HR), we tested the efficiency of HR in the double mutant rmi2-2 rtel1-1 and found a synergistic enhancement (80-fold). Searching for natural target sequences we found that RTEL1 is required for stabilizing 45S rDNA repeats. In the double mutant with rmi2-2 the number of 45S rDNA repeats is further decreased sustaining independent roles of both factors in this process. Thus, loss of suppression of HR does not only lead to a destabilization of rDNA repeats but might be especially deleterious for tissues undergoing multiple cell divisions such as the male germline.

  7. Youth access to tobacco.

    Science.gov (United States)

    Rigotti, N A

    1999-01-01

    To start smoking, young people need a supply of tobacco products. Reducing youth access to tobacco is a new approach to preventing tobacco use that has been a focus of federal, state, and local tobacco control efforts over the past decade. All 50 states ban tobacco sales to minors, but compliance is poor because laws are not enforced. Consequently, young people have little trouble obtaining tobacco products. Commercial sources of tobacco (stores and vending machines) are important for underage smokers, who often purchase their own cigarettes. Underage youths also obtain tobacco from noncommercial sources such as friends, relatives, older adolescents, and adults. Educating retailers about tobacco sales laws has not produced long-term improvement in their compliance. Active enforcement of tobacco sales laws changes retailer behavior, but whether this reduces young people's access to tobacco or their tobacco use is not clear. The effectiveness of new local, state, and federal actions that aim to reduce youth access to tobacco remains to be determined. Can enforcing tobacco sales laws reduce young people's access to tobacco? If so, will this prevent or delay the onset of their tobacco use? How will youths' sources of tobacco change as commercial sources are restricted? What are the social (noncommercial) sources of tobacco for minors and how can youths' access to tobacco from these sources be reduced? What is the impact of the new federal policies aimed at reducing youth access to tobacco? Do new state and local laws that ban youth possession or use of tobacco have a net positive or negative impact on youth attitudes, access to tobacco, or tobacco use? What is the relative effectiveness and cost-effectiveness of efforts to reduce the supply of tobacco compared to those that aim to reduce demand for tobacco? Will either work alone or are both necessary to achieve reductions in youth smoking?

  8. 7 CFR 54.1014 - Accessibility of equipment and utensils; access to establishments.

    Science.gov (United States)

    2010-01-01

    ...) AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing Practices), DEPARTMENT OF AGRICULTURE (CONTINUED) REGULATIONS AND STANDARDS UNDER THE AGRICULTURAL MARKETING ACT OF 1946 AND THE EGG PRODUCTS..., Processing, and Packaging of Livestock and Poultry Products § 54.1014 Accessibility of equipment and utensils...

  9. Concerted action of the PHD, chromo and motor domains regulates the human chromatin remodelling ATPase CHD4.

    Science.gov (United States)

    Morra, Rosa; Lee, Benjamin M; Shaw, Heather; Tuma, Roman; Mancini, Erika J

    2012-07-30

    CHD4, the core subunit of the Nucleosome Remodelling and Deacetylase (NuRD) complex, is a chromatin remodelling ATPase that, in addition to a helicase domain, harbors tandem plant homeo finger and chromo domains. By using a panel of domain constructs we dissect their roles and demonstrate that DNA binding, histone binding and ATPase activities are allosterically regulated. Molecular shape reconstruction from small-angle X-ray scattering reveals extensive domain-domain interactions, which provide a structural explanation for the regulation of CHD4 activities by intramolecular domain communication. Our results demonstrate functional interdependency between domains within a chromatin remodeller. Copyright © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  10. 15 CFR 2008.15 - General restrictions on access.

    Science.gov (United States)

    2010-01-01

    ... 15 Commerce and Foreign Trade 3 2010-01-01 2010-01-01 false General restrictions on access. 2008.15 Section 2008.15 Commerce and Foreign Trade Regulations Relating to Foreign Trade Agreements OFFICE OF THE UNITED STATES TRADE REPRESENTATIVE REGULATIONS TO IMPLEMENT E.O. 12065; OFFICE OF THE UNITED...

  11. Access control system operation

    International Nuclear Information System (INIS)

    Barnes, L.D.

    1981-06-01

    An automated method for the control and monitoring of personnel movement throughout the site was developed under contract to the Department of Energy by Allied-General Nuclear Services (AGNS) at the Barnwell Nuclear Fuel Plant (BNFP). These automated features provide strict enforcement of personnel access policy without routine patrol officer involvement. Identification methods include identification by employee ID number, identification by voice verification and identification by physical security officer verification. The ability to grant each level of access authority is distributed over the organization to prevent any single individual at any level in the organization from being capable of issuing an authorization for entry into sensitive areas. Each access event is recorded. As access events occur, the inventory of both the entered and the exited control area is updated so that a current inventory is always available for display. The system has been operated since 1979 in a development mode and many revisions have been implemented in hardware and software as areas were added to the system. Recent changes have involved the installation of backup systems and other features required to achieve a high reliability. The access control system and recent operating experience are described

  12. Functional 5' UTR mRNA structures in eukaryotic translation regulation and how to find them.

    Science.gov (United States)

    Leppek, Kathrin; Das, Rhiju; Barna, Maria

    2018-03-01

    RNA molecules can fold into intricate shapes that can provide an additional layer of control of gene expression beyond that of their sequence. In this Review, we discuss the current mechanistic understanding of structures in 5' untranslated regions (UTRs) of eukaryotic mRNAs and the emerging methodologies used to explore them. These structures may regulate cap-dependent translation initiation through helicase-mediated remodelling of RNA structures and higher-order RNA interactions, as well as cap-independent translation initiation through internal ribosome entry sites (IRESs), mRNA modifications and other specialized translation pathways. We discuss known 5' UTR RNA structures and how new structure probing technologies coupled with prospective validation, particularly compensatory mutagenesis, are likely to identify classes of structured RNA elements that shape post-transcriptional control of gene expression and the development of multicellular organisms.

  13. Enterprise Dynamic Access Control (EDAC)

    National Research Council Canada - National Science Library

    Fernandez, Richard

    2005-01-01

    .... Resources can represent software applications, web services and even facility access. An effective access control model should be capable of evaluating resource access based on user characteristics and environmentals...

  14. Nuclear information access system

    International Nuclear Information System (INIS)

    Ham, C. H.; Yang, M. H.; Yoon, S. W.

    1998-01-01

    The energy supply in the countries, which have abundant energy resources, may not be affected by accepting the assertion of anti-nuclear and environment groups. Anti-nuclear movements in the countries which have little energy resources may cause serious problem in securing energy supply. Especially, it is distinct in Korea because she heavily depends on nuclear energy in electricity supply(nuclear share in total electricity supply is about 40%).The cause of social trouble surrounding nuclear energy is being involved with various circumstances. However, it is very important that we are not aware of the importance of information access and prepared for such a situation from the early stage of nuclear energy's development. In those matter, this paper analyzes the contents of nuclear information access system in France and Japan which have dynamic nuclear development program and presents the direction of the nuclear access regime through comparing Korean status and referring to progresses of the regime

  15. Sprawl and Accessibility

    Directory of Open Access Journals (Sweden)

    Robert Bruegmann

    2008-07-01

    Full Text Available This essay argues that many of the assumptions that have been made about sprawl are misleading or just wrong. Nowhere has this been more the case than in debates about transportation and access. Because of this, it is not surprising that a good many of the policies advocated by proponents of Smart Growth would almost certainly lead to reduced mobility and impaired accessibility for a large part of the population. At very least, the debates over sprawl have pitted private vs. public transportation in a way that has contributed to serious underfunding of transportation infrastructure of all kinds.

  16. Quantum random access memory

    OpenAIRE

    Giovannetti, Vittorio; Lloyd, Seth; Maccone, Lorenzo

    2007-01-01

    A random access memory (RAM) uses n bits to randomly address N=2^n distinct memory cells. A quantum random access memory (qRAM) uses n qubits to address any quantum superposition of N memory cells. We present an architecture that exponentially reduces the requirements for a memory call: O(log N) switches need be thrown instead of the N used in conventional (classical or quantum) RAM designs. This yields a more robust qRAM algorithm, as it in general requires entanglement among exponentially l...

  17. Disruption - Access cards service

    CERN Multimedia

    2014-01-01

    We would like to inform you that between 10 November and 15 December 2014, the access cards service in Building 55 will be disrupted, as the GS Department has decided to improve the facilities for users of this building. During the work, you will find the registration, biometric registration and dosimeter exchange services on the second floor of Building 55 and the vehicle sticker service on the ground floor along with the access cards service. We thank you for your understanding and apologise for any inconvenience caused.

  18. Central Venous Access

    International Nuclear Information System (INIS)

    Ganeshan, Arul; Warakaulle, Dinuke R.; Uberoi, Raman

    2007-01-01

    Central venous access plays an important role in the management of an ever-increasing population of patients ranging from those that are critically ill to patients with difficult clinical access. Interventional radiologists are key in delivering this service and should be familiar with the wide range of techniques and catheters now available to them. A comprehensive description of these catheters with regard to indications, technical aspects of catheterization, success rates, and associated early and late complications, as well as a review of various published guidelines on central venous catheter insertion are given in this article

  19. Editorial - Open Access and accessing openness

    Directory of Open Access Journals (Sweden)

    Yngve Nordkvelle

    2008-12-01

    Full Text Available Seminar.net enters it’s fourth year, and has reached a state of maturity in a number of meanings: it receives manuscripts from all continents, the articles are read from 134 countries, of which India represents the highest number of readers, a number of articles have been read by more than 10 000 interested persons, and the frequency of issues is now three per year, and will reach four by next year. Interested parties now approach us in order to learn about our policies and practices.It takes time to become established and influential in the sense that articles are cited and referred to in prestigious publications. Still, the most prestigious publications are on paper. Many countries now embark on a policy that rewards researchers that publish in international journals, preferably in English. National languages are rendered less significant. In the UK, the research assessment exercise (RAE, and several other countries with a publication or citation based reward system in research, tend to favour quantitative dimensions at the expense of the quality of the publication. International publishing houses are huge profit-making companies that over years have increased their profit rates, charging increasingly economically pressured higher education institution with high subscription rates. With the advent of electronic publishing their position is severely challenged. It has been noted that the most significant publication of the last couple of decades was an electronic publication: Tim Berners Lee published the protocol for the World Wide Web in 1990. It was never refereed, nor was controlled by appointed gatekeepers of the “establishment”. The number of Open Access publications is rising every day, and the number of e-journals for academic publishing is reaching higher and higher numbers. In a recent case The Faculty of Arts and Sciences at Harvard University decided, that Harvard employees must publish all their material simultaneously on the

  20. Complication with intraosseous access

    DEFF Research Database (Denmark)

    Hallas, Peter; Brabrand, Mikkel; Folkestad, Lars

    2013-01-01

    with aspiration of bone marrow (12.3%), and bended/broken needle (4.0%). When using an established IO access the reported complications were difficulties with injection fluid and drugs after IO insertion (7.4%), slow infusion (despite use of pressure bag) (8.8%), displacement after insertion (8...

  1. College Access Marketing

    Science.gov (United States)

    Tremblay, Christopher W.

    2011-01-01

    College Access Marketing (CAM) is a relatively new phenomenon that seeks to positively influence the college-going rate. This report defines CAM, describes CAM examples, and discusses how CAM seeks to counter barriers to college. It explores four main elements of CAM: information, marketing, advocacy, and social mobilization. Further, it…

  2. Funding scientific open access

    International Nuclear Information System (INIS)

    Canessa, E.; Fonda, C.; Zennaro, M.

    2006-11-01

    In order to reduce the knowledge divide, more Open Access Journals (OAJ) are needed in all languages and scholarly subject areas that exercise peer-review or editorial quality control. To finance needed costs, it is discussed why and how to sell target specific advertisement by associating ads to given scientific keywords. (author)

  3. Accessing vs Sourcing Knowledge

    DEFF Research Database (Denmark)

    Awate, Snehal; Larsen, Marcus M.; Mudambi, Ram

    2015-01-01

    to get on par with industry leaders. An in-depth comparison of knowledge flows reveals that within AMNEs, headquarters often serves the primary source of knowledge for R&D subsidiaries. In contrast, within EMNEs, headquarters accesses knowledge from R&D subsidiaries in advanced economies for innovation...

  4. KAUST Open Access policy

    KAUST Repository

    Baessa, Mohamed A.

    2017-03-07

    The transition to open access (OA) is being driven by funders, libraries, researchers and publishers around the world, and is having an impact on us all. It is inevitable that different countries, organisations and disciplines are moving at different rates towards an OA model, and it is this that we will focus on in this session. Drawing on experiences from across Europe and the Middle East we will provide perspectives from both a global publisher and institutions based in the region. Taylor & Francis take a flexible, evidence-based approach to open access, providing a choice of publication routes for our authors, and a choice of agreements for our library customers. Carolyn will outline some of the open access developments, opportunities and challenges at Taylor & Francis. The library plays a critical role in facilitating open access for their researchers, from managing a repository to providing support and information on the OA publication process to their authors. Janis Tyhurst and Dr Imad Bachir will each give an overview of how this is being managed by their institution. There will be an opportunity for questions and discussion with the panel.

  5. The Open Access Dilemma

    Science.gov (United States)

    Pratt, Timothy

    2017-01-01

    Community colleges, with their commitment to open access, admit millions of students each year who are unprepared for college-level work, even though they have earned a high-school diploma. For decades the schools had a built-in base of students attracted to their open doors and relative affordability. But enrollment at public two-year college has…

  6. Access Rights Distribution

    Directory of Open Access Journals (Sweden)

    T. M. Pestunova

    2011-03-01

    Full Text Available The main aspects of the development of the information system, which grants users the rights for access to automated information system resources on the basis of the organization business-processes, are considered. The conceptual, informational, functional models of the system, as well as a model, which allows to realize control while using various formal models (RBAC, DAC, MAC, are described.

  7. Coded Random Access

    DEFF Research Database (Denmark)

    Paolini, Enrico; Stefanovic, Cedomir; Liva, Gianluigi

    2015-01-01

    The rise of machine-to-machine communications has rekindled the interest in random access protocols as a support for a massive number of uncoordinatedly transmitting devices. The legacy ALOHA approach is developed under a collision model, where slots containing collided packets are considered as ...

  8. Planning for Accessibility.

    Science.gov (United States)

    Spoor, Dana L.

    1997-01-01

    Argues that barrier-free designs should be incorporated in the first steps of school facility planning to avoid the difficulties in meeting Americans with Disabilities Act (ADA) guidelines during renovations. Explains why not all barriers need be removed to make a facility accessible to everyone. Discusses issues involving ADA guidelines and child…

  9. ACCESS Project: Final Report

    Energy Technology Data Exchange (ETDEWEB)

    Weller, Heiko [Robert Bosch, LLC, Farmington Hills, MI (United States)

    2015-04-01

    The ACCESS project addressed the development, testing, and demonstration of the proposed advanced technologies and the associated emission and fuel economy improvement at an engine dynamometer and on a full-scale vehicle. Improve fuel economy by 25% with minimum performance penalties Achieve SULEV level emissions with gasoline Demonstrate multi-mode combustion engine management system

  10. Multiple Access Communications

    DEFF Research Database (Denmark)

    This book constitutes the proceedings of the 9th International Workshop on Multiple Access Communications, MACOM 2016, held in Aalborg, Denmark, in November 2016. The 10 full papers presented in this volume were carefully reviewed and selected from 12 submissions. They were organized in topical...

  11. Kinds of Access

    DEFF Research Database (Denmark)

    Overgaard, Morten; Sandberg, Kristian

    2014-01-01

    that there is not only a theoretical, but also an empirical difference between different methods of reporting. We hypothesise that differences in the sensitivity of different scales may reveal that different types of access are used to issue direct reports about experiences and metacognitive reports about...

  12. Fixed Access Network Sharing

    Science.gov (United States)

    Cornaglia, Bruno; Young, Gavin; Marchetta, Antonio

    2015-12-01

    Fixed broadband network deployments are moving inexorably to the use of Next Generation Access (NGA) technologies and architectures. These NGA deployments involve building fiber infrastructure increasingly closer to the customer in order to increase the proportion of fiber on the customer's access connection (Fibre-To-The-Home/Building/Door/Cabinet… i.e. FTTx). This increases the speed of services that can be sold and will be increasingly required to meet the demands of new generations of video services as we evolve from HDTV to "Ultra-HD TV" with 4k and 8k lines of video resolution. However, building fiber access networks is a costly endeavor. It requires significant capital in order to cover any significant geographic coverage. Hence many companies are forming partnerships and joint-ventures in order to share the NGA network construction costs. One form of such a partnership involves two companies agreeing to each build to cover a certain geographic area and then "cross-selling" NGA products to each other in order to access customers within their partner's footprint (NGA coverage area). This is tantamount to a bi-lateral wholesale partnership. The concept of Fixed Access Network Sharing (FANS) is to address the possibility of sharing infrastructure with a high degree of flexibility for all network operators involved. By providing greater configuration control over the NGA network infrastructure, the service provider has a greater ability to define the network and hence to define their product capabilities at the active layer. This gives the service provider partners greater product development autonomy plus the ability to differentiate from each other at the active network layer.

  13. Digital Scholarship and Open Access

    Science.gov (United States)

    Losoff, Barbara; Pence, Harry E.

    2010-01-01

    Open access publications provide scholars with unrestricted access to the "conversation" that is the basis for the advancement of knowledge. The large number of open access journals, archives, and depositories already in existence demonstrates the technical and economic viability of providing unrestricted access to the literature that is the…

  14. Environmental Regulation

    Data.gov (United States)

    Kansas Data Access and Support Center — KR305b 1994 coverage, used to support water quality programs, references water quality use designations and support levels. The KR305b 1994 coverage is available for...

  15. Radiation regulation

    International Nuclear Information System (INIS)

    Braithwaite, J.; Grabosky, P.

    1985-01-01

    The five main areas of radiation regulation considered are radiation exposure in the mining of uranium and other minerals, exposure in the use of uranium in nuclear reactors, risks in the transport of radioactive materials and hazards associated with the disposal of used materials. In Australia these problems are regulated by mines departments, the Australian Atomic Energy Commission and radiation control branches in state health departments. Each of these instutional areas of regulation is examined

  16. Libraries and Accessibility: Istanbul Public Libraries Case

    Directory of Open Access Journals (Sweden)

    Gül Yücel

    2016-12-01

    Full Text Available In the study; the assessment of accessibility has been conducted in Istanbul public libraries within the scope of public area. Public libraries commonly serve with its user of more than 20 million in total, spread to the general of Turkey, having more than one thousand branches in the centrums and having more than one million registered members. The building principles and standards covering the subjects such as the selection of place, historical and architectural specification of the region, distance to the centre of population and design in a way that the disabled people could benefit from the library services fully have been determined with regulations in the construction of new libraries. There are works for the existent libraries such as access for the disabled, fire safety precautions etc. within the scope of the related standards. Easy access by everyone is prioritized in the public libraries having a significant role in life-long learning. The purpose of the study is to develop solution suggestions for the accessibility problems in the public libraries. The study based on the eye inspection and assessments carried out within the scope of accessibility in the public libraries subsidiary to Istanbul Culture and Tourism Provincial Directorate Library and Publications Department within the provincial borders of Istanbul. The arrangements such as reading halls, study areas, book shelves etc. have been examined within the frame of accessible building standards. Building entrances, ramps and staircases, horizontal and vertical circulation of building etc. have been taken into consideration within the scope of accessible building standards. The subjects such as the reading and studying areas and book shelf arrangements for the library have been assessed within the scope of specific buildings. There are a total of 34 public libraries subsidiary to Istanbul Culture and Tourism Provincial Directorate on condition that 20 ea. of them are in the

  17. Dynamic Spectrum Access: regulations, standards and green radio policy considerations

    CSIR Research Space (South Africa)

    Mfupe, L

    2012-09-01

    Full Text Available of ICT-related emissions globally. The overarching goal is to reach consensus within the global ICT sector on a common methodological framework for the measurement of energy consumption and carbon emissions arising from the production, and operation... of the standards that are of interest in the context of this paper include: (Note all IEEE standards are defined in [39]): ETSI reconfigurable radio systems (RRS) [37]: ETSI considers the feasibility of possible operations of the long- term evolution (LTE...

  18. Protecting Biodiversity: National Laws Regulating Access to Genetic ...

    International Development Research Centre (IDRC) Digital Library (Canada)

    This book is the first to compare such laws and policies across a range of countries in ... and developing worlds, including Argentina, Canada, Colombia, Costa Rica, ... conservation, the environment, intellectual property, and related issues; ...

  19. Support open access publishing

    DEFF Research Database (Denmark)

    Ekstrøm, Jeannette

    2013-01-01

    Projektet Support Open Access Publishing har til mål at få opdateret Sherpa/Romeo databasen (www.sherpa.ac.uk/romeo) med fagligt relevante, danske tidsskrifter. Projektet skal endvidere undersøge mulighederne for at få udviklet en database, hvor forskere på tværs af relevante tidsskriftsinformati......Projektet Support Open Access Publishing har til mål at få opdateret Sherpa/Romeo databasen (www.sherpa.ac.uk/romeo) med fagligt relevante, danske tidsskrifter. Projektet skal endvidere undersøge mulighederne for at få udviklet en database, hvor forskere på tværs af relevante...

  20. Analysing Access Control Specifications

    DEFF Research Database (Denmark)

    Probst, Christian W.; Hansen, René Rydhof

    2009-01-01

    When prosecuting crimes, the main question to answer is often who had a motive and the possibility to commit the crime. When investigating cyber crimes, the question of possibility is often hard to answer, as in a networked system almost any location can be accessed from almost anywhere. The most...... common tool to answer this question, analysis of log files, faces the problem that the amount of logged data may be overwhelming. This problems gets even worse in the case of insider attacks, where the attacker’s actions usually will be logged as permissible, standard actions—if they are logged at all....... Recent events have revealed intimate knowledge of surveillance and control systems on the side of the attacker, making it often impossible to deduce the identity of an inside attacker from logged data. In this work we present an approach that analyses the access control configuration to identify the set...