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Sample records for junction helicase ruvab

  1. Central role of the Holliday junction helicase RuvAB in vlsE recombination and infectivity of Borrelia burgdorferi.

    Tao Lin

    2009-12-01

    Full Text Available Antigenic variation plays a vital role in the pathogenesis of many infectious bacteria and protozoa including Borrelia burgdorferi, the causative agent of Lyme disease. VlsE, a 35 kDa surface-exposed lipoprotein, undergoes antigenic variation during B. burgdorferi infection of mammalian hosts, and is believed to be a critical mechanism by which the spirochetes evade immune clearance. Random, segmental recombination between the expressed vlsE gene and adjacent vls silent cassettes generates a large number of different VlsE variants within the infected host. Although the occurrence and importance of vlsE sequence variation is well established, little is known about the biological mechanism of vlsE recombination. To identify factors important in antigenic variation and vlsE recombination, we screened transposon mutants of genes known to be involved in DNA recombination and repair for their effects on infectivity and vlsE recombination. Several mutants, including those in BB0023 (ruvA, BB0022 (ruvB, BB0797 (mutS, and BB0098 (mutS-II, showed reduced infectivity in immunocompetent C3H/HeN mice. Mutants in ruvA and ruvB exhibited greatly reduced rates of vlsE recombination in C3H/HeN mice, as determined by restriction fragment polymorphism (RFLP screening and DNA sequence analysis. In severe combined immunodeficiency (C3H/scid mice, the ruvA mutant retained full infectivity; however, all recovered clones retained the 'parental' vlsE sequence, consistent with low rates of vlsE recombination. These results suggest that the reduced infectivity of ruvA and ruvB mutants is the result of ineffective vlsE recombination and underscores the important role that vlsE recombination plays in immune evasion. Based on functional studies in other organisms, the RuvAB complex of B. burgdorferi may promote branch migration of Holliday junctions during vlsE recombination. Our findings are consistent with those in the accompanying article by Dresser et al., and together

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

    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...

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

    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.

  4. 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.

    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

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

    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.

  6. RecQ Helicases

    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...

  7. Mitochondrial helicases and mitochondrial genome maintenance

    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...

  8. Resolution of Holliday junctions in Escherichia coli: identification of the ruvC gene product as a 19-kilodalton protein.

    Sharples, G J; Lloyd, R G

    1991-12-01

    The ruvC gene of Escherichia coli specifies a nuclease that resolves Holliday junction intermediates in genetic recombination (B. Connolly, C.A. Parsons, F.E. Benson, H.J. Dunderdale, G.J. Sharples, R.G. Lloyd, and S.C. West, Proc. Natl. Acad, Sci. USA 88:6063-6067, 1991). The gene was located between aspS and the ruvAB operon by DNA sequencing and deletion analysis of ruvC plasmids and was shown to encode a protein of 18,747 Da. Analysis of the DNA flanking ruvC indicated that the gene is transcribed independently of the LexA-regulated ruvAB operon and is not under direct SOS control. ruvC lies downstream of an open reading frame, orf-33, for a protein which migrates during sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a 33-kDa polypeptide. These two genes probably form an operon. However, expression of ruvC was found to be very poor relative to that of orf-33. A double ribosomal frameshift between these genes is proposed as a possible reason for the low level of RuvC. Two further open reading frames of unknown function were identified, one on either side of the orf-33-ruvC operon.

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

    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.

  10. The Dissolution of Double Holliday Junctions

    Bizard, Anna H; Hickson, Ian D

    2014-01-01

    as "double Holliday junction dissolution." This reaction requires the cooperative action of a so-called "dissolvasome" comprising a Holliday junction branch migration enzyme (Sgs1/BLM RecQ helicase) and a type IA topoisomerase (Top3/TopoIIIα) in complex with its OB (oligonucleotide/oligosaccharide binding......Double Holliday junctions (dHJS) are important intermediates of homologous recombination. The separate junctions can each be cleaved by DNA structure-selective endonucleases known as Holliday junction resolvases. Alternatively, double Holliday junctions can be processed by a reaction known......) fold containing accessory factor (Rmi1). This review details our current knowledge of the dissolution process and the players involved in catalyzing this mechanistically complex means of completing homologous recombination reactions....

  11. Purification and crystallization of Kokobera virus helicase

    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

  12. Helicase-dependent amplification of nucleic acids.

    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.

  13. Purification and crystallization of Kokobera virus helicase

    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.

  14. Overcoming natural replication barriers: differential helicase requirements.

    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.

  15. Structural mechanisms of human RecQ helicases WRN and BLM

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

    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.

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

    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.

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

    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.

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

    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.

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

    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.

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

    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.

  2. Synthesis and SAR studies of 5-(pyridin-4-yl)-1,3,4-thiadiazol-2-amine derivatives as potent inhibitors of Bloom helicase

    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....

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

    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.

  4. Gap Junctions

    Nielsen, Morten Schak; Axelsen, Lene Nygaard; Sorgen, Paul L.; Verma, Vandana; Delmar, Mario; Holstein-Rathlou, Niels-Henrik

    2013-01-01

    Gap junctions are essential to the function of multicellular animals, which require a high degree of coordination between cells. In vertebrates, gap junctions comprise connexins and currently 21 connexins are known in humans. The functions of gap junctions are highly diverse and include exchange of metabolites and electrical signals between cells, as well as functions, which are apparently unrelated to intercellular communication. Given the diversity of gap junction physiology, regulation of gap junction activity is complex. The structure of the various connexins is known to some extent; and structural rearrangements and intramolecular interactions are important for regulation of channel function. Intercellular coupling is further regulated by the number and activity of channels present in gap junctional plaques. The number of connexins in cell-cell channels is regulated by controlling transcription, translation, trafficking, and degradation; and all of these processes are under strict control. Once in the membrane, channel activity is determined by the conductive properties of the connexin involved, which can be regulated by voltage and chemical gating, as well as a large number of posttranslational modifications. The aim of the present article is to review our current knowledge on the structure, regulation, function, and pharmacology of gap junctions. This will be supported by examples of how different connexins and their regulation act in concert to achieve appropriate physiological control, and how disturbances of connexin function can lead to disease. © 2012 American Physiological Society. Compr Physiol 2:1981-2035, 2012. PMID:23723031

  5. Structural basis for the function of DEAH helicases

    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...

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

    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

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

    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.

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

    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.

  9. RecQ helicases and cellular responses to DNA damage

    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

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

    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.

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

    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

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

    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.

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

    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

  14. Heteroduplex DNA position defines the roles of the Sgs1, Srs2, and Mph1 helicases in promoting distinct recombination outcomes.

    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.

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

    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.

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

    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.

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

    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.

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

    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.

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

    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

  20. The MCM Helicase Motor of the Eukaryotic Replisome.

    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.

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

    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.

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

    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.

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

    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.

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

    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

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

    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.

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

    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...

  7. Molecular electronic junction transport

    Solomon, Gemma C.; Herrmann, Carmen; Ratner, Mark

    2012-01-01

    Whenasinglemolecule,oracollectionofmolecules,isplacedbetween two electrodes and voltage is applied, one has a molecular transport junction. We discuss such junctions, their properties, their description, and some of their applications. The discussion is qualitative rather than quantitative, and f...

  8. 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

    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...

  9. 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

    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...

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

    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

  11. Junction and circuit fabrication

    Jackel, L.D.

    1980-01-01

    Great strides have been made in Josephson junction fabrication in the four years since the first IC SQUID meeting. Advances in lithography have allowed the production of devices with planar dimensions as small as a few hundred angstroms. Improved technology has provided ultra-high sensitivity SQUIDS, high-efficiency low-noise mixers, and complex integrated circuits. This review highlights some of the new fabrication procedures. The review consists of three parts. Part 1 is a short summary of the requirements on junctions for various applications. Part 2 reviews intergrated circuit fabrication, including tunnel junction logic circuits made at IBM and Bell Labs, and microbridge radiation sources made at SUNY at Stony Brook. Part 3 describes new junction fabrication techniques, the major emphasis of this review. This part includes a discussion of small oxide-barrier tunnel junctions, semiconductor barrier junctions, and microbridge junctions. Part 3 concludes by considering very fine lithography and limitations to miniaturization. (orig.)

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

    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.

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

    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.

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

    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.

  15. Molecular Dynamics of the ZIKA Virus NS3 Helicase

    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.

  16. Molecular determinants of nucleolar translocation of RNA helicase A

    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

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

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

    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

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

    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.

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

    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.

  1. Josephson junction arrays

    Bindslev Hansen, J.; Lindelof, P.E.

    1985-01-01

    In this review we intend to cover recent work involving arrays of Josephson junctions. The work on such arrays falls naturally into three main areas of interest: 1. Technical applications of Josephson junction arrays for high-frequency devices. 2. Experimental studies of 2-D model systems (Kosterlitz-Thouless phase transition, commensurate-incommensurate transition in frustrated (flux) lattices). 3. Investigations of phenomena associated with non-equilibrium superconductivity in and around Josephson junctions (with high current density). (orig./BUD)

  2. Equivalent Josephson junctions

    Boyadzhiev, T.L.; ); Semerdzhieva, E.G.; Shukrinov, Yu.M.; Fiziko-Tekhnicheskij Inst., Dushanbe

    2008-01-01

    The magnetic field dependences of critical current are numerically constructed for a long Josephson junction with a shunt- or resistor-type microscopic inhomogeneities and compared to the critical curve of a junction with exponentially varying width. The numerical results show that it is possible to replace the distributed inhomogeneity of a long Josephson junction by an inhomogeneity localized at one of its ends, which has certain technological advantages. It is also shown that the critical curves of junctions with exponentially varying width and inhomogeneities localized at the ends are unaffected by the mixed fluxon-antifluxon distributions of the magnetic flux [ru

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

    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.

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

    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.

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

    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.

  6. Nucleolin inhibits G4 oligonucleotide unwinding by Werner helicase.

    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.

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

    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...

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

    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.

  9. Supramolecular tunneling junctions

    Wimbush, K.S.

    2012-01-01

    In this study a variety of supramolecular tunneling junctions were created. The basis of these junctions was a self-assembled monolayer of heptathioether functionalized ß-cyclodextrin (ßCD) formed on an ultra-flat Au surface, i.e., the bottom electrode. This gave a well-defined hexagonally packed

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

    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.

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

    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.

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

    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.

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

    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.

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

    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.

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

    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.

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

    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

  17. Primary Tunnel Junction Thermometry

    Pekola, Jukka P.; Holmqvist, Tommy; Meschke, Matthias

    2008-01-01

    We describe the concept and experimental demonstration of primary thermometry based on a four-probe measurement of a single tunnel junction embedded within four arrays of junctions. We show that in this configuration random sample specific and environment-related errors can be avoided. This method relates temperature directly to Boltzmann constant, which will form the basis of the definition of temperature and realization of official temperature scales in the future

  18. Quantum Junction Solar Cells

    Tang, Jiang

    2012-09-12

    Colloidal quantum dot solids combine convenient solution-processing with quantum size effect tuning, offering avenues to high-efficiency multijunction cells based on a single materials synthesis and processing platform. The highest-performing colloidal quantum dot rectifying devices reported to date have relied on a junction between a quantum-tuned absorber and a bulk material (e.g., TiO 2); however, quantum tuning of the absorber then requires complete redesign of the bulk acceptor, compromising the benefits of facile quantum tuning. Here we report rectifying junctions constructed entirely using inherently band-aligned quantum-tuned materials. Realizing these quantum junction diodes relied upon the creation of an n-type quantum dot solid having a clean bandgap. We combine stable, chemically compatible, high-performance n-type and p-type materials to create the first quantum junction solar cells. We present a family of photovoltaic devices having widely tuned bandgaps of 0.6-1.6 eV that excel where conventional quantum-to-bulk devices fail to perform. Devices having optimal single-junction bandgaps exhibit certified AM1.5 solar power conversion efficiencies of 5.4%. Control over doping in quantum solids, and the successful integration of these materials to form stable quantum junctions, offers a powerful new degree of freedom to colloidal quantum dot optoelectronics. © 2012 American Chemical Society.

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

    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.

  20. RNases and Helicases in Gram-Positive Bacteria.

    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.

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

    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.

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

    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.

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

    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.

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

    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.

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

    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.

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

    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.

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

    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.

  8. Four-junction superconducting circuit

    Qiu, Yueyin; Xiong, Wei; He, Xiao-Ling; Li, Tie-Fu; You, J. Q.

    2016-01-01

    We develop a theory for the quantum circuit consisting of a superconducting loop interrupted by four Josephson junctions and pierced by a magnetic flux (either static or time-dependent). In addition to the similarity with the typical three-junction flux qubit in the double-well regime, we demonstrate the difference of the four-junction circuit from its three-junction analogue, including its advantages over the latter. Moreover, the four-junction circuit in the single-well regime is also investigated. Our theory provides a tool to explore the physical properties of this four-junction superconducting circuit. PMID:27356619

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

    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....

  10. A Rad53 independent function of Rad9 becomes crucial for genome maintenance in the absence of the Recq helicase Sgs1.

    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.

  11. Mycobacterium tuberculosis RecG binds and unwinds model DNA substrates with a preference for Holliday junctions.

    Zegeye, Ephrem Debebe; Balasingham, Seetha V; Laerdahl, Jon K; Homberset, Håvard; Tønjum, Tone

    2012-08-01

    The RecG enzyme, a superfamily 2 helicase, is present in nearly all bacteria. Here we report for the first time that the recG gene is also present in the genomes of most vascular plants as well as in green algae, but is not found in other eukaryotes or archaea. The precise function of RecG is poorly understood, although ample evidence shows that it plays critical roles in DNA repair, recombination and replication. We further demonstrate that Mycobacterium tuberculosis RecG (RecG(Mtb)) DNA binding activity had a broad substrate specificity, whereas it only unwound branched-DNA substrates such as Holliday junctions (HJs), replication forks, D-loops and R-loops, with a strong preference for the HJ as a helicase substrate. In addition, RecG(Mtb) preferentially bound relatively long (≥40 nt) ssDNA, exhibiting a higher affinity for the homopolymeric nucleotides poly(dT), poly(dG) and poly(dC) than for poly(dA). RecG(Mtb) helicase activity was supported by hydrolysis of ATP or dATP in the presence of Mg(2+), Mn(2+), Cu(2+) or Fe(2+). Like its Escherichia coli orthologue, RecG(Mtb) is also a strictly DNA-dependent ATPase.

  12. The UL5 and UL52 subunits of the herpes simplex virus type 1 helicase-primase subcomplex exhibit a complex interdependence for DNA binding.

    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. Tunable Nitride Josephson Junctions.

    Missert, Nancy A. [Sandia National Lab. (SNL-NM), Albuquerque, NM (United States); Henry, Michael David [Sandia National Lab. (SNL-NM), Albuquerque, NM (United States); Lewis, Rupert M. [Sandia National Lab. (SNL-NM), Albuquerque, NM (United States); Howell, Stephen W. [Sandia National Lab. (SNL-NM), Albuquerque, NM (United States); Wolfley, Steven L. [Sandia National Lab. (SNL-NM), Albuquerque, NM (United States); Brunke, Lyle Brent [Sandia National Lab. (SNL-NM), Albuquerque, NM (United States); Wolak, Matthaeus [Sandia National Lab. (SNL-NM), Albuquerque, NM (United States)

    2017-12-01

    We have developed an ambient temperature, SiO2/Si wafer - scale process for Josephson junctions based on Nb electrodes and Ta x N barriers with tunable electronic properties. The films are fabricated by magnetron sputtering. The electronic properties of the TaxN barriers are controlled by adjusting the nitrogen flow during sputtering. This technology offers a scalable alternative to the more traditional junctions based on AlOx barriers for low - power, high - performance computing.

  14. Quantum Junction Solar Cells

    Tang, Jiang; Liu, Huan; Zhitomirsky, David; Hoogland, Sjoerd; Wang, Xihua; Furukawa, Melissa; Levina, Larissa; Sargent, Edward H.

    2012-01-01

    -performing colloidal quantum dot rectifying devices reported to date have relied on a junction between a quantum-tuned absorber and a bulk material (e.g., TiO 2); however, quantum tuning of the absorber then requires complete redesign of the bulk acceptor, compromising

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

    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

  16. The DNA repair endonuclease XPG interacts directly and functionally with the WRN helicase defective in Werner syndrome

    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.

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

    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...

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

    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

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

    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

  20. Human RecQL4 helicase plays critical roles in prostate carcinogenesis

    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...

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

    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.

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

    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.

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

    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

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

    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

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

    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

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

    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...

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

    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

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

    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.

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

    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.

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

    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.

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

    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.

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

    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.

  13. Josephson junctions array resonators

    Gargiulo, Oscar; Muppalla, Phani; Mirzaei, Iman; Kirchmair, Gerhard [Institute for Quantum Optics and Quantum Information, Innsbruck (Austria)

    2016-07-01

    We present an experimental analysis of the self- and cross-Kerr effect of extended plasma resonances in Josephson junction chains. The chain consists of 1600 individual junctions and we can measure quality factors in excess of 10000. The Kerr effect manifests itself as a frequency shift that depends linearly on the number of photons in a resonant mode. By changing the input power we are able to measure this frequency shift on a single mode (self-kerr). By changing the input power on another mode while measuring the same one, we are able to evaluate the cross-kerr effect. We can measure the cross-Kerr effect by probing the resonance frequency of one mode while exciting another mode of the array with a microwave drive.

  14. Curved Josephson junction

    Dobrowolski, Tomasz

    2012-01-01

    The constant curvature one and quasi-one dimensional Josephson junction is considered. On the base of Maxwell equations, the sine–Gordon equation that describes an influence of curvature on the kink motion was obtained. It is showed that the method of geometrical reduction of the sine–Gordon model from three to lower dimensional manifold leads to an identical form of the sine–Gordon equation. - Highlights: ► The research on dynamics of the phase in a curved Josephson junction is performed. ► The geometrical reduction is applied to the sine–Gordon model. ► The results of geometrical reduction and the fundamental research are compared.

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

    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.

  16. The human myotendinous junction

    Knudsen, A B; Larsen, M; Mackey, Abigail

    2015-01-01

    The myotendinous junction (MTJ) is a specialized structure in the musculotendinous system, where force is transmitted from muscle to tendon. Animal models have shown that the MTJ takes form of tendon finger-like processes merging with muscle tissue. The human MTJ is largely unknown and has never...... been described in three dimensions (3D). The aim of this study was to describe the ultrastructure of the human MTJ and render 3D reconstructions. Fourteen subjects (age 25 ± 3 years) with isolated injury of the anterior cruciate ligament (ACL), scheduled for reconstruction with a semitendinosus...

  17. Tight junctions and human diseases.

    Sawada, Norimasa; Murata, Masaki; Kikuchi, Keisuke; Osanai, Makoto; Tobioka, Hirotoshi; Kojima, Takashi; Chiba, Hideki

    2003-09-01

    Tight junctions are intercellular junctions adjacent to the apical end of the lateral membrane surface. They have two functions, the barrier (or gate) function and the fence function. The barrier function of tight junctions regulates the passage of ions, water, and various macromolecules, even of cancer cells, through paracellular spaces. The barrier function is thus relevant to edema, jaundice, diarrhea, and blood-borne metastasis. On the other hand, the fence function maintains cell polarity. In other words, tight junctions work as a fence to prevent intermixing of molecules in the apical membrane with those in the lateral membrane. This function is deeply involved in cancer cell biology, in terms of loss of cell polarity. Of the proteins comprising tight junctions, integral membrane proteins occludin, claudins, and JAMs have been recently discovered. Of these molecules, claudins are exclusively responsible for the formation of tight-junction strands and are connected with the actin cytoskeleton mediated by ZO-1. Thus, both functions of tight junctions are dependent on the integrity of the actin cytoskeleton as well as ATP. Mutations in the claudin14 and the claudin16 genes result in hereditary deafness and hereditary hypomagnesemia, respectively. Some pathogenic bacteria and viruses target and affect the tight-junction function, leading to diseases. In this review, the relationship between tight junctions and human diseases is summarized.

  18. Gap junctions and motor behavior

    Kiehn, Ole; Tresch, Matthew C.

    2002-01-01

    The production of any motor behavior requires coordinated activity in motor neurons and premotor networks. In vertebrates, this coordination is often assumed to take place through chemical synapses. Here we review recent data suggesting that electrical gap-junction coupling plays an important role...... in coordinating and generating motor outputs in embryonic and early postnatal life. Considering the recent demonstration of a prevalent expression of gap-junction proteins and gap-junction structures in the adult mammalian spinal cord, we suggest that neuronal gap-junction coupling might also contribute...... to the production of motor behavior in adult mammals....

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

    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

    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

    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.

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

    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.

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

    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

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

    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

  6. Distinct functions of human RecQ helicases during DNA replication

    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

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

    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

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

    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.

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

    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.

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

    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...

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

    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

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

    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.

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

    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.

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

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

    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.

  16. Helicase properties of the Escherichia coli UvrAb protein complex

    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

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

    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.

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

    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.

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

    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.

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

    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.

  1. Junction detection and pathway selection

    Peck, Alex N.; Lim, Willie Y.; Breul, Harry T.

    1992-02-01

    The ability to detect junctions and make choices among the possible pathways is important for autonomous navigation. In our script-based navigation approach where a journey is specified as a script of high-level instructions, actions are frequently referenced to junctions, e.g., `turn left at the intersection.' In order for the robot to carry out these kind of instructions, it must be able (1) to detect an intersection (i.e., an intersection of pathways), (2) know that there are several possible pathways it can take, and (3) pick the pathway consistent with the high level instruction. In this paper we describe our implementation of the ability to detect junctions in an indoor environment, such as corners, T-junctions and intersections, using sonar. Our approach uses a combination of partial scan of the local environment and recognition of sonar signatures of certain features of the junctions. In the case where the environment is known, we use additional sensor information (such as compass bearings) to help recognize the specific junction. In general, once a junction is detected and its type known, the number of possible pathways can be deduced and the correct pathway selected. Then the appropriate behavior for negotiating the junction is activated.

  2. Mixing in T-junctions

    Kok, Jacobus B.W.; van der Wal, S.

    1996-01-01

    The transport processes that are involved in the mixing of two gases in a T-junction mixer are investigated. The turbulent flow field is calculated for the T-junction with the k- turbulence model by FLOW3D. In the mathematical model the transport of species is described with a mixture fraction

  3. Dynamics of Josephson junction arrays

    Hadley, P.

    1989-01-01

    The dynamics of Josephson junction arrays is a topic that lies at the intersection of the fields of nonlinear dynamics and Josephson junction technology. The series arrays considered here consist of several rapidly oscillating Josephson junctions where each junction is coupled equally to every other junction. The purpose of this study is to understand phaselocking and other cooperative dynamics of this system. Previously, little was known about high dimensional nonlinear systems of this sort. Numerical simulations are used to study the dynamics of these arrays. Three distinct types of periodic solutions to the array equations were observed as well as period doubled and chaotic solutions. One of the periodic solutions is the symmetric, in-phase solution where all of the junctions oscillate identically. The other two periodic solutions are symmetry-broken solutions where all of the junction do not oscillate identically. The symmetry-broken solutions are highly degenerate. As many as (N - 1) stable solutions can coexist for an array of N junctions. Understanding the stability of these several solutions and the transitions among them is vital to the design of useful devices

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

    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.

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

    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.

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

    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

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

    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

  8. 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.

    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

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

    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.

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

    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.

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

    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.

  12. 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.

    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.

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

    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....

  14. Instabilities in thin tunnel junctions

    Konkin, M.K.; Adler, J.G.

    1978-01-01

    Tunnel junctions prepared for inelastic electron tunneling spectroscopy are often plagued by instabilities in the 0-500-meV range. This paper relates the bias at which the instability occurs to the barrier thickness

  15. The Control of Junction Flows

    Smith, Charles

    1997-01-01

    An experimental study of the effects of spatially-limited (i.e. localized) surface suction on unsteady laminar and turbulent junction flows was performed using hydrogen bubble flow visualization and Particle Image Velocimetry (PIV...

  16. Josephson junctions with ferromagnetic interlayer

    Wild, Georg Hermann

    2012-01-01

    We report on the fabrication of superconductor/insulator/ferromagnetic metal/superconductor (Nb/AlO x /Pd 0.82 Ni 0.18 /Nb) Josephson junctions (SIFS JJs) with high critical current densities, large normal resistance times area products, and high quality factors. For these junctions, a transition from 0- to π-coupling is observed for a thickness d F =6 nm of the ferromagnetic Pd 0.82 Ni 0.18 interlayer. The magnetic field dependence of the critical current of the junctions demonstrates good spatial homogeneity of the tunneling barrier and ferromagnetic interlayer. Magnetic characterization shows that the Pd 0.82 Ni 0.18 has an out-of-plane anisotropy and large saturation magnetization indicating negligible dead layers at the interfaces. A careful analysis of Fiske modes up to about 400 GHz provides valuable information on the junction quality factor and the relevant damping mechanisms. Whereas losses due to quasiparticle tunneling dominate at low frequencies, at high frequencies the damping is explained by the finite surface resistance of the junction electrodes. High quality factors of up to 30 around 200 GHz have been achieved. They allow to study the junction dynamics, in particular the switching probability from the zero-voltage into the voltage state with and without microwave irradiation. The experiments with microwave irradiation are well explained within semi-classical models and numerical simulations. In contrast, at mK temperature the switching dynamics without applied microwaves clearly shows secondary quantum effects. Here, we could observe for the first time macroscopic quantum tunneling in Josephson junctions with a ferromagnetic interlayer. This observation excludes fluctuations of the critical current as a consequence of an unstable magnetic domain structure of the ferromagnetic interlayer and affirms the suitability of SIFS Josephson junctions for quantum information processing.

  17. Josephson junctions with ferromagnetic interlayer

    Wild, Georg Hermann

    2012-03-04

    We report on the fabrication of superconductor/insulator/ferromagnetic metal/superconductor (Nb/AlO{sub x}/Pd{sub 0.82}Ni{sub 0.18}/Nb) Josephson junctions (SIFS JJs) with high critical current densities, large normal resistance times area products, and high quality factors. For these junctions, a transition from 0- to {pi}-coupling is observed for a thickness d{sub F}=6 nm of the ferromagnetic Pd{sub 0.82}Ni{sub 0.18} interlayer. The magnetic field dependence of the critical current of the junctions demonstrates good spatial homogeneity of the tunneling barrier and ferromagnetic interlayer. Magnetic characterization shows that the Pd{sub 0.82}Ni{sub 0.18} has an out-of-plane anisotropy and large saturation magnetization indicating negligible dead layers at the interfaces. A careful analysis of Fiske modes up to about 400 GHz provides valuable information on the junction quality factor and the relevant damping mechanisms. Whereas losses due to quasiparticle tunneling dominate at low frequencies, at high frequencies the damping is explained by the finite surface resistance of the junction electrodes. High quality factors of up to 30 around 200 GHz have been achieved. They allow to study the junction dynamics, in particular the switching probability from the zero-voltage into the voltage state with and without microwave irradiation. The experiments with microwave irradiation are well explained within semi-classical models and numerical simulations. In contrast, at mK temperature the switching dynamics without applied microwaves clearly shows secondary quantum effects. Here, we could observe for the first time macroscopic quantum tunneling in Josephson junctions with a ferromagnetic interlayer. This observation excludes fluctuations of the critical current as a consequence of an unstable magnetic domain structure of the ferromagnetic interlayer and affirms the suitability of SIFS Josephson junctions for quantum information processing.

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

    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.

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

    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.

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

    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.

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

    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.

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

    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

  3. Electronic thermometry in tunable tunnel junction

    Maksymovych, Petro

    2016-03-15

    A tunable tunnel junction thermometry circuit includes a variable width tunnel junction between a test object and a probe. The junction width is varied and a change in thermovoltage across the junction with respect to the change in distance across the junction is determined. Also, a change in biased current with respect to a change in distance across the junction is determined. A temperature gradient across the junction is determined based on a mathematical relationship between the temperature gradient, the change in thermovoltage with respect to distance and the change in biased current with respect to distance. Thermovoltage may be measured by nullifying a thermoelectric tunneling current with an applied voltage supply level. A piezoelectric actuator may modulate the probe, and thus the junction width, to vary thermovoltage and biased current across the junction. Lock-in amplifiers measure the derivatives of the thermovoltage and biased current modulated by varying junction width.

  4. Peltier cooling in molecular junctions

    Cui, Longji; Miao, Ruijiao; Wang, Kun; Thompson, Dakotah; Zotti, Linda Angela; Cuevas, Juan Carlos; Meyhofer, Edgar; Reddy, Pramod

    2018-02-01

    The study of thermoelectricity in molecular junctions is of fundamental interest for the development of various technologies including cooling (refrigeration) and heat-to-electricity conversion1-4. Recent experimental progress in probing the thermopower (Seebeck effect) of molecular junctions5-9 has enabled studies of the relationship between thermoelectricity and molecular structure10,11. However, observations of Peltier cooling in molecular junctions—a critical step for establishing molecular-based refrigeration—have remained inaccessible. Here, we report direct experimental observations of Peltier cooling in molecular junctions. By integrating conducting-probe atomic force microscopy12,13 with custom-fabricated picowatt-resolution calorimetric microdevices, we created an experimental platform that enables the unified characterization of electrical, thermoelectric and energy dissipation characteristics of molecular junctions. Using this platform, we studied gold junctions with prototypical molecules (Au-biphenyl-4,4'-dithiol-Au, Au-terphenyl-4,4''-dithiol-Au and Au-4,4'-bipyridine-Au) and revealed the relationship between heating or cooling and charge transmission characteristics. Our experimental conclusions are supported by self-energy-corrected density functional theory calculations. We expect these advances to stimulate studies of both thermal and thermoelectric transport in molecular junctions where the possibility of extraordinarily efficient energy conversion has been theoretically predicted2-4,14.

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

    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...

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

    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....

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

    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.

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

    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...

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

    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.

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

    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

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

    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.

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

    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

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

    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.

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

    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.

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

    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

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

    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.

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

    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

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

    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

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

    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.

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

    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...

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

    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...

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

    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

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

    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.

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

    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...

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

    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

  6. Transport properties of molecular junctions

    Zimbovskaya, Natalya A

    2013-01-01

    A comprehensive overview of the physical mechanisms that control electron transport and the characteristics of metal-molecule-metal (MMM) junctions is presented. As far as possible, methods and formalisms presented elsewhere to analyze electron transport through molecules are avoided. This title introduces basic concepts—a description of the electron transport through molecular junctions—and briefly describes relevant experimental methods. Theoretical methods commonly used to analyze the electron transport through molecules are presented. Various effects that manifest in the electron transport through MMMs, as well as the basics of density-functional theory and its applications to electronic structure calculations in molecules are presented. Nanoelectronic applications of molecular junctions and similar systems are discussed as well. Molecular electronics is a diverse and rapidly growing field. Transport Properties of Molecular Junctions presents an up-to-date survey of the field suitable for researchers ...

  7. NbN tunnel junctions

    Villegier, J.C.; Vieux-Rochaz, L.; Goniche, M.; Renard, P.; Vabre, M.

    1984-09-01

    All-niobium nitride Josephon junctions have been prepared successfully using a new processing called SNOP: Selective Niobium (nitride) Overlap Process. Such a process involves the ''trilayer'' deposition on the whole wafer before selective patterning of the electrodes by optically controlled dry reactive ion etching. Only two photomask levels are need to define an ''overlap'' or a ''cross-type'' junction with a good accuracy. The properties of the niobium nitride films deposited by DC-magnetron sputtering and the surface oxide growth are analysed. The most critical point to obtain high quality and high gap value junctions resides in the early stage of the NbN counterelectrode growth. Some possibilities to overcome such a handicap exist even if the fabrication needs substrate temperatures below 250 0 C

  8. 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

    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

  9. Gravitation at the Josephson Junction

    Victor Atanasov

    2018-01-01

    Full Text Available A geometric potential from the kinetic term of a constrained to a curved hyperplane of space-time quantum superconducting condensate is derived. An energy conservation relation involving the geometric field at every material point in the superconductor is demonstrated. At a Josephson junction the energy conservation relation implies the possibility of transforming electric energy into geometric field energy, that is, curvature of space-time. Experimental procedures to verify that the Josephson junction can act as a voltage-to-curvature converter are discussed.

  10. Electronic noise of superconducting tunnel junction detectors

    Jochum, J.; Kraus, H.; Gutsche, M.; Kemmather, B.; Feilitzsch, F. v.; Moessbauer, R.L.

    1994-01-01

    The optimal signal to noise ratio for detectors based on superconducting tunnel junctions is calculated and compared for the cases of a detector consisting of one single tunnel junction, as well as of series and of parallel connections of such tunnel junctions. The influence of 1 / f noise and its dependence on the dynamical resistance of tunnel junctions is discussed quantitatively. A single tunnel junction yields the minimum equivalent noise charge. Such a tunnel junction exhibits the best signal to noise ratio if the signal charge is independent of detector size. In case, signal charge increases with detector size, a parallel or a series connection of tunnel junctions would provide the optimum signal to noise ratio. The equivalent noise charge and the respective signal to noise ratio are deduced as functions of tunnel junction parameters such as tunneling time, quasiparticle lifetime, etc. (orig.)

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

    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

  12. Current noise in tunnel junctions

    Frey, Moritz; Grabert, Hermann [Physikalisches Institut, Universitaet Freiburg, Hermann-Herder-Strasse 3, 79104, Freiburg (Germany)

    2017-06-15

    We study current fluctuations in tunnel junctions driven by a voltage source. The voltage is applied to the tunneling element via an impedance providing an electromagnetic environment of the junction. We use circuit theory to relate the fluctuations of the current flowing in the leads of the junction with the voltage fluctuations generated by the environmental impedance and the fluctuations of the tunneling current. The spectrum of current fluctuations is found to consist of three parts: a term arising from the environmental Johnson-Nyquist noise, a term due to the shot noise of the tunneling current and a third term describing the cross-correlation between these two noise sources. Our phenomenological theory reproduces previous results based on the Hamiltonian model for the dynamical Coulomb blockade and provides a simple understanding of the current fluctuation spectrum in terms of circuit theory and properties of the average current. Specific results are given for a tunnel junction driven through a resonator. (copyright 2016 WILEY-VCH Verlag GmbH and Co. KGaA, Weinheim)

  13. Stability of large-area molecular junctions

    Akkerman, Hylke B.; Kronemeijer, Auke J.; Harkema, Jan; van Hal, Paul A.; Smits, Edsger C. P.; de Leeuw, Dago M.; Blom, Paul W. M.

    The stability of molecular junctions is crucial for any application of molecular electronics. Degradation of molecular junctions when exposed to ambient conditions is regularly observed. In this report the stability of large-area molecular junctions under ambient conditions for more than two years

  14. Dynamics of pi-junction interferometer circuits

    Kornkev, V.K.; Mozhaev, P.B.; Borisenko, I.V.

    2002-01-01

    The pi-junction superconducting circuit dynamics was studied by means of numerical simulation technique. Parallel arrays consisting of Josephson junctions of both 0- and pi-type were studied as a model of high-T-c grain-boundary Josephson junction. The array dynamics and the critical current depe...

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

    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...

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

    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.

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

    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

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

    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.

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

    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.

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

    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...

  1. Ferromagnetic Josephson Junctions for Cryogenic Memory

    Niedzielski, Bethany M.; Gingrich, Eric C.; Khasawneh, Mazin A.; Loloee, Reza; Pratt, William P., Jr.; Birge, Norman O.

    2015-03-01

    Josephson junctions containing ferromagnetic materials are of interest for both scientific and technological purposes. In principle, either the amplitude of the critical current or superconducting phase shift across the junction can be controlled by the relative magnetization directions of the ferromagnetic layers in the junction. Our approach concentrates on phase control utilizing two junctions in a SQUID geometry. We will report on efforts to control the phase of junctions carrying either spin-singlet or spin-triplet supercurrent for cryogenic memory applications. Supported by Northorp Grumman Corporation and by IARPA under SPAWAR Contract N66001-12-C-2017.

  2. Method of manufacturing Josephson junction integrated circuits

    Jillie, D.W. Jr.; Smith, L.N.

    1985-01-01

    Josephson junction integrated circuits of the current injection type and magnetically controlled type utilize a superconductive layer that forms both Josephson junction electrode for the Josephson junction devices on the integrated circuit as well as a ground plane for the integrated circuit. Large area Josephson junctions are utilized for effecting contact to lower superconductive layers and islands are formed in superconductive layers to provide isolation between the groudplane function and the Josephson junction electrode function as well as to effect crossovers. A superconductor-barrier-superconductor trilayer patterned by local anodization is also utilized with additional layers formed thereover. Methods of manufacturing the embodiments of the invention are disclosed

  3. Molecular series-tunneling junctions.

    Liao, Kung-Ching; Hsu, Liang-Yan; Bowers, Carleen M; Rabitz, Herschel; Whitesides, George M

    2015-05-13

    Charge transport through junctions consisting of insulating molecular units is a quantum phenomenon that cannot be described adequately by classical circuit laws. This paper explores tunneling current densities in self-assembled monolayer (SAM)-based junctions with the structure Ag(TS)/O2C-R1-R2-H//Ga2O3/EGaIn, where Ag(TS) is template-stripped silver and EGaIn is the eutectic alloy of gallium and indium; R1 and R2 refer to two classes of insulating molecular units-(CH2)n and (C6H4)m-that are connected in series and have different tunneling decay constants in the Simmons equation. These junctions can be analyzed as a form of series-tunneling junctions based on the observation that permuting the order of R1 and R2 in the junction does not alter the overall rate of charge transport. By using the Ag/O2C interface, this system decouples the highest occupied molecular orbital (HOMO, which is localized on the carboxylate group) from strong interactions with the R1 and R2 units. The differences in rates of tunneling are thus determined by the electronic structure of the groups R1 and R2; these differences are not influenced by the order of R1 and R2 in the SAM. In an electrical potential model that rationalizes this observation, R1 and R2 contribute independently to the height of the barrier. This model explicitly assumes that contributions to rates of tunneling from the Ag(TS)/O2C and H//Ga2O3 interfaces are constant across the series examined. The current density of these series-tunneling junctions can be described by J(V) = J0(V) exp(-β1d1 - β2d2), where J(V) is the current density (A/cm(2)) at applied voltage V and βi and di are the parameters describing the attenuation of the tunneling current through a rectangular tunneling barrier, with width d and a height related to the attenuation factor β.

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

    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.

  5. Flexible 2D layered material junctions

    Balabai, R.; Solomenko, A.

    2018-03-01

    Within the framework of the methods of the electron density functional and the ab initio pseudopotential, we have obtained the valence electron density spatial distribution, the densities of electron states, the widths of band gaps, the charges on combined regions, and the Coulomb potentials for graphene-based flexible 2D layered junctions, using author program complex. It is determined that the bending of the 2D layered junctions on the angle α leads to changes in the electronic properties of these junctions. In the graphene/graphane junction, there is clear charge redistribution with different signs in the regions of junctions. The presence in the heterojunctions of charge regions with different signs leads to the formation of potential barriers. The greatest potential jump is in the graphene/fluorographene junction. The greatest value of the band gap width is in the graphene/graphane junction.

  6. Josephson junctions and circle maps

    Bak, P; Bohr, T; Jensen, M H; Christiansen, P V

    1984-01-01

    The return map of a differential equation for the current driven Josephson junction, or the damped driven pendulum, is shown numerically to be a circle map. Phase locking, noise and hysteresis, can thus be understood in a simple and coherent way. The transition to chaos is related to the development of a cubic inflection point. Recent theoretical results on universal behavior at the transition to chaos can readily be checked experimentally by studying I-V characteristics. 17 references, 1 figure.

  7. Squeezed States in Josephson Junctions.

    Hu, X.; Nori, F.

    1996-03-01

    We have studied quantum fluctuation properties of Josephson junctions in the limit of large Josephson coupling energy and small charging energy, when the eigenstates of the system can be treated as being nearly localized. We have considered(X. Hu and F. Nori, preprints.) a Josephson junction in a variety of situations, e.g., coupled to one or several of the following elements: a capacitor, an inductor (in a superconducting ring), and an applied current source. By solving an effective Shrödinger equation, we have obtained squeezed vacuum (coherent) states as the ground states of a ``free-oscillating'' (linearly-driven) Josephson junction, and calculated the uncertainties of its canonical momentum, charge, and coordinate, phase. We have also shown that the excited states of the various systems we consider are similar to the number states of a simple harmonic oscillator but with different fluctuation properties. Furthermore, we have obtained the time-evolution operators for these systems. These operators can make it easier to calculate the time-dependence of the expectation values and fluctuations of various quantities starting from an arbitrary initial state.

  8. Superconducting tunnel-junction refrigerator

    Melton, R.G.; Paterson, J.L.; Kaplan, S.B.

    1980-01-01

    The dc current through an S 1 -S 2 tunnel junction, with Δ 2 greater than Δ 1 , when biased with eV 1 +Δ 2 , will lower the energy in S 1 . This energy reduction will be shared by the phonons and electrons. This device is shown to be analogous to a thermoelectric refrigerator with an effective Peltier coefficient π* approx. Δ 1 /e. Tunneling calculations yield the cooling power P/sub c/, the electrical power P/sub e/ supplied by the bias supply, and the cooling efficiency eta=P/sub c//P/sub e/. The maximum cooling power is obtained for eV= +- (Δ 2 -Δ 1 ) and t 1 =T 1 /T/sub c/1 approx. 0.9. Estimates are made of the temperature difference T 2 -T 1 achievable in Al-Pb and Sn-Pb junctions with an Al 2 O 3 tunneling barrier. The performance of this device is shown to yield a maximum cooling efficiency eta approx. = Δ 1 /(Δ 2 -Δ 1 ) which can be compared with that available in an ideal Carnot refrigerator of eta=T 1 /(T 2 -T 1 ). The development of a useful tunnel-junction refrigerator requires a tunneling barrier with an effective thermal conductance per unit area several orders of magnitude less than that provided by the A1 2 O 3 barrier in the Al-Pb and Sn-Pb systems

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

    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.

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

    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

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

    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.

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

    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.

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

    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

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

    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...

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

    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.

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

    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

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

    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.

  18. Geodynamical simulation of the RRF triple junction

    Wang, Z.; Wei, D.; Liu, M.; Shi, Y.; Wang, S.

    2017-12-01

    Triple junction is the point at which three plate boundaries meet. Three plates at the triple junction form a complex geological tectonics, which is a natural laboratory to study the interactions of plates. This work studies a special triple junction, the oceanic transform fault intersects the collinear ridges with different-spreading rates, which is free of influence of ridge-transform faults and nearby hotspots. First, we build 3-D numerical model of this triple junction used to calculate the stead-state velocity and temperature fields resulting from advective and conductive heat transfer. We discuss in detail the influence of the velocity and temperature fields of the triple junction from viscosity, spreading rate of the ridge. The two sides of the oceanic transform fault are different sensitivities to the two factors. And, the influence of the velocity mainly occurs within 200km of the triple junction. Then, we modify the model by adding a ridge-transform fault to above model and directly use the velocity structure of the Macquarie triple junction. The simulation results show that the temperature at both sides of the oceanic transform fault decreases gradually from the triple junction, but the temperature difference between the two sides is a constant about 200°. And, there is little effect of upwelling velocity away from the triple junction 100km. The model results are compared with observational data. The heat flux and thermal topography along the oceanic transform fault of this model are consistent with the observed data of the Macquarie triple junction. The earthquakes are strike slip distributed along the oceanic transform fault. Their depths are also consistent with the zone of maximum shear stress. This work can help us to understand the interactions of plates of triple junctions and help us with the foundation for the future study of triple junctions.

  19. Hysteresis development in superconducting Josephson junctions

    Refai, T.F.; Shehata, L.N.

    1988-09-01

    The resistively and capacitive shunted junction model is used to investigate hysteresis development in superconducting Josephson junctions. Two empirical formulas that relate the hysteresis width and the quasi-particle diffusion length in terms of the junctions electrical parameters, temperature and frequency are obtained. The obtained formulas provide a simple tool to investigate the full potentials of the hysteresis phenomena. (author). 9 refs, 3 figs

  20. Josephson tunnel junction microwave attenuator

    Koshelets, V. P.; Shitov, S. V.; Shchukin, A. V.

    1993-01-01

    A new element for superconducting electronic circuitry-a variable attenuator-has been proposed, designed, and successfully tested. The principle of operation is based on the change in the microwave impedance of a superconductor-insulator-superconductor (SIS) Josephson tunnel junction when dc biased...... at different points in the current-voltage characteristic. Both numerical calculations based on the Tien-Gordon theory and 70-GHz microwave experiments have confirmed the wide dynamic range (more than 15-dB attenuation for one stage) and the low insertion loss in the ''open'' state. The performance of a fully...

  1. Loss models for long Josephson junctions

    Olsen, O. H.; Samuelsen, Mogens Rugholm

    1984-01-01

    A general model for loss mechanisms in long Josephson junctions is presented. An expression for the zero-field step is found for a junction of overlap type by means of a perturbation method. Comparison between analytic solution and perturbation result shows good agreement.......A general model for loss mechanisms in long Josephson junctions is presented. An expression for the zero-field step is found for a junction of overlap type by means of a perturbation method. Comparison between analytic solution and perturbation result shows good agreement....

  2. Harmonic synchronization in resistively coupled Josephson junctions

    Blackburn, J.A.; Gronbech-Jensen, N.; Smith, H.J.T.

    1994-01-01

    The oscillations of two resistively coupled Josephson junctions biased only by a single dc current source are shown to lock harmonically in a 1:2 mode over a significant range of bias current, even when the junctions are identical. The dependence of this locking on both junction and coupling parameters is examined, and it is found that, for this particular two-junction configuration, 1:1 locking can never occur, and also that a minimum coupling coefficient is needed to support harmonic locking. Some issues related to subharmonic locking are also discussed

  3. Superconducting flux qubits with π-junctions

    Shcherbakova, Anastasia

    2014-01-01

    In this thesis, we present a fabrication technology of Al/AlO x /Al Josephson junctions on Nb pads. The described technology gives the possibility of combining a variety of Nb-based superconducting circuits, like pi-junction phase-shifters with sub-micron Al/AlO x /Al junctions. Using this approach, we fabricated hybrid Nb/Al flux qubits with and without the SFS-junctions and studied dispersive magnetic field response of these qubits as well as their spectroscopy characteristics.

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

    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.

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

    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.

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

    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.

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

    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.

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

    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.

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

    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

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

    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.

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

    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.

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

    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.

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

    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

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

    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.

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

    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

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

    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.

  18. 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.

    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).

  19. Tolerance of Escherichia coli to fluoroquinolone antibiotics depends on specific components of the SOS response pathway.

    Theodore, Alyssa; Lewis, Kim; Vulic, Marin

    2013-12-01

    Bacteria exposed to bactericidal fluoroquinolone (FQ) antibiotics can survive without becoming genetically resistant. Survival of these phenotypically resistant cells, commonly called "persisters," depends on the SOS gene network. We have examined mutants in all known SOS-regulated genes to identify functions essential for tolerance in Escherichia coli. The absence of DinG and UvrD helicases and the Holliday junction processing enzymes RuvA and RuvB leads to a decrease in survival. Analysis of the respective mutants indicates that, in addition to repair of double-strand breaks, tolerance depends on the repair of collapsed replication forks and stalled transcription complexes. Mutation in recF results in increased survival, which identifies RecAF recombination as a poisoning mechanism not previously linked to FQ lethality. DinG acts upstream of SOS promoting its induction, whereas RuvAB participates in repair only. UvrD directly promotes all repair processes initiated by FQ-induced damage and prevents RecAF-dependent misrepair, making it one of the crucial SOS functions required for tolerance.

  20. Linker-dependent Junction Formation Probability in Single-Molecule Junctions

    Yoo, Pil Sun; Kim, Taekyeong [HankukUniversity of Foreign Studies, Yongin (Korea, Republic of)

    2015-01-15

    We compare the junction formation probabilities of single-molecule junctions with different linker molecules by using a scanning tunneling microscope-based break-junction technique. We found that the junction formation probability varies as SH > SMe > NH2 for the benzene backbone molecule with different types of anchoring groups, through quantitative statistical analysis. These results are attributed to different bonding forces according to the linker groups formed with Au atoms in the electrodes, which is consistent with previous works. Our work allows a better understanding of the contact chemistry in the metal.molecule junction for future molecular electronic devices.

  1. Cavity syncronisation of underdamped Josephson junction arrays

    Barbara, P.; Filatrella, G.; Lobb, C.

    2003-01-01

    the junctions in the array and an electromagnetic cavity. Here we show that a model of a one-dimensional array of Josephson junctions coupled to a resonator can produce many features of the coherent be havior above threshold, including coherent radiation of power and the shape of the array current...

  2. Functional anatomy of the human ureterovesical junction

    Roshani, H.; Dabhoiwala, N. F.; Verbeek, F. J.; Lamers, W. H.

    1996-01-01

    BACKGROUND: The valve function of the ureterovesical-junction (UVJ) is responsible for protection of the low pressure upper urinary tract from the refluxing of urine from the bladder. Controversy about the microanatomy of the human ureterovesical-junction persists. METHODS: Ten (3 male and 7 female)

  3. Spin, Vibrations and Radiation in Superconducting Junctions

    Padurariu, C.

    2013-01-01

    This thesis presents the theoretical study of superconducting transport in several devices based on superconducting junctions. The important feature of these devices is that the transport properties of the junction are modified by the interaction with another physical system integrated in the

  4. Gap junctions and connexin-interacting proteins

    Giepmans, Ben N G

    2004-01-01

    Gap junctions form channels between adjacent cells. The core proteins of these channels are the connexins. Regulation of gap junction communication (GJC) can be modulated by connexin-associating proteins, such as regulatory protein phosphatases and protein kinases, of which c-Src is the

  5. Multiplication in Silicon p-n Junctions

    Moll, John L.

    1965-01-01

    Multiplication values were measured in the collector junctions of silicon p-n-p and n-p-n transistors before and after bombardment by 1016 neutrons/cm2. Within experimental error there was no change either in junction fields, as deduced from capacitance measurements, or in multiplication values i...

  6. impairs gap junction function causing congenital cataract

    Navya

    2017-03-24

    Mar 24, 2017 ... experiment showed a lower dye diffusion distance of Cx46 V44M cells, ... Studies of connexins show that channel gating and permeability .... have found that connexin assembled into gap junction plaques is not soluble in 1% ..... high glucose reduces gap junction activity in microvascular endothelial cells.

  7. impairs gap junction function causing congenital cataract

    LIJUAN CHEN

    2017-12-20

    Dec 20, 2017 ... showed a lower dye diffusion distance of Cx46 V44M cells, which indicates that the gap junction intercellular ... permeability could be affected by alterations of charged residues of .... bled into gap junction plaques is not soluble in 1% Triton ..... regulation of connexin 43 expression by high glucose reduces.

  8. Fabrication of Josephson Junction without shadow evaporation

    Wu, Xian; Ku, Hsiangsheng; Long, Junling; Pappas, David

    We developed a new method of fabricating Josephson Junction (Al/AlOX/Al) without shadow evaporation. Statistics from room temperature junction resistance and measurement of qubits are presented. Unlike the traditional ``Dolan Bridge'' technique, this method requires two individual lithographies and straight evaporations of Al. Argon RF plasma is used to remove native AlOX after the first evaporation, followed by oxidation and second Al evaporation. Junction resistance measured at room temperature shows linear dependence on Pox (oxidation pressure), √{tox} (oxidation time), and inverse proportional to junction area. We have seen 100% yield of qubits made with this method. This method is promising because it eliminates angle dependence during Junction fabrication, facilitates large scale qubits fabrication.

  9. Overlap junctions for high coherence superconducting qubits

    Wu, X.; Long, J. L.; Ku, H. S.; Lake, R. E.; Bal, M.; Pappas, D. P.

    2017-07-01

    Fabrication of sub-micron Josephson junctions is demonstrated using standard processing techniques for high-coherence, superconducting qubits. These junctions are made in two separate lithography steps with normal-angle evaporation. Most significantly, this work demonstrates that it is possible to achieve high coherence with junctions formed on aluminum surfaces cleaned in situ by Ar plasma before junction oxidation. This method eliminates the angle-dependent shadow masks typically used for small junctions. Therefore, this is conducive to the implementation of typical methods for improving margins and yield using conventional CMOS processing. The current method uses electron-beam lithography and an additive process to define the top and bottom electrodes. Extension of this work to optical lithography and subtractive processes is discussed.

  10. Quantum synchronization effects in intrinsic Josephson junctions

    Machida, M.; Kano, T.; Yamada, S.; Okumura, M.; Imamura, T.; Koyama, T.

    2008-01-01

    We investigate quantum dynamics of the superconducting phase in intrinsic Josephson junctions of layered high-T c superconductors motivated by a recent experimental observation for the switching rate enhancement in the low temperature quantum regime. We pay attention to only the capacitive coupling between neighboring junctions and perform large-scale simulations for the Schroedinger equation derived from the Hamiltonian considering the capacitive coupling alone. The simulation focuses on an issue whether the switching of a junction induces those of the other junctions or not. The results reveal that the superconducting phase dynamics show synchronous behavior with increasing the quantum character, e.g., decreasing the junction plane area and effectively the temperature. This is qualitatively consistent with the experimental result

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

    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.

  12. Macroscopic quantum tunneling in Josephson tunnel junctions and Coulomb blockade in single small tunnel junctions

    Cleland, A.N.

    1991-04-01

    Experiments investigating the process of macroscopic quantum tunneling in a moderately-damped, resistively shunted, Josephson junction are described, followed by a discussion of experiments performed on very small capacitance normal-metal tunnel junctions. The experiments on the resistively-shunted Josephson junction were designed to investigate a quantum process, that of the tunneling of the Josephson phase variable under a potential barrier, in a system in which dissipation plays a major role in the dynamics of motion. All the parameters of the junction were measured using the classical phenomena of thermal activation and resonant activation. Theoretical predictions are compared with the experimental results, showing good agreement with no adjustable parameters; the tunneling rate in the moderately damped (Q ∼ 1) junction is seen to be reduced by a factor of 300 from that predicted for an undamped junction. The phase is seen to be a good quantum-mechanical variable. The experiments on small capacitance tunnel junctions extend the measurements on the larger-area Josephson junctions from the region in which the phase variable has a fairly well-defined value, i.e. its wavefunction has a narrow width, to the region where its value is almost completely unknown. The charge on the junction becomes well-defined and is predicted to quantize the current through the junction, giving rise to the Coulomb blockade at low bias. I present the first clear observation of the Coulomb blockade in single junctions. The electrical environment of the tunnel junction, however, strongly affects the behavior of the junction: higher resistance leads are observed to greatly sharpen the Coulomb blockade over that seen with lower resistance leads. I present theoretical descriptions of how the environment influences the junctions; comparisons with the experimental results are in reasonable agreement

  13. Resonance Transport of Graphene Nanoribbon T-Shaped Junctions

    Xiao-Lan, Kong; Yong-Jian, Xiong

    2010-01-01

    We investigate the transport properties of T-shaped junctions composed of armchair graphene nanoribbons of different widths. Three types of junction geometries are considered. The junction conductance strongly depends on the atomic features of the junction geometry. When the shoulders of the junction have zigzag type edges, sharp conductance resonances usually appear in the low energy region around the Dirac point, and a conductance gap emerges. When the shoulders of the junction have armchair type edges, the conductance resonance behavior is weakened significantly, and the metal-metal-metal junction structures show semimetallic behaviors. The contact resistance also changes notably due to the various interface geometries of the junction

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

    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.

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

    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.

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

    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.

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

    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.

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

    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

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

    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...

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

    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

  1. Electron optics with ballistic graphene junctions

    Chen, Shaowen

    Electrons transmitted across a ballistic semiconductor junction undergo refraction, analogous to light rays across an optical boundary. A pn junction theoretically provides the equivalent of a negative index medium, enabling novel electron optics such as negative refraction and perfect (Veselago) lensing. In graphene, the linear dispersion and zero-gap bandstructure admit highly transparent pn junctions by simple electrostatic gating, which cannot be achieved in conventional semiconductors. Robust demonstration of these effects, however, has not been forthcoming. Here we employ transverse magnetic focusing to probe propagation across an electrostatically defined graphene junction. We find perfect agreement with the predicted Snell's law for electrons, including observation of both positive and negative refraction. Resonant transmission across the pn junction provides a direct measurement of the angle dependent transmission coefficient, and we demonstrate good agreement with theory. Comparing experimental data with simulation reveals the crucial role played by the effective junction width, providing guidance for future device design. Efforts toward sharper pn junction and possibility of zero field Veselago lensing will also be discussed. This work is supported by the Semiconductor Research Corporations NRI Center for Institute for Nanoelectronics Discovery and Exploration (INDEX).

  2. Valley dependent transport in graphene L junction

    Chan, K. S.

    2018-05-01

    We studied the valley dependent transport in graphene L junctions connecting an armchair lead and a zigzag lead. The junction can be used in valleytronic devices and circuits. Electrons injected from the armchair lead into the junction is not valley polarized, but they can become valley polarized in the zigzag lead. There are Fermi energies, where the current in the zigzag lead is highly valley polarized and the junction is an efficient generator of valley polarized current. The features of the valley polarized current depend sensitively on the widths of the two leads, as well as the number of dimers in the armchair lead, because this number has a sensitive effect on the band structure of the armchair lead. When an external potential is applied to the junction, the energy range with high valley polarization is enlarged enhancing its function as a generator of highly valley polarized current. The scaling behavior found in other graphene devices is also found in L junctions, which means that the results presented here can be extended to junctions with larger dimensions after appropriate scaling of the energy.

  3. Shot noise in YBCO bicrystal Josephson junctions

    Constantinian, K.Y.; Ovsyannikov, G.A.; Borisenko, I.V.

    2003-01-01

    We measured spectral noise density in YBCO symmetric bicrystal Josephson junctions on sapphire substrates at bias voltages up to 100 mV and T 4.2 K. Normal state resistance of the Josephson junctions, R-N = 20-90 Omega and ICRN up to 2.2 mV have been observed in the experimental samples. Noise...... may explain the experimentally measured linewidth broadening of Josephson oscillations at mm and submm wave frequencies in high-Tc superconducting junctions. Experimental results are discussed in terms of bound states existing at surfaces of d-wave superconducting electrodes....

  4. delta-biased Josephson tunnel junctions

    Monaco, R.; Mygind, Jesper; Koshelet, V.

    2010-01-01

    Abstract: The behavior of a long Josephson tunnel junction drastically depends on the distribution of the dc bias current. We investigate the case in which the bias current is fed in the central point of a one-dimensional junction. Such junction configuration has been recently used to detect...... the persistent currents circulating in a superconducting loop. Analytical and numerical results indicate that the presence of fractional vortices leads to remarkable differences from the conventional case of uniformly distributed dc bias current. The theoretical findings are supported by detailed measurements...

  5. Parametric frequency conversion in long Josephson junctions

    Irie, F.; Ashihara, S.; Yoshida, K.

    1976-01-01

    Current steps at voltages corresponding to the parametric coupling between an applied r.f. field and junction resonant modes have been observed in long Josephson tunnel junctions in the flux-flow state. The observed periodic variations of the step height due to the applied magnetic field are explained quantitatively by a perturbational analysis using Josephson phase equations. The present study demonstrates that the moving vortex array can serve as a coherent pump wave for signal waves propagating in the barrier region, which indicates, as a result, the possibility of traveling-wave parametric devices with long Josephson tunnel junctions. (author)

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

    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.

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

    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.

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

    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.

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

    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

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

    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.

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

    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.

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

    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.

  13. Tunnel junctions with multiferroic barriers

    Gajek, Martin; Bibes, Manuel; Fusil, Stéphane; Bouzehouane, Karim; Fontcuberta, Josep; Barthélémy, Agnès; Fert, Albert

    2007-04-01

    Multiferroics are singular materials that can exhibit simultaneously electric and magnetic orders. Some are ferroelectric and ferromagnetic and provide the opportunity to encode information in electric polarization and magnetization to obtain four logic states. However, such materials are rare and schemes allowing a simple electrical readout of these states have not been demonstrated in the same device. Here, we show that films of La0.1Bi0.9MnO3 (LBMO) are ferromagnetic and ferroelectric, and retain both ferroic properties down to a thickness of 2nm. We have integrated such ultrathin multiferroic films as barriers in spin-filter-type tunnel junctions that exploit the magnetic and ferroelectric degrees of freedom of LBMO. Whereas ferromagnetism permits read operations reminiscent of magnetic random access memories (MRAM), the electrical switching evokes a ferroelectric RAM write operation. Significantly, our device does not require the destructive ferroelectric readout, and therefore represents an advance over the original four-state memory concept based on multiferroics.

  14. Chirality effect in disordered graphene ribbon junctions

    Long Wen

    2012-01-01

    We investigate the influence of edge chirality on the electronic transport in clean or disordered graphene ribbon junctions. By using the tight-binding model and the Landauer-Büttiker formalism, the junction conductance is obtained. In the clean sample, the zero-magnetic-field junction conductance is strongly chirality-dependent in both unipolar and bipolar ribbons, whereas the high-magnetic-field conductance is either chirality-independent in the unipolar or chirality-dependent in the bipolar ribbon. Furthermore, we study the disordered sample in the presence of magnetic field and find that the junction conductance is always chirality-insensitive for both unipolar and bipolar ribbons with adequate disorders. In addition, the disorder-induced conductance plateaus can exist in all chiral bipolar ribbons provided the disorder strength is moderate. These results suggest that we can neglect the effect of edge chirality in fabricating electronic devices based on the magnetotransport in a disordered graphene ribbon. (paper)

  15. Josephson tunnel junctions in niobium films

    Wiik, Tapio.

    1976-12-01

    A method of fabricating stable Josephson tunnel junctions with reproducible characteristics is described. The junctions have a sandwich structure consisting of a vacuum evaporated niobium film, a niobium oxide layer produced by the glow discharge method and a lead film deposited by vacuum evaporation. Difficulties in producing thin-film Josephson junctions are discussed. Experimental results suggest that the lower critical field of the niobium film is the most essential parameter when evaluating the quality of these junctions. The dependence of the lower critical field on the film thickness and on the Ginzburg-Landau parameter of the film is studied analytically. Comparison with the properties of the evaporated films and with the previous calculations for bulk specimens shows that the presented model is applicable for most of the prepared samples. (author)

  16. Transparency of atom-sized superconducting junctions

    Van-der-Post, N.; Peters, E.T.; Van Ruitenbeek, J.M.; Yanson, I.K.

    1995-01-01

    We discuss the transparency of atom-size superconducting tunnel junctions by comparing experimental values of the normal resistance and Subgap Structure with the theoretical predictions for these phenomena by Landauer's formula and Multiple Andreev Reflection, respectively

  17. Josephson junction arrays and superconducting wire networks

    Lobb, C.J.

    1992-01-01

    Techniques used to fabricate integrated circuits make it possible to construct superconducting networks containing as many as 10 6 wires or Josephson junctions. Such networks undergo phase transitions from resistive high-temperature states to ordered low-resistance low-temperature states. The nature of the phase transition depends strongly on controllable parameters such as the strength of the superconductivity in each wire or junction and the external magnetic field. This paper will review the physics of these phase transitions, starting with the simplest zero-magnetic field case. This leads to a Kosterlitz-Thouless transition when the junctions or wires are weak, and a simple mean-field fransition when the junctions or wires are strong. Rich behavior, resulting from frustration, occurs in the presence of a magnetic field. (orig.)

  18. Neutron induced permanent damage in Josephson junctions

    Mueller, G.P.; Rosen, M.

    1982-01-01

    14 MeV neutron induced permanent changes in the critical current density of Josephson junctions due to displacement damage in the junction barrier are estimated using a worst case model and the binary collision simulation code MARLOWE. No likelihood of single event hard upsets is found in this model. It is estimated that a fluence of 10 18 -10 19 neutrons/cm 2 are required to change the critical current density by 5%

  19. Exotic hadron and string junction model

    Imachi, Masahiro

    1978-01-01

    Hadron structure is investigated adopting string junction model as a realization of confinement. Besides exotic hadrons (M 4 , B 5 etc.), unconventional hadrons appear. A mass formula for these hadrons is proposed. New selection rule is introduced which requires the covalence of constituent line at hadron vertex. New duality appears due to the freedom of junction, especially in anti BB→anti BB reaction. A possible assignment of exotic and unconventional hadrons to recently observed narrow meson states is presented. (auth.)

  20. Construction of tunable peptide nucleic acid junctions.

    Duan, Tanghui; He, Liu; Tokura, Yu; Liu, Xin; Wu, Yuzhou; Shi, Zhengshuang

    2018-03-15

    We report here the construction of 3-way and 4-way peptide nucleic acid (PNA) junctions as basic structural units for PNA nanostructuring. The incorporation of amino acid residues into PNA chains makes PNA nanostructures with more structural complexity and architectural flexibility possible, as exemplified by building 3-way PNA junctions with tunable nanopores. Given that PNA nanostructures have good thermal and enzymatic stabilities, they are expected to have broad potential applications in biosensing, drug delivery and bioengineering.

  1. Molecular Diffusion through Cyanobacterial Septal Junctions.

    Nieves-Morión, Mercedes; Mullineaux, Conrad W; Flores, Enrique

    2017-01-03

    Heterocyst-forming cyanobacteria grow as filaments in which intercellular molecular exchange takes place. During the differentiation of N 2 -fixing heterocysts, regulators are transferred between cells. In the diazotrophic filament, vegetative cells that fix CO 2 through oxygenic photosynthesis provide the heterocysts with reduced carbon and heterocysts provide the vegetative cells with fixed nitrogen. Intercellular molecular transfer has been traced with fluorescent markers, including calcein, 5-carboxyfluorescein, and the sucrose analogue esculin, which are observed to move down their concentration gradient. In this work, we used fluorescence recovery after photobleaching (FRAP) assays in the model heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120 to measure the temperature dependence of intercellular transfer of fluorescent markers. We find that the transfer rate constants are directly proportional to the absolute temperature. This indicates that the "septal junctions" (formerly known as "microplasmodesmata") linking the cells in the filament allow molecular exchange by simple diffusion, without any activated intermediate state. This constitutes a novel mechanism for molecular transfer across the bacterial cytoplasmic membrane, in addition to previously characterized mechanisms for active transport and facilitated diffusion. Cyanobacterial septal junctions are functionally analogous to the gap junctions of metazoans. Although bacteria are frequently considered just as unicellular organisms, there are bacteria that behave as true multicellular organisms. The heterocyst-forming cyanobacteria grow as filaments in which cells communicate. Intercellular molecular exchange is thought to be mediated by septal junctions. Here, we show that intercellular transfer of fluorescent markers in the cyanobacterial filament has the physical properties of simple diffusion. Thus, cyanobacterial septal junctions are functionally analogous to metazoan gap junctions

  2. Spinal Gap Junction Channels in Neuropathic Pain

    Jeon, Young Hoon; Youn, Dong Ho

    2015-01-01

    Damage to peripheral nerves or the spinal cord is often accompanied by neuropathic pain, which is a complex, chronic pain state. Increasing evidence indicates that alterations in the expression and activity of gap junction channels in the spinal cord are involved in the development of neuropathic pain. Thus, this review briefly summarizes evidence that regulation of the expression, coupling, and activity of spinal gap junction channels modulates pain signals in neuropathic pain states induced...

  3. Ballistic Josephson junctions based on CVD graphene

    Li, Tianyi; Gallop, John; Hao, Ling; Romans, Edward

    2018-04-01

    Josephson junctions with graphene as the weak link between superconductors have been intensely studied in recent years, with respect to both fundamental physics and potential applications. However, most of the previous work was based on mechanically exfoliated graphene, which is not compatible with wafer-scale production. To overcome this limitation, we have used graphene grown by chemical vapour deposition (CVD) as the weak link of Josephson junctions. We demonstrate that very short, wide CVD-graphene-based Josephson junctions with Nb electrodes can work without any undesirable hysteresis in their electrical characteristics from 1.5 K down to a base temperature of 320 mK, and their gate-tuneable critical current shows an ideal Fraunhofer-like interference pattern in a perpendicular magnetic field. Furthermore, for our shortest junctions (50 nm in length), we find that the normal state resistance oscillates with the gate voltage, consistent with the junctions being in the ballistic regime, a feature not previously observed in CVD-graphene-based Josephson junctions.

  4. Junction depth measurement using carrier illumination

    Borden, Peter

    2001-01-01

    Carrier Illumination [trade mark] (CI) is a new method recently developed to meet the need for a non-destructive, high throughput junction depth measurement on patterned wafers. A laser beam creates a quasi-static excess carrier profile in the semiconductor underlying the activated junction. The excess carrier profile is fairly constant below the junction, and drops rapidly in the junction, creating a steep index of refraction gradient at the junction edge. Interference with light reflected from this index gradient provides a signal that is analyzed to determine the junction depth. The paper summarizes evaluation of performance in full NMOS and PMOS process flows, on both bare and patterned wafers. The aims have been to validate (1) performance in the presence of underlying layers typically found at the source/drain (S/D) process steps and (2) measurement on patterned wafers. Correlation of CI measurements to SIMS and transistor drive current are shown. The data were obtained from NMOS structures using As S/D and LDD implants. Correlations to SRP, SIMS and sheet resistance are shown for PMOS structures using B 11 LDD implants. Gage capability measurements are also presented

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

    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

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

    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.

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

    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.

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

    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...

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

    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

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

    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.

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

    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.

  12. Josephson junctions of multiple superconducting wires

    Deb, Oindrila; Sengupta, K.; Sen, Diptiman

    2018-05-01

    We study the spectrum of Andreev bound states and Josephson currents across a junction of N superconducting wires which may have s - or p -wave pairing symmetries and develop a scattering matrix based formalism which allows us to address transport across such junctions. For N ≥3 , it is well known that Berry curvature terms contribute to the Josephson currents; we chart out situations where such terms can have relatively large effects. For a system of three s -wave or three p -wave superconductors, we provide analytic expressions for the Andreev bound-state energies and study the Josephson currents in response to a constant voltage applied across one of the wires; we find that the integrated transconductance at zero temperature is quantized to integer multiples of 4 e2/h , where e is the electron charge and h =2 π ℏ is Planck's constant. For a sinusoidal current with frequency ω applied across one of the wires in the junction, we find that Shapiro plateaus appear in the time-averaged voltage across that wire for any rational fractional multiple (in contrast to only integer multiples in junctions of two wires) of 2 e /(ℏ ω ) . We also use our formalism to study junctions of two p -wave and one s -wave wires. We find that the corresponding Andreev bound-state energies depend on the spin of the Bogoliubov quasiparticles; this produces a net magnetic moment in such junctions. The time variation of these magnetic moments may be controlled by an external voltage applied across the junction. We discuss experiments which may test our theory.

  13. Josephson tunnel junctions with ferromagnetic interlayer

    Weides, M.P.

    2006-01-01

    Superconductivity and ferromagnetism are well-known physical properties of solid states that have been widely studied and long thought about as antagonistic phenomena due to difference in spin ordering. It turns out that the combination of both superconductor and ferromagnet leads to a very rich and interesting physics. One particular example, the phase oscillations of the superconducting order parameter inside the ferromagnet, will play a major role for the devices discussed in this work. In this thesis, I present Josephson junctions with a thin Al 2 O 3 tunnel barrier and a ferromagnetic interlayer, i.e. superconductor-insulator-ferromagnet-superconductor (SIFS) stacks. The fabrication of junctions was optimized regarding the insulation of electrodes and the homogeneity of the current transport. The junctions were either in the 0 or π coupled ground state, depending on the thickness of the ferromagnetic layer and on temperature. The influence of ferromagnetic layer thickness on the transport properties and the coupling (0, π) of SIFS tunnel junctions was studied. Furthermore, using a stepped ferromagnetic layer with well-chosen thicknesses, I obtained the so-called 0-π Josephson junction. At a certain temperature this 0-π junction can be made perfectly symmetric. In this case the ground state corresponds to a vortex of supercurrent creating a magnetic flux which is a fraction of the magnetic flux quantum Φ 0 . Such structures allow to study the physics of fractional vortices and to build various electronic circuits based on them. The SIFS junctions presented here have an exponentially vanishing damping at T → 0. The SIFS technology developed within the framework of this work may be used to construct classical and quantum devices such as oscillators, memory cells and qubits. (orig.)

  14. Josephson tunnel junctions with ferromagnetic interlayer

    Weides, M.P.

    2006-07-01

    Superconductivity and ferromagnetism are well-known physical properties of solid states that have been widely studied and long thought about as antagonistic phenomena due to difference in spin ordering. It turns out that the combination of both superconductor and ferromagnet leads to a very rich and interesting physics. One particular example, the phase oscillations of the superconducting order parameter inside the ferromagnet, will play a major role for the devices discussed in this work. In this thesis, I present Josephson junctions with a thin Al{sub 2}O{sub 3} tunnel barrier and a ferromagnetic interlayer, i.e. superconductor-insulator-ferromagnet-superconductor (SIFS) stacks. The fabrication of junctions was optimized regarding the insulation of electrodes and the homogeneity of the current transport. The junctions were either in the 0 or {pi} coupled ground state, depending on the thickness of the ferromagnetic layer and on temperature. The influence of ferromagnetic layer thickness on the transport properties and the coupling (0, {pi}) of SIFS tunnel junctions was studied. Furthermore, using a stepped ferromagnetic layer with well-chosen thicknesses, I obtained the so-called 0-{pi} Josephson junction. At a certain temperature this 0-{pi} junction can be made perfectly symmetric. In this case the ground state corresponds to a vortex of supercurrent creating a magnetic flux which is a fraction of the magnetic flux quantum {phi}{sub 0}. Such structures allow to study the physics of fractional vortices and to build various electronic circuits based on them. The SIFS junctions presented here have an exponentially vanishing damping at T {yields} 0. The SIFS technology developed within the framework of this work may be used to construct classical and quantum devices such as oscillators, memory cells and qubits. (orig.)

  15. Macroscopic quantum tunneling in Josephson tunnel junctions and Coulomb blockade in single small tunnel junctions

    Cleland, A.N.

    1991-01-01

    Experiments investigated the process of macroscopic quantum tunneling in a moderately-damped, resistively shunted, Josephson junction are described, followed by a discussion of experiments performed on very-small-capacitance normal-metal tunnel junctions. The experiments on the resistively-shunted Josephson junction were designed to investigate a quantum process, that of the tunneling of the Josephson-phase variable under a potential barrier, in a system in which dissipation plays a major role in the dynamics of motion. All the parameters of the junction were measured using the classical phenomena of thermal activation and resonant activation. Theoretical predictions are compared with the experimental results, showing good agreement with no adjustable parameters. The experiments on small-capacitance tunnel junctions extend the measurements on the large-area Josephson junctions from the region in which the phase variable has a fairly well-defined value, i.e. its wave function has a narrow width, to the region where its value is almost completely unknown. The charge on the junction becomes well-defined and is predicted to quantize the current through the junction, giving rise to the Coulomb blockade at low bias

  16. Ballistic Graphene Josephson Junctions from the Short to the Long Junction Regimes.

    Borzenets, I V; Amet, F; Ke, C T; Draelos, A W; Wei, M T; Seredinski, A; Watanabe, K; Taniguchi, T; Bomze, Y; Yamamoto, M; Tarucha, S; Finkelstein, G

    2016-12-02

    We investigate the critical current I_{C} of ballistic Josephson junctions made of encapsulated graphene-boron-nitride heterostructures. We observe a crossover from the short to the long junction regimes as the length of the device increases. In long ballistic junctions, I_{C} is found to scale as ∝exp(-k_{B}T/δE). The extracted energies δE are independent of the carrier density and proportional to the level spacing of the ballistic cavity. As T→0 the critical current of a long (or short) junction saturates at a level determined by the product of δE (or Δ) and the number of the junction's transversal modes.

  17. Increasing gap junctional coupling: a tool for dissecting the role of gap junctions

    Axelsen, Lene Nygaard; Haugan, Ketil; Stahlhut, Martin

    2007-01-01

    Much of our current knowledge about the physiological and pathophysiological role of gap junctions is based on experiments where coupling has been reduced by either chemical agents or genetic modification. This has brought evidence that gap junctions are important in many physiological processes....... In a number of cases, gap junctions have been implicated in the initiation and progress of disease, and experimental uncoupling has been used to investigate the exact role of coupling. The inverse approach, i.e., to increase coupling, has become possible in recent years and represents a new way of testing...... the role of gap junctions. The aim of this review is to summarize the current knowledge obtained with agents that selectively increase gap junctional intercellular coupling. Two approaches will be reviewed: increasing coupling by the use of antiarrhythmic peptide and its synthetic analogs...

  18. A single-gradient junction technique to replace multiple-junction shifts for craniospinal irradiation treatment

    Hadley, Austin; Ding, George X.

    2014-01-01

    Craniospinal irradiation (CSI) requires abutting fields at the cervical spine. Junction shifts are conventionally used to prevent setup error–induced overdosage/underdosage from occurring at the same location. This study compared the dosimetric differences at the cranial-spinal junction between a single-gradient junction technique and conventional multiple-junction shifts and evaluated the effect of setup errors on the dose distributions between both techniques for a treatment course and single fraction. Conventionally, 2 lateral brain fields and a posterior spine field(s) are used for CSI with weekly 1-cm junction shifts. We retrospectively replanned 4 CSI patients using a single-gradient junction between the lateral brain fields and the posterior spine field. The fields were extended to allow a minimum 3-cm field overlap. The dose gradient at the junction was achieved using dose painting and intensity-modulated radiation therapy planning. The effect of positioning setup errors on the dose distributions for both techniques was simulated by applying shifts of ± 3 and 5 mm. The resulting cervical spine doses across the field junction for both techniques were calculated and compared. Dose profiles were obtained for both a single fraction and entire treatment course to include the effects of the conventional weekly junction shifts. Compared with the conventional technique, the gradient-dose technique resulted in higher dose uniformity and conformity to the target volumes, lower organ at risk (OAR) mean and maximum doses, and diminished hot spots from systematic positioning errors over the course of treatment. Single-fraction hot and cold spots were improved for the gradient-dose technique. The single-gradient junction technique provides improved conformity, dose uniformity, diminished hot spots, lower OAR mean and maximum dose, and one plan for the entire treatment course, which reduces the potential human error associated with conventional 4-shifted plans

  19. Particle detection with superconducting tunnel junctions

    Jany, P.

    1990-08-01

    At the Institute of Experimental Nuclear Physics of the University of Karlsruhe (TH) and at the Institute for Nuclear Physics of the Kernforschungszentrum Karlsruhe we started to produce superconducting tunnel junctions and to investigate them for their suitability as particle detectors. The required facilities for the production of tunnel junctions and the experimental equipments to carry out experiments with them were erected. Experiments are presented in which radiations of different kinds of particles could successfully be measured with the tunnel junctions produced. At first we succeeded in detectioning light pulses of a laser. In experiments with alpha-particles of an energy of 4,6 MeV the alpha-particles were detected with an energy resolution of 1,1%, and it was shown in specific experiments that the phonons originating from the deposition of energy by an alpha-particle in the substrate can be detected with superconducting tunnel junctions at the surface. On that occasion it turned out that the signals could be separated with respect to their point of origin (tunnel junction, contact leads, substrate). Finally X-rays with an energy of 6 keV were detected with an energy resolution of 8% in a test arrangement that makes use of the so-called trapping effect to read out a larger absorber volume. (orig.) [de

  20. Joint diseases: from connexins to gap junctions.

    Donahue, Henry J; Qu, Roy W; Genetos, Damian C

    2017-12-19

    Connexons form the basis of hemichannels and gap junctions. They are composed of six tetraspan proteins called connexins. Connexons can function as individual hemichannels, releasing cytosolic factors (such as ATP) into the pericellular environment. Alternatively, two hemichannel connexons from neighbouring cells can come together to form gap junctions, membrane-spanning channels that facilitate cell-cell communication by enabling signalling molecules of approximately 1 kDa to pass from one cell to an adjacent cell. Connexins are expressed in joint tissues including bone, cartilage, skeletal muscle and the synovium. Indicative of their importance as gap junction components, connexins are also known as gap junction proteins, but individual connexin proteins are gaining recognition for their channel-independent roles, which include scaffolding and signalling functions. Considerable evidence indicates that connexons contribute to the function of bone and muscle, but less is known about the function of connexons in other joint tissues. However, the implication that connexins and gap junctional channels might be involved in joint disease, including age-related bone loss, osteoarthritis and rheumatoid arthritis, emphasizes the need for further research into these areas and highlights the therapeutic potential of connexins.

  1. Structure and Function of a Novel ATPase that Interacts with Holliday Junction Resolvase Hjc and Promotes Branch Migration.

    Zhai, Binyuan; DuPrez, Kevin; Doukov, Tzanko I; Li, Huan; Huang, Mengting; Shang, Guijun; Ni, Jinfeng; Gu, Lichuan; Shen, Yulong; Fan, Li

    2017-04-07

    Holliday junction (HJ) is a hallmark intermediate in DNA recombination and must be processed by dissolution (for double HJ) or resolution to ensure genome stability. Although HJ resolvases have been identified in all domains of life, there is a long-standing effort to search in prokaryotes and eukarya for proteins promoting HJ migration. Here, we report the structural and functional characterization of a novel ATPase, Sulfolobus islandicusPilT N-terminal-domain-containing ATPase (SisPINA), encoded by the gene adjacent to the resolvase Hjc coding gene. PINA is conserved in archaea and vital for S. islandicus viability. Purified SisPINA forms hexameric rings in the crystalline state and in solution, similar to the HJ migration helicase RuvB in Gram-negative bacteria. Structural analysis suggests that ATP binding and hydrolysis cause conformational changes in SisPINA to drive branch migration. Further studies reveal that SisPINA interacts with SisHjc and coordinates HJ migration and cleavage. Copyright © 2017 Elsevier Ltd. All rights reserved.

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

    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.

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

    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...

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

    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.

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

    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.

  6. Terahertz Responses of Intrinsic Josephson Junctions in High TC Superconductors

    Wang, H. B.; Wu, P. H.; Yamashita, T.

    2001-01-01

    High frequency responses of intrinsic Josephson junctions up to 2.5THz, including the observation of Shapiro steps under various conditions, are reported and discussed in this Letter. The sample was an array of intrinsic Josephson junctions singled out from inside a high T C superconducting Bi 2 Sr 2 CaCu 2 O 8+x single crystal, with a bow-tie antenna integrated to it. The number of junctions in the array was controllable, the junctions were homogeneous, the distribution of applied irradiation among the junctions was even, and the junctions could synchronously respond to high frequency irradiation

  7. Phase-dependent noise in Josephson junctions

    Sheldon, Forrest; Peotta, Sebastiano; Di Ventra, Massimiliano

    2018-03-01

    In addition to the usual superconducting current, Josephson junctions (JJs) support a phase-dependent conductance related to the retardation effect of tunneling quasi-particles. This introduces a dissipative current with a memory-resistive (memristive) character that should also affect the current noise. By means of the microscopic theory of tunnel junctions we compute the complete current autocorrelation function of a Josephson tunnel junction and show that this memristive component gives rise to both a previously noted phase-dependent thermal noise, and an undescribed non-stationary, phase-dependent dynamic noise. As experiments are approaching ranges in which these effects may be observed, we examine the form and magnitude of these processes. Their phase dependence can be realized experimentally as a hysteresis effect and may be used to probe defects present in JJ based qubits and in other superconducting electronics applications.

  8. Phonon spectroscopy with superconducting tunnel junctions

    Grimshaw, J.M.

    1984-02-01

    Superconducting tunnel junctions can be used as generators and detectors of monochromatic phonons of frequency larger than 80 GHz, as was first devised by Eisenmenger and Dayem (1967) and Kinder (1972a, 1973). In this report, we intend to give a general outline of this type of spectroscopy and to present the results obtained so far. The basic physics underlying phonon generation and detection are described in chapter I, a wider approach being given in the references therein. In chapter II, the different types of junctions are considered with respect to their use. Chapter III deals with the evaporation technique for the superconducting junctions. The last part of this report is devoted to the results that we have obtained on γ-irradiated LiF, pure Si and Phosphorous implanted Si. In these chapters, the limitations of the spectrometer are brought out and suggestions for further work are given [fr

  9. Silicon fiber with p-n junction

    Homa, D.; Cito, A.; Pickrell, G.; Hill, C.; Scott, B.

    2014-01-01

    In this study, we fabricated a p-n junction in a fiber with a phosphorous doped silicon core and fused silica cladding. The fibers were fabricated via a hybrid process of the core-suction and melt-draw techniques and maintained overall diameters ranging from 200 to 900 μm and core diameters of 20–800 μm. The p-n junction was formed by doping the fiber with boron and confirmed via the current-voltage characteristic. The demonstration of a p-n junction in a melt-drawn silicon core fiber paves the way for the seamless integration of optical and electronic devices in fibers.

  10. Tunnel magnetoresistance in double spin filter junctions

    Saffarzadeh, Alireza

    2003-01-01

    We consider a new type of magnetic tunnel junction, which consists of two ferromagnetic tunnel barriers acting as spin filters (SFs), separated by a nonmagnetic metal (NM) layer. Using the transfer matrix method and the free-electron approximation, the dependence of the tunnel magnetoresistance (TMR) on the thickness of the central NM layer, bias voltage and temperature in the double SF junction are studied theoretically. It is shown that the TMR and electron-spin polarization in this structure can reach very large values under suitable conditions. The highest value of the TMR can reach 99%. By an appropriate choice of the thickness of the central NM layer, the degree of spin polarization in this structure will be higher than that of the single SF junctions. These results may be useful in designing future spin-polarized tunnelling devices

  11. Josephson junctions with ferromagnetic alloy interlayer

    Himmel, Nico

    2015-07-23

    Josephson junctions are used as active devices in superconducting electronics and quantum information technology. Outstanding properties are their distinct non-linear electrical characteristics and a usually sinusoidal relation between the current and the superconducting phase difference across the junction. In general the insertion of ferromagnetic material in the barrier of a Josephson junction is associated with a suppression of superconducting correlations. But also new phenomena can arise which may allow new circuit layouts and enhance the performance of applications. This thesis presents a systematic investigation for two concepts to fabricate Josephson junctions with a rather uncommon negative critical current. Such devices exhibit an intrinsic phase slip of π between the electrodes, so they are also known as π junctions. Both studies go well beyond existing experiments and in one system a π junction is shown for the first time. All the thin film junctions are based on superconducting Nb electrodes. In a first approach, barriers made from Si and Fe were investigated with respect to the realisation of π junctions by spin-flip processes. The distribution of Fe in the Si matrix was varied from pure layers to disperse compounds. The systematic fabrication of alloy barriers was facilitated by the development of a novel timing-based combinatorial sputtering technique for planetary deposition systems. An orthogonal gradient approach allowed to create binary layer libraries with independent variations of thickness and composition. Second, Nb vertical stroke AlO{sub x} vertical stroke Nb vertical stroke Ni{sub 60}Cu{sub 40} vertical stroke Nb (SIsFS) double barrier junctions were experimentally studied for the occurrence of proximity effect induced order parameter oscillations. Detailed dependencies of the critical current density on the thickness of s-layer and F-layer were acquired and show a remarkable agreement to existing theoretical predictions. Especially

  12. Electrical analog of a Josephson junction

    Goldman, A.M.

    1979-01-01

    It is noted that a mathematical description of the phase-coupling of two oscillators synchronized by a phase-lock-loop under the influence of thermal white noise is analogous to that of the phase coupling of two superconductors in a Josephson junction also under the influence of noise. This analogy may be useful in studying threshold instabilities of the Josephson junction in regimes not restricted to the case of large damping. This is of interest because the behavior of the mean voltage near the threshold current can be characterized by critical exponents which resemble those exhibited by an order parameter of a continuous phase transition. As it is possible to couple a collection of oscillators together in a chain, the oscillator analogy may also be useful in exploring the dynamics and statistical mechanics of coupled junctions

  13. Thermionic refrigeration at CNT-CNT junctions

    Li, C.; Pipe, K. P.

    2016-10-01

    Monte Carlo (MC) simulation is used to study carrier energy relaxation following thermionic emission at the junction of two van der Waals bonded single-walled carbon nanotubes (SWCNTs). An energy-dependent transmission probability gives rise to energy filtering at the junction, which is predicted to increase the average electron transport energy by as much as 0.115 eV, leading to an effective Seebeck coefficient of 386 μV/K. MC results predict a long energy relaxation length (˜8 μm) for hot electrons crossing the junction into the barrier SWCNT. For SWCNTs of optimal length, an analytical transport model is used to show that thermionic cooling can outweigh parasitic heat conduction due to high SWCNT thermal conductivity, leading to a significant cooling capacity (2.4 × 106 W/cm2).

  14. Josephson junctions with ferromagnetic alloy interlayer

    Himmel, Nico

    2015-01-01

    Josephson junctions are used as active devices in superconducting electronics and quantum information technology. Outstanding properties are their distinct non-linear electrical characteristics and a usually sinusoidal relation between the current and the superconducting phase difference across the junction. In general the insertion of ferromagnetic material in the barrier of a Josephson junction is associated with a suppression of superconducting correlations. But also new phenomena can arise which may allow new circuit layouts and enhance the performance of applications. This thesis presents a systematic investigation for two concepts to fabricate Josephson junctions with a rather uncommon negative critical current. Such devices exhibit an intrinsic phase slip of π between the electrodes, so they are also known as π junctions. Both studies go well beyond existing experiments and in one system a π junction is shown for the first time. All the thin film junctions are based on superconducting Nb electrodes. In a first approach, barriers made from Si and Fe were investigated with respect to the realisation of π junctions by spin-flip processes. The distribution of Fe in the Si matrix was varied from pure layers to disperse compounds. The systematic fabrication of alloy barriers was facilitated by the development of a novel timing-based combinatorial sputtering technique for planetary deposition systems. An orthogonal gradient approach allowed to create binary layer libraries with independent variations of thickness and composition. Second, Nb vertical stroke AlO x vertical stroke Nb vertical stroke Ni 60 Cu 40 vertical stroke Nb (SIsFS) double barrier junctions were experimentally studied for the occurrence of proximity effect induced order parameter oscillations. Detailed dependencies of the critical current density on the thickness of s-layer and F-layer were acquired and show a remarkable agreement to existing theoretical predictions. Especially a variation of

  15. 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.

    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.

  16. Mechanically controllable break junctions for molecular electronics.

    Xiang, Dong; Jeong, Hyunhak; Lee, Takhee; Mayer, Dirk

    2013-09-20

    A mechanically controllable break junction (MCBJ) represents a fundamental technique for the investigation of molecular electronic junctions, especially for the study of the electronic properties of single molecules. With unique advantages, the MCBJ technique has provided substantial insight into charge transport processes in molecules. In this review, the techniques for sample fabrication, operation and the various applications of MCBJs are introduced and the history, challenges and future of MCBJs are discussed. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. Microscopic tunneling theory of long Josephson junctions

    Grønbech-Jensen, N.; Hattel, Søren A.; Samuelsen, Mogens Rugholm

    1992-01-01

    We present a numerical scheme for solving a nonlinear partial integro-differential equation with nonlocal time dependence. The equation describes the dynamics in a long Josephson junction modeled by use of the microscopic theory for tunneling between superconductors. We demonstrate that the detai......We present a numerical scheme for solving a nonlinear partial integro-differential equation with nonlocal time dependence. The equation describes the dynamics in a long Josephson junction modeled by use of the microscopic theory for tunneling between superconductors. We demonstrate...

  18. Fluctuation of heat current in Josephson junctions

    P. Virtanen

    2015-02-01

    Full Text Available We discuss the statistics of heat current between two superconductors at different temperatures connected by a generic weak link. As the electronic heat in superconductors is carried by Bogoliubov quasiparticles, the heat transport fluctuations follow the Levitov–Lesovik relation. We identify the energy-dependent quasiparticle transmission probabilities and discuss the resulting probability density and fluctuation relations of the heat current. We consider multichannel junctions, and find that heat transport in diffusive junctions is unique in that its statistics is independent of the phase difference between the superconductors.

  19. Superconductive junctions for x-ray spectroscopy

    Grand, J.B. le; Bruijn, M.P.; Frericks, M.; Korte, P.A.J. de; Houwman, E.P.; Flokstra, J.

    1992-01-01

    Biasing of SIS-junctions for the purpose of high energy resolution x-ray detection is complicated by the presence of a DC Josephson current and AC Josephson current resonances, so that a large magnetic field is normally used for the suppression of these Josephson features. A transimpedance amplifier is proposed for biasing and signal amplification at low magnetic field. X-ray spectroscopy detectors for astronomy require a high detection efficiency in the 0.5-10 keV energy band and a reasonable (∼1 cm 2 ) detector area. Calculations on absorber-junctions combinations which might meet these requirements are presented. (author) 9 refs.; 10 figs

  20. Electroplated Ni on the PN Junction Semiconductor

    Kim, Jin Joo; Uhm, Young Rang; Son, Kwang Jae; Kim, Jong Bum; Choi, Sang Moo; Park, Jong Han; Hong, Jintae [Korea Atomic Energy Research Institute, Daejeon (Korea, Republic of)

    2015-05-15

    Nickel (Ni) electroplating was implemented by using a metal Ni powder in order to establish a Ni-63 plating condition on the PN junction semiconductor needed for production of betavoltaic battery. PN junction semiconductors with a Ni seed layer of 500 and 1000 A were coated with Ni at current density from 10 to 50 mA cm{sup 2}. The surface roughness and average grain size of Ni deposits were investigated by XRD and SEM techniques. The roughness of Ni deposit was increased as the current density was increased, and decreased as the thickness of Ni seed layer was increased.

  1. Electroplated Ni on the PN Junction Semiconductor

    Kim, Jin Joo; Uhm, Young Rang; Son, Kwang Jae; Kim, Jong Bum; Choi, Sang Moo; Park, Jong Han; Hong, Jintae

    2015-01-01

    Nickel (Ni) electroplating was implemented by using a metal Ni powder in order to establish a Ni-63 plating condition on the PN junction semiconductor needed for production of betavoltaic battery. PN junction semiconductors with a Ni seed layer of 500 and 1000 A were coated with Ni at current density from 10 to 50 mA cm 2 . The surface roughness and average grain size of Ni deposits were investigated by XRD and SEM techniques. The roughness of Ni deposit was increased as the current density was increased, and decreased as the thickness of Ni seed layer was increased

  2. Gap junctions-guards of excitability

    Stroemlund, Line Waring; Jensen, Christa Funch; Qvortrup, Klaus

    2015-01-01

    Cardiomyocytes are connected by mechanical and electrical junctions located at the intercalated discs (IDs). Although these structures have long been known, it is becoming increasingly clear that their components interact. This review describes the involvement of the ID in electrical disturbances...... of the heart and focuses on the role of the gap junctional protein connexin 43 (Cx43). Current evidence shows that Cx43 plays a crucial role in organizing microtubules at the intercalated disc and thereby regulating the trafficking of the cardiac sodium channel NaV1.5 to the membrane....

  3. Common features of a vortex structure in long exponentially shaped Josephson junctions and Josephson junctions with inhomogeneities

    Boyadjiev, T.L.; Semerdjieva, E.G.; Shukrinov, Yu.M.

    2007-01-01

    We study the vortex structure in three different models of the long Josephson junction: the exponentially shaped Josephson junction and the Josephson junctions with the resistor and the shunt inhomogeneities in the barrier layer. For these three models the critical curves 'critical current-magnetic field' are numerically constructed. We develop the idea of the equivalence of the exponentially shaped Josephson junction and the rectangular junction with the distributed inhomogeneity and demonstrate that at some parameters of the shunt and the resistor inhomogeneities in the ends of the junction the corresponding critical curves are very close to the exponentially shaped one

  4. Atomic-scaled characterization of graphene PN junctions

    Zhou, Xiaodong; Wang, Dennis; Dadgar, Ali; Agnihotri, Pratik; Lee, Ji Ung; Reuter, Mark C.; Ross, Frances M.; Pasupathy, Abhay N.

    Graphene p-n junctions are essential devices for studying relativistic Klein tunneling and the Veselago lensing effect in graphene. We have successfully fabricated graphene p-n junctions using both lithographically pre-patterned substrates and the stacking of vertical heterostructures. We then use our 4-probe STM system to characterize the junctions. The ability to carry out scanning electron microscopy (SEM) in our STM instrument is essential for us to locate and measure the junction interface. We obtain both the topography and dI/dV spectra at the junction area, from which we track the shift of the graphene chemical potential with position across the junction interface. This allows us to directly measure the spatial width and roughness of the junction and its potential barrier height. We will compare the junction properties of devices fabricated by the aforementioned two methods and discuss their effects on the performance as a Veselago lens.

  5. Structure modeling and mutational analysis of gap junction beta 2 ...

    Yomi

    2012-04-03

    Apr 3, 2012 ... Three dimensional (3 D) structure is very useful for understanding biological functions. Gap junction beta 2 (GJB2), human gene encoding for gap junction beta 2 protein is involved in ... Research in deafness became real.

  6. Systematic optimization of quantum junction colloidal quantum dot solar cells

    Liu, Huan; Zhitomirsky, David; Hoogland, Sjoerd; Tang, Jiang; Kramer, Illan J.; Ning, Zhijun; Sargent, Edward H.

    2012-01-01

    The recently reported quantum junction architecture represents a promising approach to building a rectifying photovoltaic device that employs colloidal quantum dot layers on each side of the p-n junction. Here, we report an optimized quantum

  7. Macroscopic Refrigeration Using Superconducting Tunnel Junctions

    Lowell, Peter; O'Neil, Galen; Underwood, Jason; Zhang, Xiaohang; Ullom, Joel

    2014-03-01

    Sub-kelvin temperatures are often a prerequisite for modern scientific experiments, such as quantum information processing, astrophysical missions looking for dark energy signatures and tabletop time resolved x-ray spectroscopy. Existing methods of reaching these temperatures, such as dilution refrigerators, are bulky and costly. In order to increase the accessibility of sub-Kelvin temperatures, we have developed a new method of refrigeration using normal-metal/insulator/superconductor (NIS) tunnel junctions. NIS junctions cool the electrons in the normal metal since the hottest electrons selectively tunnel from the normal metal into the superconductor. By extending the normal metal onto a thermally isolated membrane, the cold electrons can cool the phonons through the electron-phonon coupling. When these junctions are combined with a pumped 3He system, they provide a potentially inexpensive method of reaching these temperatures. Using only three devices, each with a junction area of approximately 3,500 μm2, we have cooled a 2 cm3 Cu plate from 290 mK to 256 mK. We will present these experimental results along with recent modeling predictions that strongly suggest that further refinements will allow cooling from 300 mK to 120 mK. This work is supported by the NASA APRA program.

  8. Characterization of magnetic tunnel junction test pads

    Østerberg, Frederik Westergaard; Kjær, Daniel; Nielsen, Peter Folmer

    2015-01-01

    We show experimentally as well as theoretically that patterned magnetic tunnel junctions can be characterized using the current-in-plane tunneling (CIPT) method, and the key parameters, the resistance-area product (RA) and the tunnel magnetoresistance (TMR), can be determined. The CIPT method...

  9. Double-barrier junction based dc SQUID

    Bartolomé, M.E.; Brinkman, Alexander; Flokstra, Jakob; Golubov, Alexandre Avraamovitch; Rogalla, Horst

    2000-01-01

    dc SQUIDs based on double-barrier Nb/Al/AlOx/Al/AlOx/Al/Nb junctions (DBSQs) have been fabricated and tested for the first time. The current–voltage curves have been measured at temperatures down to 1.4 K. The critical current, Ic, dependence on the temperature T is partially described by the

  10. Anatomy of the human atrioventricular junctions revisited

    Anderson, R. H.; Ho, S. Y.; Becker, A. E.

    2000-01-01

    There have been suggestions made recently that our understanding of the atrioventricular junctions of the heart is less than adequate, with claims for several new findings concerning the arrangement of the ordinary working myocardium and the specialised pathways for atrioventricular conduction. In

  11. CANFLEX fuel bundle junction pressure drop

    Chung, H. J.; Chung, C. H.; Jun, J. S.; Hong, S. D.; Chang, S. K.; Kim, B. D.

    1996-11-01

    This report describes the junction pressure drop test results which are to used to determine the alignment angle between bundles to achieve the most probable fuel string pressure drop for randomly aligned bundles for use in the fuel string total pressure drop test. (author). 4 tabs., 17 figs

  12. Axial p-n-junctions in nanowires.

    Fernandes, C; Shik, A; Byrne, K; Lynall, D; Blumin, M; Saveliev, I; Ruda, H E

    2015-02-27

    The charge distribution and potential profile of p-n-junctions in thin semiconductor nanowires (NWs) were analyzed. The characteristics of screening in one-dimensional systems result in a specific profile with large electric field at the boundary between the n- and p- regions, and long tails with a logarithmic drop in the potential and charge density. As a result of these tails, the junction properties depend sensitively on the geometry of external contacts and its capacity has an anomalously large value and frequency dispersion. In the presence of an external voltage, electrons and holes in the NWs can not be described by constant quasi-Fermi levels, due to small values of the average electric field, mobility, and lifetime of carriers. Thus, instead of the classical Sah-Noice-Shockley theory, the junction current-voltage characteristic was described by an alternative theory suitable for fast generation-recombination and slow diffusion-drift processes. For the non-uniform electric field in the junction, this theory predicts the forward branch of the characteristic to have a non-ideality factor η several times larger than the values 1 < η < 2 from classical theory. Such values of η have been experimentally observed by a number of researchers, as well as in the present work.

  13. Gap junction diseases of the skin.

    Steensel, M.A.M. van

    2004-01-01

    Gap junctions are intercellular channels that allow the passage of water, ions, and small molecules. They are involved in quick, short-range messaging between cells and are found in skin, nervous tissue, heart, and muscle. An increasing number of hereditary skin disorders appear to be caused by

  14. Fluxon density waves in long Josephson junctions

    Olsen, O. H.; Ustinov, A. V.; Pedersen, Niels Falsig

    1993-01-01

    Numerical simulations of the multiple fluxon dynamics stimulated by an external oscillating force applied at a boundary of a long Josephson junction are presented. The calculated IV characteristics agree well with a recent experimental observation of rf-induced satellite flux-flow steps. The volt...... density waves....

  15. Soliton excitations in Josephson tunnel junctions

    Lomdahl, P. S.; Sørensen, O. H.; Christiansen, Peter Leth

    1982-01-01

    A detailed numerical study of a sine-Gordon model of the Josephson tunnel junction is compared with experimental measurements on junctions with different L / λJ ratios. The soliton picture is found to apply well on both relatively long (L / λJ=6) and intermediate (L / λJ=2) junctions. We find good...... agreement for the current-voltage characteristics, power output, and for the shape and height of the zero-field steps (ZFS). Two distinct modes of soliton oscillations are observed: (i) a bunched or congealed mode giving rise to the fundamental frequency f1 on all ZFS's and (ii) a "symmetric" mode which...... on the Nth ZFS yields the frequency Nf1 Coexistence of two adjacent frequencies is found on the third ZFS of the longer junction (L / λJ=6) in a narrow range of bias current as also found in the experiments. Small asymmetries in the experimental environment, a weak magnetic field, e.g., is introduced via...

  16. Dissipative current in SIFS Josephson junctions

    Vasenko, A.; Kawabata, S.; Golubov, Alexandre Avraamovitch; Kupriyanov, M. Yu; Hekking, F.W.J.

    2010-01-01

    We investigate superconductor/insulator/ferromagnet/superconductor (SIFS) tunnel Josephson junctions in the dirty limit, using the quasiclassical theory. We consider the case of a strong tunnel barrier such that the left S layer and the right FS bilayer are decoupled. We calculate quantitatively the

  17. CANFLEX fuel bundle junction pressure drop

    Chung, H. J.; Chung, C. H.; Jun, J. S.; Hong, S. D.; Chang, S. K.; Kim, B. D.

    1996-11-01

    This report describes the junction pressure drop test results which are to used to determine the alignment angle between bundles to achieve the most probable fuel string pressure drop for randomly aligned bundles for use in the fuel string total pressure drop test. (author). 4 tabs., 17 figs.

  18. The functional anatomy of the ureterovesical junction

    Thomson, A. S.; Dabhoiwala, N. F.; Verbeek, F. J.; Lamers, W. H.

    1994-01-01

    To obtain a new insight into the anti-reflux mechanism of the ureterovesical junction by studying the topographical anatomy of the juxta- and intravesical ureter and its relationship to the surrounding bladder musculature. Fresh pig bladders were fixed, frozen and serially sectioned. Enzyme

  19. Critical current of pure SNS junctions

    Golub, A.A.; Bezzub, O.P.

    1982-01-01

    Boundary conditions at the superconductor-normal metal interface are determined, taking into account the differences in the effective masses and the density of states of the metals constituting the transition and assumed to be pure. The potential barrier of the interface is chosen to be zero. The critical current of the junction is calculated [ru

  20. Improving transition voltage spectroscopy of molecular junctions

    Markussen, Troels; Chen, Jingzhe; Thygesen, Kristian Sommer

    2011-01-01

    Transition voltage spectroscopy (TVS) is a promising spectroscopic tool for molecular junctions. The principles in TVS is to find the minimum on a Fowler-Nordheim plot where ln(I/V2) is plotted against 1/V and relate the voltage at the minimum Vmin to the closest molecular level. Importantly, Vmin...

  1. Fractional Solitons in Excitonic Josephson Junctions

    Su, Jung-Jung; Hsu, Ya-Fen

    The Josephson effect is especially appealing because it reveals macroscopically the quantum order and phase. Here we study this effect in an excitonic Josephson junction: a conjunct of two exciton condensates with a relative phase ϕ0 applied. Such a junction is proposed to take place in the quantum Hall bilayer (QHB) that makes it subtler than in superconductor because of the counterflow of excitonic supercurrent and the interlayer tunneling in QHB. We treat the system theoretically by first mapping it into a pseudospin ferromagnet then describing it by the Landau-Lifshitz-Gilbert equation. In the presence of interlayer tunneling, the excitonic Josephson junction can possess a family of fractional sine-Gordon solitons that resemble the static fractional Josephson vortices in the extended superconducting Josephson junctions. Interestingly, each fractional soliton carries a topological charge Q which is not necessarily a half/full integer but can vary continuously. The resultant current-phase relation (CPR) shows that solitons with Q =ϕ0 / 2 π are the lowest energy states for small ϕ0. When ϕ0 > π , solitons with Q =ϕ0 / 2 π - 1 take place - the polarity of CPR is then switched.

  2. Constructing carbon nanotube junctions by Ar ion beam irradiation

    Ishaq, Ahmad; Ni Zhichun; Yan Long; Gong Jinlong; Zhu Dezhang

    2010-01-01

    Carbon nanotubes (CNTs) irradiated by Ar ion beams at elevated temperature were studied. The irradiation-induced defects in CNTs are greatly reduced by elevated temperature. Moreover, the two types of CNT junctions, the crossing junction and the parallel junction, were formed. And the CNT networks may be fabricated by the two types of CNT junctions. The formation process and the corresponding mechanism of CNT networks are discussed.

  3. Absolute migration and the evolution of the Rodriguez triple junction ...

    The Rodriguez Triple Junction (RTJ) is a junction connecting three mid-ocean ridges in the Indian Ocean: the Southwest Indian Ridge (SWIR), the Central Indian Ridge (CIR) and the Southeast Indian Ridge (SEIR). The evolution of the RTJ has been studied extensively for the past 10 Ma and the triple junction is believed to ...

  4. Junction depth dependence of breakdown in silicon detector diodes

    Beck, G.A.; Carter, A.A.; Carter, J.R.; Greenwood, N.M.; Lucas, A.D.; Munday, D.J.; Pritchard, T.W.; Robinson, D.; Wilburn, C.D.; Wyllie, K.

    1996-01-01

    The high voltage capability of detector diodes fabricated in the planar process is limited by the high field generated at the edge of the junction.We have fabricated diodes with increased junction depth with respect to our standard process and find a significantly higher breakdown voltage,in reasonable agreement with previous studies of junction breakdown. (orig.)

  5. Long Josephson Junction Stack Coupled to a Cavity

    Madsen, Søren Peder; Pedersen, Niels Falsig; Groenbech-Jensen, N.

    2007-01-01

    A stack of inductively coupled long Josephson junctions are modeled as a system of coupled sine-Gordon equations. One boundary of the stack is coupled electrically to a resonant cavity. With one fluxon in each Josephson junction, the inter-junction fluxon forces are repulsive. We look at a possible...... transition, induced by the cavity, to a bunched state....

  6. Phenomenological approach to bistable behavior of Josephson junctions

    Nishi, K.; Nara, S.; Hamanaka, K.

    1985-01-01

    The interaction of unbiased Josephson junction with external electromagnetic field in the presence of externally applied uniform magnetic field is theoretically examined by means of phenomenological treatment. It is proposed that an irradiated junction with suitably chosen parameters shows a bistable behavior of voltage across the junction as a function of the radiation intensity

  7. Systematic study of shallow junction formation on germanium substrates

    Hellings, Geert; Rosseel, Erik; Clarysse, Trudo

    2011-01-01

    Published results on Ge junctions are benchmarked systematically using RS–XJ plots. The electrical activation level required to meet the ITRS targets is calculated. Additionally, new results are presented on shallow furnace-annealed B junctions and shallow laser-annealed As junctions. Co-implanting...

  8. RECQ5 helicase associates with the C-terminal repeat domain of RNA polymerase II during productive elongation phase of transcription

    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

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

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

    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.

  12. The anatomical locus of T-junction processing.

    Schirillo, James A

    2009-07-01

    Inhomogeneous surrounds can produce either asymmetrical or symmetrical increment/decrement induction by orienting T-junctions to selectively group a test patch with surrounding regions [Melfi, T., & Schirillo, J. (2000). T-junctions in inhomogeneous surrounds. Vision Research, 40, 3735-3741]. The current experiments aimed to determine where T-junctions are processed by presenting each eye with a different image so that T-junctions exist only in the fused percept. Only minor differences were found between retinal and cortical versus cortical-only conditions, indicating that T-junctions are processed cortically.

  13. Planar Josephson tunnel junctions in a transverse magnetic field

    Monacoa, R.; Aarøe, Morten; Mygind, Jesper

    2007-01-01

    demagnetization effects imposed by the tunnel barrier and electrodes geometry are important. Measurements of the junction critical current versus magnetic field in planar Nb-based high-quality junctions with different geometry, size, and critical current density show that it is advantageous to use a transverse......Traditionally, since the discovery of the Josephson effect in 1962, the magnetic diffraction pattern of planar Josephson tunnel junctions has been recorded with the field applied in the plane of the junction. Here we discuss the static junction properties in a transverse magnetic field where...

  14. Phase diagrams of particles with dissimilar patches: X-junctions and Y-junctions

    Tavares, J M; Teixeira, P I C

    2012-01-01

    We use Wertheim’s first-order perturbation theory to investigate the phase behaviour and the structure of coexisting fluid phases for a model of patchy particles with dissimilar patches (two patches of type A and f B patches of type B). A patch of type α = {A,B} can bond to a patch of type β = {A,B} in a volume v αβ , thereby decreasing the internal energy by ε αβ . We analyse the range of model parameters where AB bonds, or Y-junctions, are energetically disfavoured (ε AB AA /2) but entropically favoured (v AB ≫ v αα ), and BB bonds, or X-junctions, are energetically favoured (ε BB > 0). We show that, for low values of ε BB /ε AA , the phase diagram has three different regions: (i) close to the critical temperature a low-density liquid composed of long chains and rich in Y-junctions coexists with a vapour of chains; (ii) at intermediate temperatures there is coexistence between a vapour of short chains and a liquid of very long chains with X- and Y-junctions; (iii) at low temperatures an ideal gas coexists with a high-density liquid with all possible AA and BB bonds formed. It is also shown that in region (i) the liquid binodal is reentrant (its density decreases with decreasing temperature) for the lower values of ε BB /ε AA . The existence of these three regions is a consequence of the competition between the formation of X- and Y-junctions: X-junctions are energetically favoured and thus dominate at low temperatures, whereas Y-junctions are entropically favoured and dominate at higher temperatures. (paper)

  15. Unwinding after high salinity stress: Pea DNA helicase 45 over- expression in tobacco confers high salinity tolerance without affecting yield (abstract)

    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

  16. Single-electron tunnel junction array

    Likharev, K.K.; Bakhvalov, N.S.; Kazacha, G.S.; Serdyukova, S.I.

    1989-01-01

    The authors have carried out an analysis of statics and dynamics of uniform one-dimensional arrays of ultrasmall tunnel junctions. The correlated single-electron tunneling in the junctions of the array results in its behavior qualitatively similar to that of the Josephson transmission line. In particular, external electric fields applied to the array edges can inject single-electron-charged solitons into the array interior. Shape of such soliton and character of its interactions with other solitons and the array edges are very similar to those of the Josephson vortices (sine-Gordon solitons) in the Josephson transmission line. Under certain conditions, a coherent motion of the soliton train along the array is possible, resulting in generation of narrowband SET oscillations with frequency f/sub s/ = /e where is the dc current flowing along the array

  17. Electron transport in doped fullerene molecular junctions

    Kaur, Milanpreet; Sawhney, Ravinder Singh; Engles, Derick

    The effect of doping on the electron transport of molecular junctions is analyzed in this paper. The doped fullerene molecules are stringed to two semi-infinite gold electrodes and analyzed at equilibrium and nonequilibrium conditions of these device configurations. The contemplation is done using nonequilibrium Green’s function (NEGF)-density functional theory (DFT) to evaluate its density of states (DOS), transmission coefficient, molecular orbitals, electron density, charge transfer, current, and conductance. We conclude from the elucidated results that Au-C16Li4-Au and Au-C16Ne4-Au devices behave as an ordinary p-n junction diode and a Zener diode, respectively. Moreover, these doped fullerene molecules do not lose their metallic nature when sandwiched between the pair of gold electrodes.

  18. Charge Transport Phenomena in Peptide Molecular Junctions

    Luchini, A.; Petricoin, E.F.; Geho, D.H.; Liotta, L.A.; Long, D.P.; Vaisman, I.I.

    2008-01-01

    Inelastic electron tunneling spectroscopy (IETS) is a valuable in situ spectroscopic analysis technique that provides a direct portrait of the electron transport properties of a molecular species. In the past, IETS has been applied to small molecules. Using self-assembled nano electronic junctions, IETS was performed for the first time on a large polypeptide protein peptide in the phosphorylated and native form, yielding interpretable spectra. A reproducible 10-fold shift of the I/V characteristics of the peptide was observed upon phosphorylation. Phosphorylation can be utilized as a site-specific modification to alter peptide structure and thereby influence electron transport in peptide molecular junctions. It is envisioned that kinases and phosphatases may be used to create tunable systems for molecular electronics applications, such as biosensors and memory devices.

  19. Electron and Phonon Transport in Molecular Junctions

    Li, Qian

    Molecular electronics provide the possibility to investigate electron and phonon transport at the smallest imaginable scale, where quantum effects can be investigated and exploited directly in the design. In this thesis, we study both electron transport and phonon transport in molecular junctions....... The system we are interested in here are π-stacked molecules connected with two semi-infinite leads. π-stacked aromatic rings, connected via π-π electronic coupling, provides a rather soft mechanical bridge while maintaining high electronic conductivity. We investigate electron transport...... transmission at the Fermi energy. We propose and analyze a way of using π   stacking to design molecular junctions to control heat transport. We develop a simple model system to identify optimal parameter regimes and then use density functional theory (DFT) to extract model parameters for a number of specific...

  20. Non-Lagrangian theories from brane junctions

    Bao, Ling [Chalmers Univ. of Technology, Goeteborg (Sweden); Mitev, Vladimir [Humboldt Univ., Berlin (Germany). Inst. fuer Mathematik und Inst. fuer Physik; Pomoni, Elli [Deutsches Elektronen-Synchrotron DESY, Hamburg (Germany). Theory Group; Taki, Masato [RIKEN Nishina Center, Saitama (Japan). Mathematical Physics Lab.; Yagi, Futoshi [International School of Advanced Studies (SISSA), Trieste (Italy); INFN, Trieste (Italy); Korea Institute for Advanced Study (KIAS), Seoul (Korea, Republic of)

    2013-10-15

    In this article we use 5-brane junctions to study the 5D T{sub N} SCFTs corresponding to the 5D N=1 uplift of the 4D N=2 strongly coupled gauge theories, which are obtained by compactifying N M5 branes on a sphere with three full punctures. Even though these theories have no Lagrangian description, by using the 5-brane junctions proposed by Benini, Benvenuti and Tachikawa, we are able to derive their Seiberg-Witten curves and Nekrasov partition functions. We cross-check our results with the 5D superconformal index proposed by Kim, Kim and Lee. Through the AGTW correspondence, we discuss the relations between 5D superconformal indices and n-point functions of the q-deformed W{sub N} Toda theories.

  1. Non-Lagrangian theories from brane junctions

    Bao, Ling; Mitev, Vladimir

    2013-10-01

    In this article we use 5-brane junctions to study the 5D T N SCFTs corresponding to the 5D N=1 uplift of the 4D N=2 strongly coupled gauge theories, which are obtained by compactifying N M5 branes on a sphere with three full punctures. Even though these theories have no Lagrangian description, by using the 5-brane junctions proposed by Benini, Benvenuti and Tachikawa, we are able to derive their Seiberg-Witten curves and Nekrasov partition functions. We cross-check our results with the 5D superconformal index proposed by Kim, Kim and Lee. Through the AGTW correspondence, we discuss the relations between 5D superconformal indices and n-point functions of the q-deformed W N Toda theories.

  2. Fractional flux quanta in Josephson junctions

    Goldobin, E.; Buckenmaier, K.; Gaber, T.; Kemmler, M.; Pfeiffer, J.; Koelle, D.; Kleiner, R. [Physikalisches Inst. - Experimentalphysik II, Univ. Tuebingen (Germany); Weides, M.; Kohlstedt, H. [Center of Nanoelectronic Systems for Information Technology (CNI), Research Centre Juelich (Germany); Siegel, M. [Inst. fuer Mikro- und Nanoelektronische Systeme, Univ. Karlsruhe (Germany)

    2007-07-01

    Fractional Josephson vortices may appear in the so-called 0-{kappa} Josephson junctions ({kappa} is an arbitrary number) and carry magnetic flux {phi}, which is a fraction of the magnetic flux quantum {phi}{sub 0}{approx}2.07 x 10{sup -15} Wb. Their properties are very different from the usual integer fluxons: they are pinned, and often represent the ground state of the system with spontaneous circulating supercurrent. They behave as well controlled macroscopic spins and can be used to construct bits, qubits, tunable photonic crystals and to study the (quantum) physics of spin systems. In this talk we discuss recent advances in 0-{pi} junction technology and present recent experimental results: evidence of the spontaneous flux in the ground state, spectroscopy of the fractional vortex eigenfrequencies and observation of dynamics effects related to the flipping of the fractional vortices. (orig.)

  3. Relaxation oscillation logic in Josephson junction circuits

    Fulton, T.A.

    1981-01-01

    A dc powered, self-resetting Josephson junction logic circuit relying on relaxation oscillations is described. A pair of Josephson junction gates are connected in series, a first shunt is connected in parallel with one of the gates, and a second shunt is connected in parallel with the series combination of gates. The resistance of the shunts and the dc bias current bias the gates so that they are capable of undergoing relaxation oscillations. The first shunt forms an output line whereas the second shunt forms a control loop. The bias current is applied to the gates so that, in the quiescent state, the gate in parallel with the second shunt is at V O, and the other gate is undergoing relaxation oscillations. By controlling the state of the first gate with the current in the output loop of another identical circuit, the invert function is performed

  4. Junctional epidermolysis bullosa(non-herlitz type)

    Bhinder, M. A.; Arshad, M. W.; Shabbir, M. I.; Zahoor, M. Y.; Shehzad, W.; Tariq, M.

    2017-01-01

    Junctional epidermolysis bullosa (JEB) is a recessively inherited skin blistering disease and is caused due to abnormalities in proteins that hold layers of the skin. Herlitz JEB is the severe form and non-Herlitz JEB is the milder form. This report describes a case of congenitally affected male child aged 5 years, with skin blistering. He has mitten-like hands and soft skin blistering on hands, legs and knees. Symptoms almost disappeared at the age of 3 years but reappeared with increased severity after 6 months. Histopathological examination showed epidermal detachment with intact basal cell layer and sparse infiltrate of lymphocytes with few eosinophils in the dermis. There was no blistering on the moist lining of the mouth and digestive tract. Localized symptoms with less lethality and histopathological examination indicated the presence of non-Herlitz type of JEB. This is the first report which confirms the presence of non-Herlitz junctional epidermolysis bullosa in Pakistan. (author)

  5. Junctional Epidermolysis Bullosa (Non-Herlitz Type).

    Bhinder, Munir Ahmad; Arshad, Muhammad Waqar; Zahoor, Muhammad Yasir; Shehzad, Wasim; Tariq, Muhammad; Shabbir, Muhammad Imran

    2017-05-01

    Junctional epidermolysis bullosa (JEB) is a recessively inherited skin blistering disease and is caused due to abnormalities in proteins that hold layers of the skin. Herlitz JEB is the severe form and non-Herlitz JEB is the milder form. This report describes a case of congenitally affected male child aged 5 years, with skin blistering. He has mitten-like hands and soft skin blistering on hands, legs and knees. Symptoms almost disappeared at the age of 3 years but reappeared with increased severity after 6 months. Histopathological examination showed epidermal detachment with intact basal cell layer and sparse infiltrate of lymphocytes with few eosinophils in the dermis. There was no blistering on the moist lining of the mouth and digestive tract. Localized symptoms with less lethality and histopathological examination indicated the presence of non-Herlitz type of JEB. This is the first report which confirms the presence of non-Herlitz junctional epidermolysis bullosa in Pakistan.

  6. Defect formation in long Josephson junctions

    Gordeeva, Anna; Pankratov, Andrey

    2010-01-01

    We study numerically a mechanism of vortex formation in a long Josephson junction within the framework of the one-dimensional sine-Gordon model. This mechanism is switched on below the critical temperature. It is shown that the number of fluxons versus velocity of cooling roughly scales according...... to the power law with the exponent of either 0.25 or 0.5 depending on the temperature variation in the critical current density....

  7. Syringomyelia and Craniocervical Junction Abnormalities in Chihuahuas

    Kiviranta, A.‐M.; Rusbridge, C.; Laitinen‐Vapaavuori, O.; Hielm‐Björkman, A.; Lappalainen, A.K.; Knowler, S.P.; Jokinen, T.S.

    2017-01-01

    Background: Chiari-like malformation (CM) and syringomyelia (SM) are widely reported in Cavalier King Charles Spaniels and Griffon Bruxellois dogs. Increasing evidence indicates that CM and SM also occur in other small and toy breed dogs, such as Chihuahuas. Objectives: To describe the presence of SM and craniocervical junction (CCJ) abnormalities in Chihuahuas and to evaluate the possible association of CCJ abnormalities with SM. To describe CM/SM-related clinical signs and neuro...

  8. Nonlinearity in superconductivity and Josephson junctions

    Lazarides, N.

    1995-01-01

    Within the framework of the Bardeen, Cooper and Schrieffers (BCS) theory, the influence of anisotropy on superconducting states are investigated. Crystal anisotropy exists in un-conventional low temperature superconductors as e.g. U 1-x Th x Be 13 and in high temperature superconductors. Starting from a phenomenological pairing interaction of the electrons or holes, the BCS approach is used to derive a set of coupled nonlinear algebraic equations for the momentum dependent gap parameter. The emphasis is put on bifurcation phenomena between s-, d-wave and mixed s- and d-wave symmetry and the influence on measurable quantities as the electron specific heat, spin susceptibility and Josephson tunnelling. Pitch-fork and perturbed pitch-fork bifurcations have been found separating s- and d-wave superconducting states from mixed s- and d-wave states. The additional superconducting states give rise to jumps in the electron specific heat below the transition temperature. These jumps are rounded in the case of perturbed pitch-fork bifurcations. An experiment to measure the sign of the interlayer interaction using dc SQUIDS is suggested. The Ambegaokar-Baratoff formalism has been used for calculating the quasiparticle current and the two phase coherent tunnelling currents in a Josephson junction made of anisotropic superconductors. It is shown that anisotropy can lead to a reduction in the product of the normal resistance and the critical current. For low voltages across the junction the usual resistively shunted Josephson model can be used. Finally, bunching in long circular Josephson junctions and suppression of chaos in point junctions have been investigated. (au) 113 refs

  9. Fractional Solitons in Excitonic Josephson Junctions

    Hsu, Ya-Fen; Su, Jung-Jung

    2015-01-01

    The Josephson effect is especially appealing to physicists because it reveals macroscopically the quantum order and phase. In excitonic bilayers the effect is even subtler due to the counterflow of supercurrent as well as the tunneling between layers (interlayer tunneling). Here we study, in a quantum Hall bilayer, the excitonic Josephson junction: a conjunct of two exciton condensates with a relative phase ? 0 applied. The system is mapped into a pseudospin ferromagnet then described numeric...

  10. A semiconductor nanowire Josephson junction microwave laser

    Cassidy, Maja; Uilhoorn, Willemijn; Kroll, James; de Jong, Damaz; van Woerkom, David; Nygard, Jesper; Krogstrup, Peter; Kouwenhoven, Leo

    We present measurements of microwave lasing from a single Al/InAs/Al nanowire Josephson junction strongly coupled to a high quality factor superconducting cavity. Application of a DC bias voltage to the Josephson junction results in photon emission into the cavity when the bias voltage is equal to a multiple of the cavity frequency. At large voltage biases, the strong non-linearity of the circuit allows for efficient down conversion of high frequency microwave photons down to multiple photons at the fundamental frequency of the cavity. In this regime, the emission linewidth narrows significantly below the bare cavity linewidth to 50%. The junction-cavity coupling and laser emission can be tuned rapidly via an external gate, making it suitable to be integrated into a scalable qubit architecture as a versatile source of coherent microwave radiation. This work has been supported by the Netherlands Organisation for Scientific Research (NWO/OCW), Foundation for Fundamental Research on Matter (FOM), European Research Council (ERC), and Microsoft Corporation Station Q.

  11. Structure based modification of Bluetongue virus helicase protein VP6 to produce a viable VP6-truncated BTV

    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.

  12. Relationship between osteosarcoma and ionizing radiation hypersensitive human B lymphocyte cells lacking RecQL4 helicase

    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)

  13. Non-Watson–Crick interactions between PNA and DNA inhibit the ATPase activity of bacteriophage T4 Dda helicase

    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

  14. Characterization of papillomavirus E1 helicase mutants defective for interaction with the SUMO-conjugating enzyme Ubc9

    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.

  15. DNA unwinding by ASCC3 helicase is coupled to ALKBH3 dependent DNA alkylation repair and cancer cell proliferation

    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

  16. Development of Reverse Transcription Thermostable Helicase-Dependent DNA Amplification for the Detection of Tomato Spotted Wilt Virus.

    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.

  17. Helicase-Dependent Isothermal Amplification of DNA and RNA by Using Self-Avoiding Molecular Recognition Systems.

    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.

  18. DHX9 helicase is involved in preventing genomic instability induced by alternatively structured DNA in human cells.

    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.

  19. Electron Transport through Porphyrin Molecular Junctions

    Zhou, Qi

    The goal of this work is to study the properties that would affect the electron transport through a porphyrin molecular junction. This work contributes to the field of electron transport in molecular junctions in the following 3 aspects. First of all, by carrying out experiments comparing the conductance of the iron (III) porphyrin (protected) and the free base porphyrin (protected), it is confirmed that the molecular energy level broadening and shifting occurs for porphyrin molecules when coupled with the metal electrodes, and this level broadening and shifting plays an important role in the electron transport through molecular junctions. Secondly, by carrying out an in-situ deprotection of the acetyl-protected free base porphyrin molecules, it is found out that the presence of acetyl groups reduces the conductance. Thirdly, by incorporating the Matrix-assisted laser desorption/ionization (MALDI) spectrum and the in-situ deprotection prior to formation of molecular junctions, it allows a more precise understanding of the molecules involved in the formation of molecular junctions, and therefore allows an accurate analysis of the conductance histogram. The molecules are prepared by self-assembly and the junctions are formed using a Scanning Tunneling Microscopy (STM) molecular break junction technique. The porphyrin molecules are characterized by MALDI in solution before self-assembly to a gold/mica substrate. The self-assembled monolayers (SAMs) of porphyrins on gold are characterized by Ultraviolet-visible (UV-Vis) reflection spectroscopy to confirm that the molecules are attached to the substrate. The SAMs are then characterized by Angle-Resolved X-ray photoelectron spectroscopy (ARXPS) to determine the thickness and the average molecular orientation of the molecular layer. The electron transport is measured by conductance-displacement (G-S) experiments under a given bias (-0.4V). The conductance value of a single molecule is identified by a statistical analysis

  20. Single P-N junction tandem photovoltaic device

    Walukiewicz, Wladyslaw [Kensington, CA; Ager, III, Joel W.; Yu, Kin Man [Lafayette, CA

    2011-10-18

    A single P-N junction solar cell is provided having two depletion regions for charge separation while allowing the electrons and holes to recombine such that the voltages associated with both depletion regions of the solar cell will add together. The single p-n junction solar cell includes an alloy of either InGaN or InAlN formed on one side of the P-N junction with Si formed on the other side in order to produce characteristics of a two junction (2J) tandem solar cell through only a single P-N junction. A single P-N junction solar cell having tandem solar cell characteristics will achieve power conversion efficiencies exceeding 30%.

  1. Electromagnetic waves in single- and multi-Josephson junctions

    Matsumoto, Hideki; Koyama, Tomio; Machida, Masahiko

    2008-01-01

    The terahertz wave emission from the intrinsic Josephson junctions is one of recent topics in high T c superconductors. We investigate, by numerical simulation, properties of the electromagnetic waves excited by a constant bias current in the single- and multi-Josephson junctions. Nonlinear equations of phase-differences are solved numerically by treating the effects of the outside electromagnetic fields as dynamical boundary conditions. It is shown that the emitted power of the electromagnetic wave can become large near certain retrapping points of the I-V characteristics. An instability of the inside phase oscillation is related to large amplitude of the oscillatory waves. In the single- (or homogeneous mutli-) Josephson junctions, electromagnetic oscillations can occur either in a form of standing waves (shorter junctions) or by formation of vortex-antivortex pairs (longer junctions). How these two effects affects the behavior of electromagnetic waves in the intrinsic Josephson junctions is discussed

  2. Shunted-Josephson-junction model. I. The autonomous case

    Belykh, V. N.; Pedersen, Niels Falsig; Sørensen, O. H.

    1977-01-01

    The shunted-Josephson-junction model: the parallel combination of a capacitance, a phase-dependent conductance, and an ideal junction element biased by a constant current, is discussed for arbitrary values of the junction parameters. The main objective is to provide a qualitative understanding...... current-voltage curves are presented. The case with a time-dependent monochromatic bias current is treated in a similar fashion in the companion paper....

  3. Magnetic interaction between spatially extended superconducting tunnel junctions

    Grønbech-Jensen, Niels; Samuelsen, Mogens Rugholm

    2002-01-01

    A general description of magnetic interactions between superconducting tunnel junctions is given. The description covers a wide range of possible experimental systems, and we explicitly explore two experimentally relevant limits of coupled junctions. One is the limit of junctions with tunneling...... been considered through arrays of superconducting weak links based on semiconductor quantum wells with superconducting electrodes. We use the model to make direct interpretations of the published experiments and thereby propose that long-range magnetic interactions are responsible for the reported...

  4. Critical current fluctuation in a microwave-driven Josephson junction

    Dong Ning; Sun Guozhu; Wang Yiwen; Cao Junyu; Yu Yang; Chen Jian; Kang Lin; Xu Weiwei; Han Siyuan; Wu Peiheng

    2007-01-01

    Josephson junction devices are good candidates for quantum computation. A large energy splitting was observed in the spectroscopy of a superconducting Josephson junction. The presence of the critical current fluctuation near the energy splitting indicated coupling between the junction and a two-level system. Furthermore, we find that this fluctuation is microwave dependent. It only appears at certain microwave frequency. This relation suggested that the decoherence of qubits is influenced by the necessary computing operations

  5. ACCIDENT PREDICTION MODELS FOR UNSIGNALISED URBAN JUNCTIONS IN GHANA

    Mohammed SALIFU, MSc., PhD, MIHT, MGhIE

    2004-01-01

    The main objective of this study was to provide an improved method for safety appraisal in Ghana through the development and application of suitable accident prediction models for unsignalised urban junctions. A case study was designed comprising 91 junctions selected from the two most cosmopolitan cities in Ghana. A wide range of traffic and road data together with the corresponding accident data for each junction for the three-year period 1996-1998 was utilized in the model development p...

  6. Quasiparticle current in superconductor-semiconductor-superconductor junctions

    Tartakovskij, A.V.; Fistul', M.V.

    1988-01-01

    It is shown that the quasiparticle current in a superconductor-semiconductor-superconductor junction may significantly increase as a result of resonant passage of the quasiparticle along particular trajectories from periodically situated localized centers. A prediction of the theory is that with increasing junction resistance there should be a change from an excessive current to a insufficient current on the current-voltage characteristics (at high voltages). The effect of transparency of the boundaries on resonance tunneling in such junctions is also investigated

  7. Nature of inhomogeneous states in superconducting junctions

    Ivlev, B.I.; Kopnin, N.B.

    1982-01-01

    A superconducting structure which arises in a superconducting film under a strong injection of a current through a tunnel junction is considered. If the current density in the film exceeds the critical Ginzburg-Landau value, an inhomogeneous resistive state with phase-slip centers can arise in it. This state is charcterized by the presence of regions with different chemical potentials of the Cooper pairs. These shifts of the pair chemical potential and the nonuniform structure of the order parameter may account for the so-called multigap states which have been observed experimentally

  8. Towards ferromagnet/superconductor junctions on graphene

    Pakkayil, Shijin Babu

    2015-01-01

    Ever since A. Aspect et al. performed the famous 1982 experiment to prove the violation of Bell's inequality, there have been suggestions to conduct the same experiment in a solid state system. Some of those proposals involve superconductors as the source of entangled electron pair and spin depended interfaces as the optical analogue of polariser/filter. Semiconductors can serve as the best medium for such an experiment due to their long relaxation lengths. So far there are no reports on a ferromagnet/superconductor junctions on a semiconductor even though such junctions has been successfully realised in metallic systems. This thesis reports the successful fabrication of ferromagnet/superconductor junction along with characterising measurements in a perfectly two dimensional zero-gap semiconductor known as graphene. Since it's discovery in 2004, graphene has attracted prodigious interest from both academia and industry due to it's inimitable physical properties: very high mobility, high thermal and electrical conductivity, a high Young's modulus and impermeability. Graphene is also expected to have very long spin relaxation length and high spin life time because of it's low spin orbit coupling. For this reason and since researchers are always looking for novel materials and devices to comply with the high demands for better and faster data storage devices, graphene has emanated as a brand new material system for spin based devices. The very first spin injection and detection in graphene was realised in 2007 and ever since, the focal point of the research has been to improve the spin transport properties. A part of this thesis discusses a new fabrication recipe which has a high yield for successfully contacting graphene with a ferromagnet. A high starting yield for ferromagnetic contacts is a irremissible condition for combining superconducting contacts to the device to fabricate ferromagnet/superconductor junctions. Any fabrication recipe

  9. Powered supports for T-junctions

    von Klinggraeff, G.; Bohnes, K.

    1981-04-23

    The hydraulic self advancing support system first introduced at Niederberg colliery for a T-junction between a thin seam and a roadway with porch set supports included nearly all components for underpinning the roadway support closest to the face and for supporting the face end close to the roadway, including the rib-side. It ensures a fixed cycle of operations without the need for improvisation while providing continuous strata control during displacement of units. This support combination has proved itself in underground use. As a result, accident incidence was reduced, the number of breakdowns reduced, made the work easier and reduced the number of shifts needed.

  10. Towards ferromagnet/superconductor junctions on graphene

    Pakkayil, Shijin Babu

    2015-07-01

    Ever since A. Aspect et al. performed the famous 1982 experiment to prove the violation of Bell's inequality, there have been suggestions to conduct the same experiment in a solid state system. Some of those proposals involve superconductors as the source of entangled electron pair and spin depended interfaces as the optical analogue of polariser/filter. Semiconductors can serve as the best medium for such an experiment due to their long relaxation lengths. So far there are no reports on a ferromagnet/superconductor junctions on a semiconductor even though such junctions has been successfully realised in metallic systems. This thesis reports the successful fabrication of ferromagnet/superconductor junction along with characterising measurements in a perfectly two dimensional zero-gap semiconductor known as graphene. Since it's discovery in 2004, graphene has attracted prodigious interest from both academia and industry due to it's inimitable physical properties: very high mobility, high thermal and electrical conductivity, a high Young's modulus and impermeability. Graphene is also expected to have very long spin relaxation length and high spin life time because of it's low spin orbit coupling. For this reason and since researchers are always looking for novel materials and devices to comply with the high demands for better and faster data storage devices, graphene has emanated as a brand new material system for spin based devices. The very first spin injection and detection in graphene was realised in 2007 and ever since, the focal point of the research has been to improve the spin transport properties. A part of this thesis discusses a new fabrication recipe which has a high yield for successfully contacting graphene with a ferromagnet. A high starting yield for ferromagnetic contacts is a irremissible condition for combining superconducting contacts to the device to fabricate ferromagnet/superconductor junctions. Any fabrication recipe

  11. Gap Junctional Intercellular Communication and Breast Cancer Metastasis to Bone

    Donahue, Henry

    2001-01-01

    .... We found that: 1) expressing the metastasis suppressing gene BRMS1 in diverse cancer cell lines, including breast and melanoma, restores homotypic gap junctional intercellular communication (GJIC); 2...

  12. On simulation of local fluxes in molecular junctions

    Cabra, Gabriel; Jensen, Anders; Galperin, Michael

    2018-05-01

    We present a pedagogical review of the current density simulation in molecular junction models indicating its advantages and deficiencies in analysis of local junction transport characteristics. In particular, we argue that current density is a universal tool which provides more information than traditionally simulated bond currents, especially when discussing inelastic processes. However, current density simulations are sensitive to the choice of basis and electronic structure method. We note that while discussing the local current conservation in junctions, one has to account for the source term caused by the open character of the system and intra-molecular interactions. Our considerations are illustrated with numerical simulations of a benzenedithiol molecular junction.

  13. Spectrum of resonant plasma oscillations in long Josephson junctions

    Holst, T.

    1996-01-01

    An analysis is presented for the amplitude of the plasma oscillations in the zero-voltage state of a long and narrow Josephson tunnel junction. The calculation is valid for arbitrary normalized junction length and arbitrary bias current. The spectrum of the plasma resonance is found numerically as solutions to an analytical equation. The low-frequency part of the spectrum contains a single resonance, which is known to exist also in the limit of a short and narrow junction. Above a certain cutoff frequency, a series of high-frequency standing wave plasma resonances is excited, a special feature of long Josephson junctions. copyright 1996 The American Physical Society

  14. Field modulation of the critical current in magnetic Josephson junctions

    Blamire, M G; Smiet, C B; Banerjee, N; Robinson, J W A

    2013-01-01

    The dependence of the critical current of a simple Josephson junction on the applied magnetic field is well known and, for a rectangular junction, gives rise to the classic ‘Fraunhofer’ modulation with periodic zeros at the fields that introduce a flux quantum into the junction region. Much recent work has been performed on Josephson junctions that contain magnetic layers. The magnetization of such layers introduces additional flux into the junction and, for large junction areas or strong magnetic materials, can significantly distort the modulation of the critical current and strongly suppress the maximum critical current. The growing interest in junctions that induce odd-frequency triplet pairing in a ferromagnet, and the need to make quantitative comparisons with theory, mean that a full understanding of the role of magnetic barriers in controlling the critical current is necessary. This paper analyses the effect of magnetism and various magnetic configurations on Josephson critical currents; the overall treatment applies to junctions of general shape, but the specific cases of square and rectangular junctions are considered. (paper)

  15. Several alternative approaches to the manufacturing of HTS Josephson junctions

    Villegier , J.; Boucher , H.; Ghis , A.; Levis , M.; Méchin , Laurence; Moriceau , H.; Pourtier , F.; Vabre , M.; Nicoletti , S.; Correra , L.

    1994-01-01

    In this work we describe comparatively the fabrication and the characterization of various types of HTS Josephson junctions manufactured using different processes : grain boundary junctions have been studied both by the way of junctions on bicrystal substrates and of bi-epitaxial junctions. Ramp-edge types have been elaborated and characterized using mainly N-YBaCuO thin film as a barrier while the trilayer approach has been investigated through a-axis structures. YBaCuO or GdBaCuO supercondu...

  16. ‘Gap Junctions and Cancer: Communicating for 50 Years’

    Aasen, Trond; Mesnil, Marc; Naus, Christian C.; Lampe, Paul D.; Laird, Dale W.

    2017-01-01

    Fifty years ago, tumour cells were found to lack electrical coupling, leading to the hypothesis that loss of direct intercellular communication is commonly associated with cancer onset and progression. Subsequent studies linked this phenomenon to gap junctions composed of connexin proteins. While many studies support the notion that connexins are tumour suppressors, recent evidence suggests that, in some tumour types, they may facilitate specific stages of tumour progression through both junctional and non-junctional signalling pathways. This Timeline article highlights the milestones connecting gap junctions to cancer, and underscores important unanswered questions, controversies and therapeutic opportunities in the field. PMID:27782134

  17. Some chaotic features of intrinsically coupled Josephson junctions

    Kolahchi, M.R.; Shukrinov, Yu.M.; Hamdipour, M.; Botha, A.E.; Suzuki, M.

    2013-01-01

    Highlights: ► Intrinsically coupled Josephson junctions model a high-T c superconductor. ► Intrinsically coupled Josephson junctions can act as a chaotic nonlinear system. ► Chaos could be due to resonance overlap. ► Avoiding parameters that lead to chaos is important for the design of resonators. -- Abstract: We look for chaos in an intrinsically coupled system of Josephson junctions. This study has direct applications for the high-T c resonators which require coherence amongst the junctions

  18. Superconducting Tunnel Junction Arrays for UV Photon Detection, Phase I

    National Aeronautics and Space Administration — An innovative method is described for the fabrication of superconducting tunnel junction (STJ) detector arrays offering true "three dimensional" imaging throughout...

  19. Microwave phase locking of Josephson-junction fluxon oscillators

    Salerno, M.; Samuelsen, Mogens Rugholm; Filatrella, G.

    1990-01-01

    Application of the classic McLaughlin-Scott soliton perturbation theory to a Josephson-junction fluxon subjected to a microwave field that interacts with the fluxon only at the junction boundaries reduces the problem of phase locking of the fluxon oscillation to the study of a two-dimensional fun......Application of the classic McLaughlin-Scott soliton perturbation theory to a Josephson-junction fluxon subjected to a microwave field that interacts with the fluxon only at the junction boundaries reduces the problem of phase locking of the fluxon oscillation to the study of a two...

  20. Junction Potentials Bias Measurements of Ion Exchange Membrane Permselectivity.

    Kingsbury, Ryan S; Flotron, Sophie; Zhu, Shan; Call, Douglas F; Coronell, Orlando

    2018-04-17

    Ion exchange membranes (IEMs) are versatile materials relevant to a variety of water and waste treatment, energy production, and industrial separation processes. The defining characteristic of IEMs is their ability to selectively allow positive or negative ions to permeate, which is referred to as permselectivity. Measured values of permselectivity that equal unity (corresponding to a perfectly selective membrane) or exceed unity (theoretically impossible) have been reported for cation exchange membranes (CEMs). Such nonphysical results call into question our ability to correctly measure this crucial membrane property. Because weighing errors, temperature, and measurement uncertainty have been shown to not explain these anomalous permselectivity results, we hypothesized that a possible explanation are junction potentials that occur at the tips of reference electrodes. In this work, we tested this hypothesis by comparing permselectivity values obtained from bare Ag/AgCl wire electrodes (which have no junction) to values obtained from single-junction reference electrodes containing two different electrolytes. We show that permselectivity values obtained using reference electrodes with junctions were greater than unity for CEMs. In contrast, electrodes without junctions always produced permselectivities lower than unity. Electrodes with junctions also resulted in artificially low permselectivity values for AEMs compared to electrodes without junctions. Thus, we conclude that junctions in reference electrodes introduce two biases into results in the IEM literature: (i) permselectivity values larger than unity for CEMs and (ii) lower permselectivity values for AEMs compared to those for CEMs. These biases can be avoided by using electrodes without a junction.

  1. Effect of junction configurations on microdroplet formation in a T-junction microchannel

    Lih, F. L.; Miao, J. M.

    2015-03-01

    This study investigates the dynamic formation process of water microdroplets in a silicon oil flow in a T-junction microchannel. Segmented water microdroplets are formed at the junction when the water flow is perpendicularly injected into the silicon oil flow in a straight rectangular microchannel. This study further presents the effects of the water flow inlet geometry on hydrodynamic characteristics of water microdroplet formation. A numerical multiphase volume of fluid (VOF) scheme is coupled to solve the unsteady three-dimensional laminar Navier-Stokes equations to depict the droplet formation phenomena at the junction. Predicted results on the length and generated frequency of the microdroplets agree well with experimental results in a T-junction microchannel with straight and flat inlets (the base model) for both fluid flows. Empirical correlations are reported between the volumetric flow ratio and the dimensionless microdroplet length or dimensionless frequency of droplet generation at a fixed capillary number of 4.7 · 10-3. The results of this study indicate a reduction in the droplet length of approximately 21% if the straight inlet for the water flow is modified to a downstream sudden contraction inlet for the water flow.

  2. A Listeria monocytogenes RNA helicase essential for growth and ribosomal maturation at low temperatures uses its C terminus for appropriate interaction with the ribosome.

    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.

  3. Identification of unique interactions between the flexible linker and the RecA-like domains of DEAD-box helicase Mss116

    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)

  4. Identification of unique interactions between the flexible linker and the RecA-like domains of DEAD-box helicase Mss116

    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.

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

    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.

  6. G-quadruplex and G-rich sequence stimulate Pif1p-catalyzed downstream duplex DNA unwinding through reducing waiting time at ss/dsDNA junction

    Zhang, Bo; Wu, Wen-Qiang; Liu, Na-Nv; Duan, Xiao-Lei; Li, Ming; Dou, Shuo-Xing; Hou, Xi-Miao; Xi, Xu-Guang

    2016-01-01

    Alternative DNA structures that deviate from B-form double-stranded DNA such as G-quadruplex (G4) DNA can be formed by G-rich sequences that are widely distributed throughout the human genome. We have previously shown that Pif1p not only unfolds G4, but also unwinds the downstream duplex DNA in a G4-stimulated manner. In the present study, we further characterized the G4-stimulated duplex DNA unwinding phenomenon by means of single-molecule fluorescence resonance energy transfer. It was found that Pif1p did not unwind the partial duplex DNA immediately after unfolding the upstream G4 structure, but rather, it would dwell at the ss/dsDNA junction with a ‘waiting time’. Further studies revealed that the waiting time was in fact related to a protein dimerization process that was sensitive to ssDNA sequence and would become rapid if the sequence is G-rich. Furthermore, we identified that the G-rich sequence, as the G4 structure, equally stimulates duplex DNA unwinding. The present work sheds new light on the molecular mechanism by which G4-unwinding helicase Pif1p resolves physiological G4/duplex DNA structures in cells. PMID:27471032

  7. Fabrication of magnetic tunnel junctions with epitaxial and textured ferromagnetic layers

    Chang, Y. Austin; Yang, Jianhua Joshua

    2008-11-11

    This invention relates to magnetic tunnel junctions and methods for making the magnetic tunnel junctions. The magnetic tunnel junctions include a tunnel barrier oxide layer sandwiched between two ferromagnetic layers both of which are epitaxial or textured with respect to the underlying substrate upon which the magnetic tunnel junctions are grown. The magnetic tunnel junctions provide improved magnetic properties, sharper interfaces and few defects.

  8. Behavior of tight-junction, adherens-junction and cell polarity proteins during HNF-4α-induced epithelial polarization

    Satohisa, Seiro; Chiba, Hideki; Osanai, Makoto; Ohno, Shigeo; Kojima, Takashi; Saito, Tsuyoshi; Sawada, Norimasa

    2005-01-01

    We previously reported that expression of tight-junction molecules occludin, claudin-6 and claudin-7, as well as establishment of epithelial polarity, was triggered in mouse F9 cells expressing hepatocyte nuclear factor (HNF)-4α [H. Chiba, T. Gotoh, T. Kojima, S. Satohisa, K. Kikuchi, M. Osanai, N. Sawada. Hepatocyte nuclear factor (HNF)-4α triggers formation of functional tight junctions and establishment of polarized epithelial morphology in F9 embryonal carcinoma cells, Exp. Cell Res. 286 (2003) 288-297]. Using these cells, we examined in the present study behavior of tight-junction, adherens-junction and cell polarity proteins and elucidated the molecular mechanism behind HNF-4α-initiated junction formation and epithelial polarization. We herein show that not only ZO-1 and ZO-2, but also ZO-3, junctional adhesion molecule (JAM)-B, JAM-C and cell polarity proteins PAR-3, PAR-6 and atypical protein kinase C (aPKC) accumulate at primordial adherens junctions in undifferentiated F9 cells. In contrast, CRB3, Pals1 and PATJ appeared to exhibit distinct subcellular localization in immature cells. Induced expression of HNF-4α led to translocation of these tight-junction and cell polarity proteins to beltlike tight junctions, where occludin, claudin-6 and claudin-7 were assembled, in differentiated cells. Interestingly, PAR-6, aPKC, CRB3 and Pals1, but not PAR-3 or PATJ, were also concentrated on the apical membranes in differentiated cells. These findings indicate that HNF-4α provokes not only expression of tight-junction adhesion molecules, but also modulation of subcellular distribution of junction and cell polarity proteins, resulting in junction formation and epithelial polarization

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

    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.

  10. Targeting helicase-dependent amplification products with an electrochemical genosensor for reliable and sensitive screening of genetically modified organisms.

    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.

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

    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.

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

    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.

  13. DNA2 cooperates with the WRN and BLM RecQ helicases to mediate long-range DNA end resection in human cells

    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

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

    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.

  15. Dynamics of the Josephson multi-junction system with junctions characterized by non-sinusoidal current - phase relationship

    Abal'osheva, I.; Lewandowski, S.J.

    2004-01-01

    It is shown that the inclusion of junctions characterized by non-sinusoidal current - phase relationship in the systems composed of multiple Josephson junctions - results in the appearance of additional system phase states. Numerical simulations and stability considerations confirm that those phase states can be realized in practice. Moreover, spontaneous formation of the grain boundary junctions in high-T c superconductors with non-trivial current-phase relations due to the d-wave symmetry of the order parameter is probable. Switching between the phase states of multiple grain boundary junction systems can lead to additional 1/f noise in high-T c superconductors. (author)

  16. Theoretical and experimental investigations on synchronization in many-junction arrays of HTSC Josephson junctions. Final report

    Seidel, P.; Heinz, E.; Pfuch, A.; Machalett, F.; Krech, W.; Basler, M.

    1996-06-01

    Different many-junction arrays of Josephson junctions were studied theoretically to analyse the mechanisms of synchronization, the influence of internal and external parameters and the maximal allowed spread of parameters for the single junctions. Concepts to realize arrays using standard high-T c superconductor technology were created, e.g. the new arrangement of multijunction superconducting loops (MSL). First experimental results show the relevance of this concept. Intrinsic one-dimensional arrays in thin film technology were prepared as mesas out of Bi or Tl 2212 films. to characterize HTSC Josephson junctions methods based on the analysis of microwave-induced steps were developed. (orig.) [de

  17. Y-junction of superconducting Josephson chains

    Giuliano, Domenico; Sodano, Pasquale

    2009-01-01

    We show that, for pertinent values of the fabrication and control parameters, an attractive finite coupling fixed point emerges in the phase diagram of a Y-junction of superconducting Josephson chains. The new fixed point arises only when the dimensionless flux f piercing the central loop of the network equals π and, thus, does not break time-reversal invariance; for f≠π, only the strongly coupled fixed point survives as a stable attractive fixed point. Phase slips (instantons) have a crucial role in establishing this transition: we show indeed that, at f=π, a new set of instantons-the W-instantons-comes into play to destabilize the strongly coupled fixed point. Finally, we provide a detailed account of the Josephson current-phase relationship along the arms of the network, near each one of the allowed fixed points. Our results evidence remarkable similarities between the phase diagram accessible to a Y-junction of superconducting Josephson chains and the one found in the analysis of quantum Brownian motion on frustrated planar lattices

  18. Edge currents in frustrated Josephson junction ladders

    Marques, A. M.; Santos, F. D. R.; Dias, R. G.

    2016-09-01

    We present a numerical study of quasi-1D frustrated Josephson junction ladders with diagonal couplings and open boundary conditions, in the large capacitance limit. We derive a correspondence between the energy of this Josephson junction ladder and the expectation value of the Hamiltonian of an analogous tight-binding model, and show how the overall superconducting state of the chain is equivalent to the minimum energy state of the tight-binding model in the subspace of one-particle states with uniform density. To satisfy the constraint of uniform density, the superconducting state of the ladder is written as a linear combination of the allowed k-states of the tight-binding model with open boundaries. Above a critical value of the parameter t (ratio between the intra-rung and inter-rung Josephson couplings) the ladder spontaneously develops currents at the edges, which spread to the bulk as t is increased until complete coverage is reached. Above a certain value of t, which varies with ladder size (t = 1 for an infinite-sized ladder), the edge currents are destroyed. The value t = 1 corresponds, in the tight-binding model, to the opening of a gap between two bands. We argue that the disappearance of the edge currents with this gap opening is not coincidental, and that this points to a topological origin for these edge current states.

  19. GAP junctional communication in brain secondary organizers.

    Bosone, Camilla; Andreu, Abraham; Echevarria, Diego

    2016-06-01

    Gap junctions (GJs) are integral membrane proteins that enable the direct cytoplasmic exchange of ions and low molecular weight metabolites between adjacent cells. They are formed by the apposition of two connexons belonging to adjacent cells. Each connexon is formed by six proteins, named connexins (Cxs). Current evidence suggests that gap junctions play an important part in ensuring normal embryo development. Mutations in connexin genes have been linked to a variety of human diseases, although the precise role and the cell biological mechanisms of their action remain almost unknown. Among the big family of Cxs, several are expressed in nervous tissue but just a few are expressed in the anterior neural tube of vertebrates. Many efforts have been made to elucidate the molecular bases of Cxs cell biology and how they influence the morphogenetic signal activity produced by brain signaling centers. These centers, orchestrated by transcription factors and morphogenes determine the axial patterning of the mammalian brain during its specification and regionalization. The present review revisits the findings of GJ composed by Cx43 and Cx36 in neural tube patterning and discuss Cx43 putative enrollment in the control of Fgf8 signal activity coming from the well known secondary organizer, the isthmic organizer. © 2016 The Authors. Development, Growth & Differentiation published by John Wiley & Sons Australia, Ltd on behalf of Japanese Society of Developmental Biologists.

  20. Electrophysiological study in neuromuscular junction disorders

    Ajith Cherian

    2013-01-01

    Full Text Available This review is on ultrastructure and subcellular physiology at normal and abnormal neuromuscular junctions. The clinical and electrophysiological findings in myasthenia gravis, Lambert-Eaton myasthenic syndrome (LEMS, congenital myasthenic syndromes, and botulinum intoxication are discussed. Single fiber electromyography (SFEMG helps to explain the basis of testing neuromuscular junction function by repetitive nerve stimulation (RNS. SFEMG requires skill and patience and its availability is limited to a few centers. For RNS supramaximal stimulation is essential and so is display of the whole waveform of each muscle response at maximum amplitude. The amplitudes of the negative phase of the first and fourth responses are measured from baseline to negative peak, and the percent change of the fourth response compared with the first represents the decrement or increment. A decrement greater than 10% is accepted as abnormal and smooth progression of response amplitude train and reproducibility form the crux. In suspected LEMS the effect of fast rates of stimulation should be determined after RNS response to slow rates of stimulation. Caution is required to avoid misinterpretation of potentiation and pseudofacilitation.

  1. Development of superconducting tunnel junction radiation detectors

    Katagiri, Masaki; Kishimoto, Maki; Ukibe, Masahiro; Nakamura, Tatsuya; Nakazawa, Masaharu [Japan Atomic Energy Research Inst., Tokyo (Japan); Kurakado, Masahiko; Ishibashi, Kenji; Maehata, Keisuke

    1998-07-01

    Study on development of high energy resolution X-ray detector using superconducting tunnel junction (STJ) for radiation detection was conducted for 5 years under cooperation of University of Tokyo group and Kyushu University group by Quantum measurement research group of Advanced fundamental research center of JAERI. As the energy resolution of STJ could be obtained better results than that of Si semiconductor detector told to be actually best at present, this study aimed to actualize an X-ray detector usable for the experimental field and to elucidate radiation detection mechanism due to STJ. The STJ element used for this study was the one developed by Kurakado group of Nippon Steel Corp. As a results, some technical problems were almost resolved, which made some trouble when using the STJ element to detection element of X-ray spectrometer. In order to make the X-ray detector better, it is essential to manufacture a STJ element and develop serial junction type STJ element on the base of optimization of the element structure and selection and single crystallization of new superconducting materials such as Ta and others, activating the research results. (G.K.)

  2. Molecular Diffusion through Cyanobacterial Septal Junctions

    Mercedes Nieves-Morión

    2017-01-01

    Full Text Available Heterocyst-forming cyanobacteria grow as filaments in which intercellular molecular exchange takes place. During the differentiation of N2-fixing heterocysts, regulators are transferred between cells. In the diazotrophic filament, vegetative cells that fix CO2 through oxygenic photosynthesis provide the heterocysts with reduced carbon and heterocysts provide the vegetative cells with fixed nitrogen. Intercellular molecular transfer has been traced with fluorescent markers, including calcein, 5-carboxyfluorescein, and the sucrose analogue esculin, which are observed to move down their concentration gradient. In this work, we used fluorescence recovery after photobleaching (FRAP assays in the model heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120 to measure the temperature dependence of intercellular transfer of fluorescent markers. We find that the transfer rate constants are directly proportional to the absolute temperature. This indicates that the “septal junctions” (formerly known as “microplasmodesmata” linking the cells in the filament allow molecular exchange by simple diffusion, without any activated intermediate state. This constitutes a novel mechanism for molecular transfer across the bacterial cytoplasmic membrane, in addition to previously characterized mechanisms for active transport and facilitated diffusion. Cyanobacterial septal junctions are functionally analogous to the gap junctions of metazoans.

  3. Terbinafine inhibits gap junctional intercellular communication

    Lee, Ju Yeun; Yoon, Sei Mee; Choi, Eun Ju; Lee, Jinu

    2016-01-01

    Terbinafine is an antifungal agent that selectively inhibits fungal sterol synthesis by blocking squalene epoxidase. We evaluated the effect of terbinafine on gap junctional intercellular communication (GJIC). Fluorescence recovery after photobleaching (FRAP) and I-YFP GJIC assays revealed that terbinafine inhibits GJIC in a reversible and dose-dependent manner in FRT-Cx43 and LN215 cells. Treatment with terbinafine did not affect Cx43 phosphorylation status or intracellular Ca 2+ concentration, well-known action mechanisms of various GJIC blockers. While a structurally related chemical, naftifine, attenuated GJIC, epigallocatechin gallate, another potent squalene epoxidase inhibitor with a different structure, did not. These results suggest that terbinafine inhibits GJIC with a so far unknown mechanism of action. - Highlights: • In vitro pharmacological studies were performed on FRT-Cx43 and LN215 cells. • Terbinafine inhibits gap junctional intercellular communication in both cell lines. • The inhibitory effect of terbinafine is reversible and dose-dependent. • Treatment of terbinafine does not alter Cx43 phosphorylation or cytosolic Ca 2+ concentration. • Inhibition of squalene epoxidase is not involved in this new effect of terbinafine.

  4. Dynamics of domain wall networks with junctions

    Avelino, P. P.; Oliveira, J. C. R. E.; Martins, C. J. A. P.; Menezes, J.; Menezes, R.

    2008-01-01

    We use a combination of analytic tools and an extensive set of the largest and most accurate three-dimensional field theory numerical simulations to study the dynamics of domain wall networks with junctions. We build upon our previous work and consider a class of models which, in the limit of large number N of coupled scalar fields, approaches the so-called ''ideal'' model (in terms of its potential to lead to network frustration). We consider values of N between N=2 and N=20, and a range of cosmological epochs, and we also compare this class of models with other toy models used in the past. In all cases we find compelling evidence for a gradual approach to scaling, strongly supporting our no-frustration conjecture. We also discuss the various possible types of junctions (including cases where there is a hierarchy of them) and their roles in the dynamics of the network. Finally, we provide a cosmological Zel'dovich-type bound on the energy scale of this kind of defect network: it must be lower than 10 keV.

  5. Concept and design of super junction devices

    Zhang, Bo; Zhang, Wentong; Qiao, Ming; Zhan, Zhenya; Li, Zhaoji

    2018-02-01

    The super junction (SJ) has been recognized as the " milestone” of the power MOSFET, which is the most important innovation concept of the voltage-sustaining layer (VSL). The basic structure of the SJ is a typical junction-type VSL (J-VSL) with the periodic N and P regions. However, the conventional VSL is a typical resistance-type VSL (R-VSL) with only an N or P region. It is a qualitative change of the VSL from the R-VSL to the J-VSL, introducing the bulk depletion to increase the doping concentration and optimize the bulk electric field of the SJ. This paper firstly summarizes the development of the SJ, and then the optimization theory of the SJ is discussed for both the vertical and the lateral devices, including the non-full depletion mode, the minimum specific on-resistance optimization method and the equivalent substrate model. The SJ concept breaks the conventional " silicon limit” relationship of R on∝V B 2.5, showing a quasi-linear relationship of R on∝V B 1.03.

  6. Virus interaction with the apical junctional complex.

    Gonzalez-Mariscal, Lorenza; Garay, Erika; Lechuga, Susana

    2009-01-01

    In order to infect pathogens must breach the epithelial barriers that separate the organism from the external environment or that cover the internal cavities and ducts of the body. Epithelia seal the passage through the paracellular pathway with the apical junctional complex integrated by tight and adherens junctions. In this review we describe how viruses like coxsackie, swine vesicular disease virus, adenovirus, reovirus, feline calcivirus, herpes viruses 1 and 2, pseudorabies, bovine herpes virus 1, poliovirus and hepatitis C use as cellular receptors integral proteins present at the AJC of epithelial cells. Interaction with these proteins contributes in a significant manner in defining the particular tropism of each virus. Besides these proteins, viruses exhibit a wide range of cellular co-receptors among which proteins present in the basolateral cell surface like integrins are often found. Therefore targeting proteins of the AJC constitutes a strategy that might allow viruses to bypass the physical barrier that blocks their access to receptors expressed on the basolateral surface of epithelial cells.

  7. Terbinafine inhibits gap junctional intercellular communication

    Lee, Ju Yeun, E-mail: whitewndus@naver.com [College of Pharmacy, Yonsei Institute of Pharmaceutical Sciences, Yonsei University, 85 Songdogwahak-ro, Yeonsu-gu, Incheon 21983 (Korea, Republic of); Yoon, Sei Mee, E-mail: sei_mee@naver.com [College of Pharmacy, Yonsei Institute of Pharmaceutical Sciences, Yonsei University, 85 Songdogwahak-ro, Yeonsu-gu, Incheon 21983 (Korea, Republic of); Department of Integrated OMICS for Biomedical Sciences, Yonsei University, 50 Yonsei-ro, Seodaemun-gu, Seoul 120-749 (Korea, Republic of); Choi, Eun Ju, E-mail: yureas@naver.com [College of Pharmacy, Yonsei Institute of Pharmaceutical Sciences, Yonsei University, 85 Songdogwahak-ro, Yeonsu-gu, Incheon 21983 (Korea, Republic of); Lee, Jinu, E-mail: jinulee@yonsei.ac.kr [College of Pharmacy, Yonsei Institute of Pharmaceutical Sciences, Yonsei University, 85 Songdogwahak-ro, Yeonsu-gu, Incheon 21983 (Korea, Republic of)

    2016-09-15

    Terbinafine is an antifungal agent that selectively inhibits fungal sterol synthesis by blocking squalene epoxidase. We evaluated the effect of terbinafine on gap junctional intercellular communication (GJIC). Fluorescence recovery after photobleaching (FRAP) and I-YFP GJIC assays revealed that terbinafine inhibits GJIC in a reversible and dose-dependent manner in FRT-Cx43 and LN215 cells. Treatment with terbinafine did not affect Cx43 phosphorylation status or intracellular Ca{sup 2+} concentration, well-known action mechanisms of various GJIC blockers. While a structurally related chemical, naftifine, attenuated GJIC, epigallocatechin gallate, another potent squalene epoxidase inhibitor with a different structure, did not. These results suggest that terbinafine inhibits GJIC with a so far unknown mechanism of action. - Highlights: • In vitro pharmacological studies were performed on FRT-Cx43 and LN215 cells. • Terbinafine inhibits gap junctional intercellular communication in both cell lines. • The inhibitory effect of terbinafine is reversible and dose-dependent. • Treatment of terbinafine does not alter Cx43 phosphorylation or cytosolic Ca{sup 2+} concentration. • Inhibition of squalene epoxidase is not involved in this new effect of terbinafine.

  8. Modeling Bloch oscillations in nanoscale Josephson junctions

    Vora, Heli; Kautz, R. L.; Nam, S. W.; Aumentado, J.

    2018-01-01

    Bloch oscillations in nanoscale Josephson junctions with a Coulomb charging energy comparable to the Josephson coupling energy are explored within the context of a model previously considered by Geigenmüller and Schön that includes Zener tunneling and treats quasiparticle tunneling as an explicit shot-noise process. The dynamics of the junction quasicharge are investigated numerically using both Monte Carlo and ensemble approaches to calculate voltage-current characteristics in the presence of microwaves. We examine in detail the origin of harmonic and subharmonic Bloch steps at dc biases I = (n/m)2ef induced by microwaves of frequency f and consider the optimum parameters for the observation of harmonic (m = 1) steps. We also demonstrate that the GS model allows a detailed semiquantitative fit to experimental voltage-current characteristics previously obtained at the Chalmers University of Technology, confirming and strengthening the interpretation of the observed microwave-induced steps in terms of Bloch oscillations. PMID:29577106

  9. Charge Transport Processes in Molecular Junctions

    Smith, Christopher Eugene

    Molecular electronics (ME) has evolved into a rich area of exploration that combines the fields of chemistry, materials, electronic engineering and computational modeling to explore the physics behind electronic conduction at the molecular level. Through studying charge transport properties of single molecules and nanoscale molecular materials the field has gained the potential to bring about new avenues for the miniaturization of electrical components where quantum phenomena are utilized to achieve solid state molecular device functionality. Molecular junctions are platforms that enable these studies and consist of a single molecule or a small group of molecules directly connected to electrodes. The work presented in this thesis has built upon the current understanding of the mechanisms of charge transport in ordered junctions using self-assembled monolayer (SAM) molecular thin films. Donor and acceptor compounds were synthesized and incorporated into SAMs grown on metal substrates then the transport properties were measured with conducting probe atomic force microscopy (CP-AFM). In addition to experimentally measured current-voltage (I-V) curves, the transport properties were addressed computationally and modeled theoretically. The key objectives of this project were to 1) investigate the impact of molecular structure on hole and electron charge transport, 2) understand the nature of the charge carriers and their structure-transport properties through long (chemically gated to modulate the transport. These results help advance our understanding of transport behavior in semiconducting molecular thin films, and open opportunities to engineer improved electronic functionality into molecular devices.

  10. Stress analysis of PCV nozzle junction

    Uchiyama, Shoichi; Oikawa, Tsuneo; Hoshino, Seizo

    1976-01-01

    Most of various pressure vessels comprise each one cylindrical shell and one or more nozzles. In this study, in order to analyze the stress in the structures of this type as minutely and exactly as possible, the program for stress analysis by the finite element method was made, which is required for the strength analysis for three-dimensional structures. Especially, the problem of the stress distribution around nozzle junctions was solved theoretically with the program. The program for the analysis developed in this study is provided with various functions, such as the input generator for cylindrical, conical and spherical shells, and plotter, and is very covenient. The accuracy of analysis is very good. The method of analysis and the calculation of the rigidity matrices for the deformation in plane and bending are explained. The result of the stress analysis around the nozzle junctions of a containment vessel with this program was in good agreement with experimental data and the result with SAP-4 code, therefore the propriety of the calculated result with this program was proved. Also calculations were carried out on three cases, namely a flat plate fixed at one end with distributed load, a cylinder fixed at one end with internal pressure, and an I-beam fixed at one end with concentrated load. The calculated results agreed well with theoretical solutions in all cases. (Kako, I.)

  11. Single Molecule Nanoelectrochemistry in Electrical Junctions.

    Nichols, Richard J; Higgins, Simon J

    2016-11-15

    It is now possible to reliably measure single molecule conductance in a wide variety of environments including organic liquids, ultrahigh vacuum, water, ionic liquids, and electrolytes. The most commonly used methods deploy scanning probe microscopes, mechanically formed break junctions, or lithographically formed nanogap contacts. Molecules are generally captured between a pair of facing electrodes, and the junction current response is measured as a function of bias voltage. Gating electrodes can also be added so that the electrostatic potential at the molecular bridge can be independently controlled by this third noncontacting electrode. This can also be achieved in an electrolytic environment using a four-electrode bipotentiostatic configuration, which allows independent electrode potential control of the two contacting electrodes. This is commonly realized using an electrochemical STM and enables single molecule electrical characterization as a function of electrode potential and redox state of the molecular bridge. This has emerged as a powerful tool in modern interfacial electrochemistry and nanoelectrochemistry for studying charge transport across single molecules as a function of electrode potential and the electrolytic environments. Such measurements are possible in electrolytes ranging from aqueous buffers to nonaqueous ionic liquids. In this Account, we illustrate a number of examples of single molecule electrical measurements under electrode potential control use a scanning tunneling microscope (STM) and demonstrate how these can help in the understanding of charge transport in single molecule junctions. Examples showing charge transport following phase coherent tunneling to incoherent charge hopping across redox active molecular bridges are shown. In the case of bipyridinium (or viologen) molecular wires, it is shown how electrochemical reduction leads to an increase of the single molecule conductance, which is controlled by the liquid electrochemical

  12. Josephson junctions in high-T/sub c/ superconductors

    Falco, C.M.; Lee, T.W.

    1981-01-14

    The invention includes a high T/sub c/ Josephson sperconducting junction as well as the method and apparatus which provides the junction by application of a closely controlled and monitored electrical discharge to a microbridge region connecting two portions of a superconducting film.

  13. Water and suspended sediment division at a stratified tidal junction

    Buschman, F.A.; Vegt, van der M.; Hoitink, A.J.F.; Hoekstra, P.

    2013-01-01

    [1] Tidal junctions play a crucial role in the transport of water, salt, and sediment through a delta distributary network. Water, salt and sediment are exchanged at tidal junctions, thereby influencing the transports in the connecting branches and the overall dynamics of the system. This paper

  14. Towards molecular electronics with large-area molecular junctions

    Akkerman, HB; Blom, PWM; de Leeuw, DM; de Boer, B

    2006-01-01

    Electronic transport through single molecules has been studied extensively by academic(1-8) and industrial(9,10) research groups. Discrete tunnel junctions, or molecular diodes, have been reported using scanning probes(11,12), break junctions(13,14), metallic crossbars(6) and nanopores(8,15). For

  15. Shapiro and parametric resonances in coupled Josephson junctions

    Gaafar, Ma A; Shukrinov, Yu M; Foda, A

    2012-01-01

    The effect of microwave irradiation on the phase dynamics of intrinsic Josephson junctions in high temperature superconductors is investigated. We compare the current-voltage characteristics for a stack of coupled Josephson junctions under external irradiation calculated in the framework of CCJJ and CCJJ+DC models.

  16. Dynamic Control of Tunneling Conductance in Ferroelectric Tunnel Junctions

    Zou Ya-Yi; Zhou Yan; Chew Khian-Hooi

    2013-01-01

    We investigate the dynamic characteristics of electric polarization P(t) in a ferroelectric junction under ac applied voltage and stress, and calculate the frequency response and the cut-off frequency f 0 , which provides a reference for the upper limit of the working frequency. Our study might be significant for sensor and memory applications of nanodevices based on ferroelectric junctions

  17. Gap junction protein connexin-43 interacts directly with microtubules

    Giepmans, B N; Verlaan, I; Hengeveld, T; Janssen, H; Calafat, J; Falk, M M; Moolenaar, W H

    2001-01-01

    Gap junctions are specialized cell-cell junctions that mediate intercellular communication. They are composed of connexin proteins, which form transmembrane channels for small molecules [1, 2]. The C-terminal tail of connexin-43 (Cx43), the most widely expressed connexin member, has been implicated

  18. Water and suspended sediment division at a stratified tidal junction

    Buschman, F.A.; Vegt, M. van der; Hoitink, A.J.F.; Hoekstra, P.

    2013-01-01

    Tidal junctions play a crucial role in the transport of water, salt, and sediment through a delta distributary network. Water, salt and sediment are exchanged at tidal junctions, thereby influencing the transports in the connecting branches and the overall dynamics of the system. This paper

  19. 75 FR 76294 - Radio Broadcasting Services: Pacific Junction, IA

    2010-12-08

    ... FEDERAL COMMUNICATIONS COMMISSION 47 CFR Part 73 [DA 10-2236; MB Docket No. 10-108] Radio Broadcasting Services: Pacific Junction, IA AGENCY: Federal Communications Commission. ACTION: Final rule. SUMMARY: The staff deletes FM Channel 299C2 at Pacific Junction, Iowa, because the record in this...

  20. Vortex dynamics in Josephson ladders with II-junctions

    Kornev, Victor K.; Klenov, N. V.; Oboznov, V.A.

    2004-01-01

    Both experimental and numerical studies of a self-frustrated triangular array of pi-junctions are reported. The array of SFS Josephson junctions shows a transition to the pi-state and self-frustration with a decrease in temperature. This manifests itself in a half-period shift of the bias critica...

  1. Effect of surface losses on soliton propagation in Josephson junctions

    Davidson, A.; Pedersen, Niels Falsig; Pagano, S.

    1986-01-01

    We have explored numerically the effects on soliton propagation of a third order damping term in the modified sine-Gordon equation. In Josephson tunnel junctions such a term corresponds physically to quasiparticle losses within the metal electrodes of the junction. We find that this loss term pla...

  2. Preparation of CN /Carbon Nanotube Intramolecular Junctions by ...

    NICO

    intramolecular junctions composed of CNx with a bamboo-like structure and empty hollow carbon nanotubes were observed, ... and excellent thermal and mechanical properties.1,2 In recent .... tion of hexane, and the other segment with a curved compart- ... by an arrow lies at the interface of the junction between 'b' and.

  3. Short chain molecular junctions: Charge transport versus dipole moment

    Ikram, I. Mohamed; Rabinal, M.K.

    2015-01-01

    Graphical abstract: - Highlights: • The role of dipole moment of organic molecules on molecular junctions has been studied. • Molecular junctions constituted using propargyl molecules of different dipole moments. • The electronic properties of the molecules were calculated using Gaussian software. • Junctions show varying rectification due to their varying dipole moment and orientation. - Abstract: The investigation of the influence of dipole moment of short chain organic molecules having three carbon atoms varying in end group on silicon surface was carried on. Here, we use three different molecules of propargyl series varying in dipole moment and its orientation to constitute molecular junctions. The charge transport mechanism in metal–molecules–semiconductor (MMS) junction obtained from current–voltage (I–V) characteristics shows the rectification behavior for two junctions whereas the other junction shows a weak rectification. The electronic properties of the molecules were calculated using Gaussian software package. The observed rectification behavior of these junctions is examined and found to be accounted to the orientation of dipole moment and electron cloud density distribution inside the molecules

  4. Triple junction polymer solar cells for photoelectrochemical water splitting

    Esiner, S.; Eersel, van H.; Wienk, M.M.; Janssen, R.A.J.

    2013-01-01

    A triple junction polymer solar cell in a novel 1 + 2 type configuration provides photoelectrochemical water splitting in its maximum power point at V ˜ 1.70 V with an estimated solar to hydrogen energy conversion efficiency of 3.1%. The triple junction cell consists of a wide bandgap front cell and

  5. Manufacturing P-N junctions in germanium bodies

    Hall, R.N.

    1980-01-01

    A method of producing p-n junctions in Ge so as to facilitate their use as radiation detectors involves forming a body of high purity p-type germanium, diffusing lithium deep into the body, in the absence of electrolytic processes, to form a junction between n-type and p-type germanium greater than 1 mm depth. (UK)

  6. The critical current of point symmetric Josephson tunnel junctions

    Monaco, Roberto

    2016-01-01

    Highlights: • We disclose some geometrical properties of the critical current field dependence that apply to a large class of Josephson junctions characterized by a point symmetric shape. • The developed theory is valid for any orientation of the applied magnetic field, therefore it allows the determine the consequences of field misalignment in the experimental setups. • We also address that the threshold curves of Josephson tunnel junctions with complex shapes can be expressed as a linear combination of the threshold curves of junctions with simpler point symmetric shapes. - Abstract: The physics of Josephson tunnel junctions drastically depends on their geometrical configurations. The shape of the junction determines the specific form of the magnetic-field dependence of its Josephson current. Here we address the magnetic diffraction patterns of specially shaped planar Josephson tunnel junctions in the presence of an in-plane magnetic field of arbitrary orientations. We focus on a wide ensemble of junctions whose shape is invariant under point reflection. We analyze the implications of this type of isometry and derive the threshold curves of junctions whose shape is the union or the relative complement of two point symmetric plane figures.

  7. Nonlocal Cooper pair splitting in a pSn-junction

    Veldhorst, M.; Brinkman, Alexander

    2010-01-01

    Perfect Cooper pair splitting is proposed, based on crossed Andreev reflection (CAR) in a p-type semiconductor-superconductor-n-type semiconductor (pSn) junction. The ideal splitting is caused by the energy filtering that is enforced by the band structure of the electrodes. The pSn junction is

  8. Fast temporal fluctuations in single-molecule junctions.

    Ochs, Roif; Secker, Daniel; Elbing, Mark; Mayor, Marcel; Weber, Heiko B

    2006-01-01

    The noise within the electrical current through single-molecule junctions is studied cryogenic temperature. The organic sample molecules were contacted with the mechanically controlled break-junction technique. The noise spectra refer to a where only few Lorentzian fluctuators occur in the conductance. The frequency dependence shows qualitative variations from sample to sample.

  9. Shunted-Josephson-junction model. II. The nonautonomous case

    Belykh, V. N.; Pedersen, Niels Falsig; Sørensen, O. H.

    1977-01-01

    The shunted-Josephson-junction model with a monochromatic ac current drive is discussed employing the qualitative methods of the theory of nonlinear oscillations. As in the preceding paper dealing with the autonomous junction, the model includes a phase-dependent conductance and a shunt capacitance....... The mathematical discussion makes use of the phase-space representation of the solutions to the differential equation. The behavior of the trajectories in phase space is described for different characteristic regions in parameter space and the associated features of the junction IV curve to be expected are pointed...... out. The main objective is to provide a qualitative understanding of the junction behavior, to clarify which kinds of properties may be derived from the shunted-junction model, and to specify the relative arrangement of the important domains in the parameter-space decomposition....

  10. Magnetic field behavior of current steps in long Josephson junctions

    Costabile, G.; Cucolo, A.M.; Pace, S.; Parmentier, R.D.; Savo, B.; Vaglio, R.

    1980-01-01

    The zero-field steps, or dc current singularities, in the current-voltage characteristics of long Josephson tunnel junctions, first reported by Chen et al., continue to attract research interest both because their study can provide fundamental information on the dynamics of fluxons in such junctions and because they are accompanied by the emission of microwave radiation from the junction, which may be exploitable in practical oscillator applications. The purpose of this paper is to report some experimental observations of the magnetic field behavior of the steps in junctions fabricated in our Laboratory and to offer a qualitative explanation for this behavior. Measurements have been made both for very long (L >> lambdasub(J)) and for slightly long (L approx. >= lambdasub(J)) junctions with a view toward comparing our results with those of other workers. (orig./WRI)

  11. Two coupled Josephson junctions: dc voltage controlled by biharmonic current

    Machura, L; Spiechowicz, J; Kostur, M; Łuczka, J

    2012-01-01

    We study transport properties of two Josephson junctions coupled by an external shunt resistance. One of the junctions (say, the first) is driven by an unbiased ac current consisting of two harmonics. The device can rectify the ac current yielding a dc voltage across the first junction. For some values of coupling strength, controlled by an external shunt resistance, a dc voltage across the second junction can be generated. By variation of system parameters such as the relative phase or frequency of two harmonics, one can conveniently manipulate both voltages with high efficiency, e.g. changing the dc voltages across the first and second junctions from positive to negative values and vice versa. (paper)

  12. Visualizing supercurrents in 0-{pi} ferromagnetic Josephson tunnel junctions

    Goldobin, Edward; Guerlich, Christian; Gaber, Tobias; Koelle, Dieter; Kleiner, Reinhold [Physikalisches Institut and Center for Collective Quantum Phenomena, Universitaet Tuebingen (Germany); Weides, Martin; Kohlstedt, Hermann [Institute of Solid State Physics, Reserch Center Juelich (Germany)

    2009-07-01

    So-called 0 and {pi} Josephson junctions can be treated as having positive and negative critical currents. This implies that the same phase shift applied to a Josephson junction causes counterflow of supercurrents in 0 and in {pi} junctions connected in parallel provided they are short in comparison with Josephson penetration depth {lambda}{sub J}. We have fabricated several 0, {pi}, 0-{pi}, 0-{pi}-0 and 20 x (0-{pi}-) planar superconductor-insulator-ferromagnet-superconductor Josephson junctions and studied the spatial supercurrent density distribution j{sub s}(x,y) across the junction area using low temperature scanning electron microscopy. At zero magnetic field we clearly see counterflow of the supercurrents in 0 and {pi} regions. The picture also changes consistently in the applied magnetic field.

  13. Response of high Tc superconducting Josephson junction to nuclear radiation

    Ding Honglin; Zhang Wanchang; Zhang Xiufeng

    1992-10-01

    The development of nuclear radiation detectors and research on high T c superconducting nuclear radiation detectors are introduced. The emphases are the principle of using thin-film and thick-film Josephson junctions (bridge junction) based on high T c YBCO superconductors to detect nuclear radiation, the fabrication of thin film and thick-film Josephson junction, and response of junction to low energy gamma-rays of 59.5 keV emitted from 241 Am and beta-rays of 546 keV. The results show that a detector for measuring nuclear radiation spectrum made of high T c superconducting thin-film or thick-film, especially, thick-film Josephson junction, certainly can be developed

  14. Spatially resolved detection of mutually locked Josephson junctions in arrays

    Keck, M.; Doderer, T.; Huebener, R.P.; Traeuble, T.; Dolata, R.; Weimann, T.; Niemeyer, J.

    1997-01-01

    Mutual locking due to the internal coupling in two-dimensional arrays of Josephson junctions was investigated. The appearance of Shapiro steps in the current versus voltage curve of a coupled on-chip detector junction is used to indicate coherent oscillations in the array. A highly coherent state is observed for some range of the array bias current. By scanning the array with a low-power electron beam, mutually locked junctions remain locked while the unlocked junctions generate a beam-induced additional voltage drop at the array. This imaging technique allows the detection of the nonlocked or weakly locked Josephson junctions in a (partially) locked array state. copyright 1997 American Institute of Physics

  15. Scattering theory of superconductive tunneling in quantum junctions

    Shumeiko, V.S.; Bratus', E.N.

    1997-01-01

    A consistent theory of superconductive tunneling in single-mode junctions within a scattering formulation of Bogolyubov-de Gennes quantum mechanics is presented. The dc Josephson effect and dc quasiparticle transport in the voltage-biased junctions are considered. Elastic quasiparticle scattering by the junction determines the equilibrium Josephson current. The origin of Andreev bound states in tunnel junctions and their role in equilibrium Josephson transport are discussed. In contrast, quasiparticle tunneling in voltage-biased junctions is determined by inelastic scattering. A general expression for inelastic scattering amplitudes is derived and the quasiparticle current is calculated at all voltages with emphasis on a discussion of the properties of sub gap tunnel current and the nature of subharmonic gap structure

  16. Functional Molecular Junctions Derived from Double Self-Assembled Monolayers.

    Seo, Sohyeon; Hwang, Eunhee; Cho, Yunhee; Lee, Junghyun; Lee, Hyoyoung

    2017-09-25

    Information processing using molecular junctions is becoming more important as devices are miniaturized to the nanoscale. Herein, we report functional molecular junctions derived from double self-assembled monolayers (SAMs) intercalated between soft graphene electrodes. Newly assembled molecular junctions are fabricated by placing a molecular SAM/(top) electrode on another molecular SAM/(bottom) electrode by using a contact-assembly technique. Double SAMs can provide tunneling conjugation across the van der Waals gap between the terminals of each monolayer and exhibit new electrical functions. Robust contact-assembled molecular junctions can act as platforms for the development of equivalent contact molecular junctions between top and bottom electrodes, which can be applied independently to different kinds of molecules to enhance either the structural complexity or the assembly properties of molecules. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. Surface-Enhanced Raman Scattering in Molecular Junctions.

    Iwane, Madoka; Fujii, Shintaro; Kiguchi, Manabu

    2017-08-18

    Surface-enhanced Raman scattering (SERS) is a surface-sensitive vibrational spectroscopy that allows Raman spectroscopy on a single molecular scale. Here, we present a review of SERS from molecular junctions, in which a single molecule or molecules are made to have contact from the top to the bottom of metal surfaces. The molecular junctions are nice platforms for SERS as well as transport measurement. Electronic characterization based on the transport measurements of molecular junctions has been extensively studied for the development of miniaturized electronic devices. Simultaneous SERS and transport measurement of the molecular junctions allow both structural (geometrical) and electronic information on the single molecule scale. The improvement of SERS measurement on molecular junctions open the door toward new nanoscience and nanotechnology in molecular electronics.

  18. Observation of supercurrent in graphene-based Josephson junction

    Wang, Libin; Li, Sen; Kang, Ning [Key Laboratory for the Physics and Chemistry of Nanodevices and Department of Electronics, Peking University, Beijing 100871 (China); Xu, Chuan; Ren, Wencai [Shenyang National Laboratory for Materials Science, Institute of Metal Research, Chinese Academy of Sciences, Shenyang 110016 (China)

    2015-07-01

    Josephson junctions with a normal metal region sandwiched between two superconductors (S) are known as superconductor- normal-superconductor (SNS) structures. It has attracted significant attention especially when changing the normal metal with graphene, which allow for high tunability with the gate voltage and to study the proximity effect of the massless Dirac fermions. Here we report our work on graphene-based Josephson junction with a new two dimensional superconductor crystal, which grown directly on graphene, as superconducting electrodes. At low temperature, we observer proximity effect induced supercurrent flowing through the junction. The temperature and the magnetic field dependences of the critical current characteristics of the junction are also studied. The critical current exhibits a Fraunhofer-type diffraction pattern against magnetic field. Our experiments provided a new route of fabrication of graphene-based Josephson junction.

  19. Mycobacterial UvrD1 is a Ku-dependent DNA helicase that plays a role in multiple DNA repair events, including double-strand break repair.

    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.

  20. DndEi Exhibits Helicase Activity Essential for DNA Phosphorothioate Modification and ATPase Activity Strongly Stimulated by DNA Substrate with a GAAC/GTTC Motif.

    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.

  1. Helicase-dependent isothermal amplification: a novel tool in the development of molecular-based analytical systems for rapid pathogen detection.

    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).

  2. Formation of a Trimeric Xpo1-Ran[GTP]-Ded1 Exportin Complex Modulates ATPase and Helicase Activities of Ded1.

    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.

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

    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.

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

    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.

  5. The helicase and RNaseIIIa domains of Arabidopsis Dicer-Like1 modulate catalytic parameters during MicroRNA biogenesis

    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.

  6. Cas3 is a single-stranded DNA nuclease and ATP-dependent helicase in the CRISPR/Cas immune system.

    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.

  7. The rem mutations in the ATP-binding groove of the Rad3/XPD helicase lead to Xeroderma pigmentosum-Cockayne syndrome-like phenotypes.

    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.

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

    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.

  9. Microstructure of Josephson junctions: Effect on supercurrent transport in YBCO grain boundary and barrier layer junctions

    Merkle, K.L.; Huang, Y.

    1998-01-01

    The electric transport of high-temperature superconductors, such as YBa 2 Cu 3 O 7-x (YBCO), can be strongly restricted by the presence of high-angle grain boundaries (GB). This weak-link behavior is governed by the macroscopic GB geometry and the microscopic grain boundary structure and composition at the atomic level. Whereas grain boundaries present a considerable impediment to high current applications of high T c materials, there is considerable commercial interest in exploiting the weak-link-nature of grain boundaries for the design of microelectronic devices, such as superconducting quantum interference devices (SQUIDs). The Josephson junctions which form the basis of this technology can also be formed by introducing artificial barriers into the superconductor. The authors have examined both types of Josephson junctions by EM techniques in an effort to understand the connection between microstructure/chemistry and electrical transport properties. This knowledge is a valuable resource for the design and production of improved devices

  10. RF assisted switching in magnetic Josephson junctions

    Caruso, R.; Massarotti, D.; Bolginov, V. V.; Ben Hamida, A.; Karelina, L. N.; Miano, A.; Vernik, I. V.; Tafuri, F.; Ryazanov, V. V.; Mukhanov, O. A.; Pepe, G. P.

    2018-04-01

    We test the effect of an external RF field on the switching processes of magnetic Josephson junctions (MJJs) suitable for the realization of fast, scalable cryogenic memories compatible with Single Flux Quantum logic. We show that the combined application of microwaves and magnetic field pulses can improve the performances of the device, increasing the separation between the critical current levels corresponding to logical "0" and "1." The enhancement of the current level separation can be as high as 80% using an optimal set of parameters. We demonstrate that external RF fields can be used as an additional tool to manipulate the memory states, and we expect that this approach may lead to the development of new methods of selecting MJJs and manipulating their states in memory arrays for various applications.

  11. Ultimately short ballistic vertical graphene Josephson junctions

    Lee, Gil-Ho; Kim, Sol; Jhi, Seung-Hoon; Lee, Hu-Jong

    2015-01-01

    Much efforts have been made for the realization of hybrid Josephson junctions incorporating various materials for the fundamental studies of exotic physical phenomena as well as the applications to superconducting quantum devices. Nonetheless, the efforts have been hindered by the diffusive nature of the conducting channels and interfaces. To overcome the obstacles, we vertically sandwiched a cleaved graphene monoatomic layer as the normal-conducting spacer between superconducting electrodes. The atomically thin single-crystalline graphene layer serves as an ultimately short conducting channel, with highly transparent interfaces with superconductors. In particular, we show the strong Josephson coupling reaching the theoretical limit, the convex-shaped temperature dependence of the Josephson critical current and the exceptionally skewed phase dependence of the Josephson current; all demonstrate the bona fide short and ballistic Josephson nature. This vertical stacking scheme for extremely thin transparent spacers would open a new pathway for exploring the exotic coherence phenomena occurring on an atomic scale. PMID:25635386

  12. Quasiparticle dynamics in superconducting tunnel junctions

    Kozorezov, A.G.; Brammertz, G.; Hijmering, R.A.; Wigmore, J.K.; Peacock, A.; Martin, D.; Verhoeve, P.; Golubov, A.A.; Rogalla, H.

    2006-01-01

    Superconducting Tunnel Junctions (STJs) used as single photon detectors possess extreme sensitivity and excellent resolving power. However, like many other cryogenic detectors they operate under extremely non-equilibrium conditions. In order to understand the physics of the non-equilibrium, non-stationary state, to interpret experimental data adequately, and to optimize the STJs unique performance, it is necessary to use a fully kinetic approach. We have developed the detailed theory of interactions between quasiparticles (qps) and the two types of phonons, sub-gap and pair-breaking, in STJ photon detectors. We discuss the results of extensive sets of experiments to study the non-equilibrium state in Al-based STJs. For the first time we are capable of explaining all available data systematically using a single set of parameters determined from STJ diagnostics and independent experiments

  13. Field-effect P-N junction

    Regan, William; Zettl, Alexander

    2015-05-05

    This disclosure provides systems, methods, and apparatus related to field-effect p-n junctions. In one aspect, a device includes an ohmic contact, a semiconductor layer disposed on the ohmic contact, at least one rectifying contact disposed on the semiconductor layer, a gate including a layer disposed on the at least one rectifying contact and the semiconductor layer and a gate contact disposed on the layer. A lateral width of the rectifying contact is less than a semiconductor depletion width of the semiconductor layer. The gate contact is electrically connected to the ohmic contact to create a self-gating feedback loop that is configured to maintain a gate electric field of the gate.

  14. Operating modes of superconducting tunnel junction device

    Maehata, Keisuke [Kyushu Univ., Fukuoka (Japan). Faculty of Engineering

    1998-07-01

    In the Electrotechnical Laboratory, an Nb type superconducting tunnel junction (STJ) device with 200 x 200 sq. micron in area and super high quality was manufactured. By using 55-fe source, response of this large area STJ to X-ray was measured. In this measurement, two action modes with different output wave height from front amplifier were observed. Then, in this study, current-voltage feature of the element in each action mode was analyzed to elucidate a mechanism to form such two action modes. The feature was analyzed by using first order approximate solution on cavity resonance mode of Sine-Gordon equation. From the analytical results, it could be supposed that direction and magnitude of effective magnetic field penetrating into jointed area changed by an induction current effect owing to impressing speed of the magnetic field, which brings two different current-voltage features to make possible to observe two action modes with different pulse wave height. (G.K.)

  15. Electronic transport properties of phenylacetylene molecular junctions

    Liu Wen; Cheng Jie; Yan Cui-Xia; Li Hai-Hong; Wang Yong-Juan; Liu De-Sheng

    2011-01-01

    Electronic transport properties of a kind of phenylacetylene compound— (4-mercaptophenyl)-phenylacetylene are calculated by the first-principles method in the framework of density functional theory and the nonequilibrium Green's function formalism. The molecular junction shows an obvious rectifying behaviour at a bias voltage larger than 1.0 V. The rectification effect is attributed to the asymmetry of the interface contacts. Moreover, at a bias voltage larger than 2.0 V, which is not referred to in a relevant experiment [Fang L, Park J Y, Ma H, Jen A K Y and Salmeron M 2007 Langmuir 23 11522], we find a negative differential resistance phenomenon. The negative differential resistance effect may originate from the change of the delocalization degree of the molecular orbitals induced by the bias. (condensed matter: electronic structure, electrical, magnetic, and optical properties)

  16. Terbinafine inhibits gap junctional intercellular communication.

    Lee, Ju Yeun; Yoon, Sei Mee; Choi, Eun Ju; Lee, Jinu

    2016-09-15

    Terbinafine is an antifungal agent that selectively inhibits fungal sterol synthesis by blocking squalene epoxidase. We evaluated the effect of terbinafine on gap junctional intercellular communication (GJIC). Fluorescence recovery after photobleaching (FRAP) and I-YFP GJIC assays revealed that terbinafine inhibits GJIC in a reversible and dose-dependent manner in FRT-Cx43 and LN215 cells. Treatment with terbinafine did not affect Cx43 phosphorylation status or intracellular Ca(2+) concentration, well-known action mechanisms of various GJIC blockers. While a structurally related chemical, naftifine, attenuated GJIC, epigallocatechin gallate, another potent squalene epoxidase inhibitor with a different structure, did not. These results suggest that terbinafine inhibits GJIC with a so far unknown mechanism of action. Copyright © 2016 Elsevier Inc. All rights reserved.

  17. Pentacene-based photodiode with Schottky junction

    Lee, Jiyoul; Hwang, D.K.; Park, C.H.; Kim, S.S.; Im, Seongil

    2004-01-01

    We have fabricated a metal/organic semiconductor Schottky photodiode based on Al/pentacene junction. Since the energy band gap of thin solid pentacene was determined to be 1.82 eV, as characterized by direct absorption spectroscopy, we measured spectral photoresponses on our Schottky photodiode in the monochromatic light illumination range of 325-650 nm applying a reverse bias of -2 V. The main features of photo-response spectra were found to shift from those of direct absorption spectra toward higher photon energies. It is because the direct absorption spectra mainly show exciton level peaks rather than the true highest occupied molecular orbital (HOMO)-lowest unoccupied molecular orbital (LUMO) gaps while the photo-response spectra clearly represents the true HOMO-LUMO gap. Our photo-response spectra reveal 1.97 eV as the HOMO-LUMO gap

  18. Intercellular coupling mediated by potassium accumulation in peg-and-socket junctions

    Vigmond, Edward J.; Bardakjian, Berj L.; Thuneberg, Lars

    2000-01-01

    Physiology, peg-and-socket junctions, smooth muscle, boundary element method, coupling, morphology......Physiology, peg-and-socket junctions, smooth muscle, boundary element method, coupling, morphology...

  19. NS3 from Hepatitis C Virus Strain JFH-1 Is an Unusually Robust Helicase That Is Primed To Bind and Unwind Viral RNA

    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

  20. Heated junction thermocouple level measurement apparatus

    Bevilacqua, F.; Burger, J.M.

    1984-01-01

    A liquid level sensing apparatus senses the level of liquid surrounding the apparatus. A plurality of axially spaced sensors are enclosed in a separator tube. The separator tube tends to collapse the level of a two-phase fluid within the separator tube into essentially a liquid phase and a gaseous phase where the collapsed level bears a relationship to the coolant inventory outside the separator tube. The level of the liquid phase is sensed by level sensing apparatus. The separator tube contains inlet-outlet ports near the top and bottom thereof to equalize the liquid level inside and outside the separator tube when the level fluctuates or the water within the separator tube flashes to steam. Each sensor is comprised of a heater, a heated thermocouple junction and an unheated thermocouple junction within an elongated heat conductive housing. The heated portion of housing is enclosed in a splash guard with inlet-outlet ports near the top and bottom to equalize the liquid level inside and outside the splash guardand to eliminate the spurious indications of liquid level change which may arise if water droplets contact the housing in the region of the heater. To prevent steam bubbles entrained in a two-phase fluid cross flow from entering the lateral inlet-outlet ports of the separator tube, the separator tube is enclosed in support tube which may in turn be enclosed in an otherwise unused control element assembly shroud. The lateral inlet-outlet ports of separator tube are axially offset from lateral inlet-outlet ports of support tube at least where support tube is subjected to cross flow. The shroud is open on the bottom and has lateral inlet-outlet ports to facilitate liquid level fluctuations to equalize inside and outside shroud

  1. High Tc Josephson Junctions, SQUIDs and magnetometers

    Clarke, J.

    1991-01-01

    There has recently been considerable progress in the state-of-the-art of high-T c magnetometers based on dc SQUIDs (Superconducting Quantum Interference Devices). This progress is due partly to the development of more manufacturable Josephson junctions, making SQUIDs easier to fabricate, and partly to the development of multiturn flux transformers that convert the high sensitivity of SQUIDs to magnetic flux to a correspondingly high sensitivity to magnetic field. Needless to say, today's high-T c SQUIDs are still considerably less sensitive than their low-T c counterparts, particularly at low frequencies (f) where their level of 1/f noise remains high. Nonetheless, the performance of the high-T c devices has now reached the point where they are adequate for a number of the less demanding applications; furthermore, as we shall see, at least modest improvements in performance are expected in the near future. In this article, the author outlines these various developments. This is far from a comprehensive review of the field, however, and, apart from Sec. 2, he describes largely his own work. He begins in Sec. 2 with an overview of the various types of Josephson junctions that have been investigated, and in Sec. 3, he describes some of the SQUIDs that have been tested, and assess their performance. Section 4 discuss the development of the multilayer structures essential for an interconnect technology, and, in particular, for crossovers and vias. Section 5 shows how this technology enables one to fabricate multiturn flux transformers which, in turn, can be coupled to SQUIDs to make magnetometers. The performance and possible future improvements in these magnetometers are assessed, and some applications mentioned

  2. Ligament augmentation for prevention of proximal junctional kyphosis and proximal junctional failure in adult spinal deformity.

    Safaee, Michael M; Deviren, Vedat; Dalle Ore, Cecilia; Scheer, Justin K; Lau, Darryl; Osorio, Joseph A; Nicholls, Fred; Ames, Christopher P

    2018-05-01

    OBJECTIVE Proximal junctional kyphosis (PJK) is a well-recognized, yet incompletely defined, complication of adult spinal deformity surgery. There is no standardized definition for PJK, but most studies describe PJK as an increase in the proximal junctional angle (PJA) of greater than 10°-20°. Ligament augmentation is a novel strategy for PJK reduction that provides strength to the upper instrumented vertebra (UIV) and adjacent segments while also reducing junctional stress at those levels. METHODS In this study, ligament augmentation was used in a consecutive series of adult spinal deformity patients at a single institution. Patient demographics, including age; sex; indication for surgery; revision surgery; surgical approach; and use of 3-column osteotomies, vertebroplasty, or hook fixation at the UIV, were collected. The PJA was measured preoperatively and at last follow-up using 36-inch radiographs. Data on change in PJA and need for revision surgery were collected. Univariate and multivariate analyses were performed to identify factors associated with change in PJA and proximal junctional failure (PJF), defined as PJK requiring surgical correction. RESULTS A total of 200 consecutive patients were included: 100 patients before implementation of ligament augmentation and 100 patients after implementation of this technique. The mean age of the ligament augmentation cohort was 66 years, and 67% of patients were women. Over half of these cases (51%) were revision surgeries, with 38% involving a combined anterior or lateral and posterior approach. The mean change in PJA was 6° in the ligament augmentation group compared with 14° in the control group (p historical cohort, ligament augmentation is associated with a significant decrease in PJK and PJF. These data support the implementation of ligament augmentation in surgery for adult spinal deformity, particularly in patients with a high risk of developing PJK and PJF.

  3. 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

    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...

  4. 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

    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

  5. Poster - Thur Eve - 57: Craniospinal irradiation with jagged-junction IMRT approach without beam edge matching for field junctions.

    Cao, F; Ramaseshan, R; Corns, R; Harrop, S; Nuraney, N; Steiner, P; Aldridge, S; Liu, M; Carolan, H; Agranovich, A; Karva, A

    2012-07-01

    Craniospinal irradiation were traditionally treated the central nervous system using two or three adjacent field sets. A intensity-modulated radiotherapy (IMRT) plan (Jagged-Junction IMRT) which overcomes problems associated with field junctions and beam edge matching, improves planning and treatment setup efficiencies with homogenous target dose distribution was developed. Jagged-Junction IMRT was retrospectively planned on three patients with prescription of 36 Gy in 20 fractions and compared to conventional treatment plans. Planning target volume (PTV) included the whole brain and spinal canal to the S3 vertebral level. The plan employed three field sets, each with a unique isocentre. One field set with seven fields treated the cranium. Two field sets treated the spine, each set using three fields. Fields from adjacent sets were overlapped and the optimization process smoothly integrated the dose inside the overlapped junction. For the Jagged-Junction IMRT plans vs conventional technique, average homogeneity index equaled 0.08±0.01 vs 0.12±0.02, and conformity number equaled 0.79±0.01 vs 0.47±0.12. The 95% isodose surface covered (99.5±0.3)% of the PTV vs (98.1±2.0)%. Both Jagged-Junction IMRT plans and the conventional plans had good sparing of the organs at risk. Jagged-Junction IMRT planning provided good dose homogeneity and conformity to the target while maintaining a low dose to the organs at risk. Jagged-Junction IMRT optimization smoothly distributed dose in the junction between field sets. Since there was no beam matching, this treatment technique is less likely to produce hot or cold spots at the junction in contrast to conventional techniques. © 2012 American Association of Physicists in Medicine.

  6. Advance of Mechanically Controllable Break Junction for Molecular Electronics.

    Wang, Lu; Wang, Ling; Zhang, Lei; Xiang, Dong

    2017-06-01

    Molecular electronics stands for the ultimate size of functional elements, keeping up with an unstoppable trend over the past few decades. As a vital component of molecular electronics, single molecular junctions have attracted significant attention from research groups all over the world. Due to its pronounced superiority, the mechanically controllable break junctions (MCBJ) technique has been widely applied to characterize the dynamic performance of single molecular junctions. This review presents a system analysis for single-molecule junctions and offers an overview of four test-beds for single-molecule junctions, thus offering more insight into the mechanisms of electron transport. We mainly focus on the development of state-of-the-art mechanically controlled break junctions. The three-terminal gated MCBJ approaches are introduced to manipulate the electron transport of molecules, and MCBJs are combined with characterization techniques. Additionally, applications of MCBJs and remarkable properties of single molecules are addressed. Finally, the challenges and perspective for the mechanically controllable break junctions technique are provided.

  7. Contour junctions defined by dynamic image deformations enhance perceptual transparency.

    Kawabe, Takahiro; Nishida, Shin'ya

    2017-11-01

    The majority of work on the perception of transparency has focused on static images with luminance-defined contour junctions, but recent work has shown that dynamic image sequences with dynamic image deformations also provide information about transparency. The present study demonstrates that when part of a static image is dynamically deformed, contour junctions at which deforming and nondeforming contours are connected facilitate the deformation-based perception of a transparent layer. We found that the impression of a transparent layer was stronger when a dynamically deforming area was adjacent to static nondeforming areas than when presented alone. When contour junctions were not formed at the dynamic-static boundaries, however, the impression of a transparent layer was not facilitated by the presence of static surrounding areas. The effect of the deformation-defined junctions was attenuated when the spatial pattern of luminance contrast at the junctions was inconsistent with the perceived transparency related to luminance contrast, while the effect did not change when the spatial luminance pattern was consistent with it. In addition, the results showed that contour completions across the junctions were required for the perception of a transparent layer. These results indicate that deformation-defined junctions that involve contour completion between deforming and nondeforming regions enhance the perception of a transparent layer, and that the deformation-based perceptual transparency can be promoted by the simultaneous presence of appropriately configured luminance and contrast-other features that can also by themselves produce the sensation of perceiving transparency.

  8. Flicker (1/f) noise in tunnel junction DC SQUIDS

    Koch, R.H.; Clarke, J.; Goubau, W.M.; Martinis, J.M.; Pegrum, C.M.; Van Harlingen, D.J.

    1983-01-01

    We have measured the spectral density of the 1/f voltage noise in current-biased resistively shunted Josephson tunnel junctions and dc SQUIDs. A theory in which fluctuations in the temperature give rise to fluctuations in the critical current and hence in the voltage predicts the magnitude of the noise quite accurately for junctions with areas of about 2 x 10 4 μm 2 , but significantly overestimates the noise for junctions with areas of about 6 μm 2 . DC SQUIDs fabricated from these two types of junctions exhibit substantially more 1/f voltage noise than would be predicted from a model in which the noise arises from critical current fluctuations in the junctions. This result was confirmed by an experiment involving two different bias current and flux modulation schemes, which demonstrated that the predominant 1/f voltage noise arises not from critical current fluctuations, but from some unknown source that can be regarded as an apparent 1/f flux noise. Measurements on five different configurations of dc SQUIDs fabricated with thin-film tunnel junctions and with widely varying areas, inductances, and junction capacitances show that the spectral density of the 1/f equivalent flux noise is roughtly constant, within a factor of three of (10 -10 /f)phi 2 0 Hz -1 . It is emphasized that 1/f flux noise may not be the predominant source of 1/f noise in SQUIDS fabricated with other technologies

  9. Ileocolic junction resection in dogs and cats: 18 cases.

    Fernandez, Yordan; Seth, Mayank; Murgia, Daniela; Puig, Jordi

    2017-12-01

    There is limited veterinary literature about dogs or cats with ileocolic junction resection and its long-term follow-up. To evaluate the long-term outcome in a cohort of dogs and cats that underwent resection of the ileocolic junction without extensive (≥50%) small or large bowel resection. Medical records of dogs and cats that had the ileocolic junction resected were reviewed. Follow-up information was obtained either by telephone interview or e-mail correspondence with the referring veterinary surgeons. Nine dogs and nine cats were included. The most common cause of ileocolic junction resection was intussusception in dogs (5/9) and neoplasia in cats (6/9). Two dogs with ileocolic junction lymphoma died postoperatively. Only 2 of 15 animals, for which long-term follow-up information was available, had soft stools. However, three dogs with suspected chronic enteropathy required long-term treatment with hypoallergenic diets alone or in combination with medical treatment to avoid the development of diarrhoea. Four of 6 cats with ileocolic junction neoplasia were euthanised as a consequence of progressive disease. Dogs and cats undergoing ileocolic junction resection and surviving the perioperative period may have a good long-term outcome with mild or absent clinical signs but long-term medical management may be required.

  10. Dilute Nitrides For 4-And 6- Junction Space Solar Cells

    Essig, S.; Stammler, E.; Ronsch, S.; Oliva, E.; Schachtner, M.; Siefer, G.; Bett, A. W.; Dimroth, F.

    2011-10-01

    According to simulations the efficiency of conventional, lattice-matched GaInP/GaInAs/Ge triple-junction space solar cells can be strongly increased by the incorporation of additional junctions. In this way the existing excess current of the Germanium bottom cell can be reduced and the voltage of the stack can be increased. In particular, the use of 1.0 eV materials like GaInNAs opens the door for solar cells with significantly improved conversion efficiency. We have investigated the material properties of GaInNAs grown by metal organic vapour phase epitaxy (MOVPE) and its impact on the quantum efficiency of solar cells. Furthermore we have developed a GaInNAs subcell with a bandgap energy of 1.0 eV and integrated it into a GaInP/GaInAs/GaInNAs/Ge 4-junction and a AlGaInP/GaInP/AlGaInAs/GaInAs/GaInNAs/Ge 6- junction space solar cell. The material quality of the dilute nitride junction limits the current density of these devices to 9.3 mA/cm2 (AM0). This is not sufficient for a 4-junction cell but may lead to current matched 6- junction devices in the future.

  11. Association of visceral adiposity with oesophageal and junctional adenocarcinomas.

    Beddy, P

    2012-02-01

    BACKGROUND: Obesity is associated with an increased incidence of oesophageal and oesophagogastric junction adenocarcinoma, in particular Siewert types I and II. This study compared abdominal fat composition in patients with oesophageal\\/junctional adenocarcinoma with that in patients with oesophageal squamous cell carcinoma and gastric adenocarcinoma, and in controls. METHOD: In total, 194 patients (110 with oesophageal\\/junctional adenocarcinoma, 38 with gastric adenocarcinoma and 46 with oesophageal squamous cell carcinoma) and 90 matched control subjects were recruited. The abdominal fat area was assessed using computed tomography (CT), and the total fat area (TFA), visceral fat area (VFA) and subcutaneous fat area (SFA) were calculated. RESULTS: Patients with oesophageal\\/junctional adenocarcinoma had significantly higher TFA and VFA values compared with controls (both P < 0.001), patients with gastric adenocarcinoma (P = 0.013 and P = 0.006 respectively) and patients with oesophageal squamous cell carcinoma (both P < 0.001). For junctional tumours, the highest TFA and VFA values were seen in patients with Siewert type I tumours (respectively P = 0.041 and P = 0.033 versus type III; P = 0.332 and P = 0.152 versus type II). CONCLUSION: Patients with oesophageal\\/junctional adenocarcinoma, in particular oesophageal and Siewert type I junctional tumours, have greater CT-defined visceral adiposity than patients with gastric adenocarcinoma or oesophageal squamous cell carcinoma, or controls.

  12. Solar cell junction temperature measurement of PV module

    Huang, B.J.

    2011-02-01

    The present study develops a simple non-destructive method to measure the solar cell junction temperature of PV module. The PV module was put in the environmental chamber with precise temperature control to keep the solar PV module as well as the cell junction in thermal equilibrium with the chamber. The open-circuit voltage of PV module Voc is then measured using a short pulse of solar irradiation provided by a solar simulator. Repeating the measurements at different environment temperature (40-80°C) and solar irradiation S (200-1000W/m2), the correlation between the open-circuit voltage Voc, the junction temperature Tj, and solar irradiation S is derived.The fundamental correlation of the PV module is utilized for on-site monitoring of solar cell junction temperature using the measured Voc and S at a short time instant with open circuit. The junction temperature Tj is then determined using the measured S and Voc through the fundamental correlation. The outdoor test results show that the junction temperature measured using the present method, Tjo, is more accurate. The maximum error using the average surface temperature Tave as the junction temperature is 4.8 °C underestimation; while the maximum error using the present method is 1.3 °C underestimation. © 2010 Elsevier Ltd.

  13. Electron-beam damaged high-temperature superconductor Josephson junctions

    Pauza, A.J.; Booij, W.E.; Herrmann, K.; Moore, D.F.; Blamire, M.G.; Rudman, D.A.; Vale, L.R.

    1997-01-01

    Results are presented on the fabrication and characterization of high critical temperature Josephson junctions in thin films of YBa 2 Cu 3 O 7-δ produced by the process of focused electron-beam irradiation using 350 keV electrons. The junctions so produced have uniform spatial current densities, can be described in terms of the resistive shunted junction model, and their current densities can be tailored for a given operating temperature. The physical properties of the damaged barrier can be described as a superconducting material of either reduced or zero critical temperature (T c ), which has a length of ∼15nm. The T c reduction is caused primarily by oxygen Frenkel defects in the Cu - O planes. The large beam currents used in the fabrication of the junctions mean that the extent of the barrier is limited by the incident electron-beam diameter, rather than by scattering within the film. The properties of the barrier can be calculated using a superconductor/normal/superconductor (SNS) junction model with no boundary resistance. From the SNS model, we can predict the scaling of the critical current resistance (I c R n ) product and gain insight into the factors controlling the junction properties, T c , and reproducibility. From the measured I c R n scaling data, we can predict the I c R n product of a junction at a given operating temperature with a given current density. I c R n products of ∼2mV can be achieved at 4.2 K. The reproducibility of several junctions in a number of samples can be characterized by the ratio of the maximum-to-minimum critical currents on the same substrate of less than 1.4. Stability over several months has been demonstrated at room and refrigerator temperatures (297 and 281 K) for junctions that have been initially over damaged and then annealed at temperatures ∼380K. (Abstract Truncated)

  14. The cranial-spinal junction in medulloblastoma: does it matter?

    Narayana, Ashwatha; Jeswani, Sam; Paulino, Arnold C.

    1999-01-01

    Purpose: Late effects of treatment in children and young adults with medulloblastoma can be influenced by the technique employed in radiating the craniospinal axis. The purpose of this study is to determine whether the placement of the cranial-spinal junction has an impact on dose to the cervical spinal cord and surrounding organs. Methods and Materials: Five patients underwent computed tomography (CT) simulation in the prone position for craniospinal irradiation. A dose of 36 Gy was prescribed to the entire neuraxis. The doses to the cervical spinal cord and surrounding organs were calculated using a cranial-spinal junction at the C1-C2 vertebral interspace (high junction) or at the lowest point in the neck, with exclusion of the shoulders in the lateral cranial fields (low junction).The volume of critical organs at risk, as well as dose to these structures using the cranial and spinal field(s) were outlined and calculated using the CMS FOCUS 3-dimensional treatment planning system. Results: The average dose to the cervical spinal cord was 11.9% higher than the prescribed dose with the low junction, and 6.7% higher with the high junction. However, doses to the thyroid gland, mandible, pharynx, and larynx were increased by an average of 29.6%, 75.8%, 70.6%, and 227.7%, respectively, by the use of the high junction compared to the low junction. Conclusion: A higher dose to the cervical spinal cord can be minimized by using a high junction. However, this would be at the cost of substantially increased doses to surrounding organs such as the thyroid gland, mandible, pharynx, and larynx. This can be critical in children and young adults, where hypothyroidism, mandibular hypoplasia, and development of second malignancies may be a late sequela of radiation therapy

  15. Mycobacterium smegmatis RqlH defines a novel clade of bacterial RecQ-like DNA helicases with ATP-dependent 3'-5' translocase and duplex unwinding activities.

    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.

  16. Internal resonances in periodically modulated long Josephson junctions

    Larsen, Britt Hvolbæk; Mygind, Jesper; Ustinov, Alexey V.

    1995-01-01

    Current-voltage (I-V) characteristics of long Josephson junctions with a periodic lattice of localized inhomogeneities are studied. The interaction between the moving fluxons and the inhomogeneities causes resonant steps in the IV-curve. Some of these steps are due to a synchronization to resonant...... Fiske modes in the sub-junctions formed between the inhomogeneities. The voltage positions of the resonant steps oscillate as function of the applied magnetic field with a period corresponding to the inclusion of one magnetic flux quantum, Φ0=h/2e, per sub-junction. A qualitative explanation that takes...

  17. Temporal correlations and structural memory effects in break junction measurements

    Magyarkuti, A.; Lauritzen, Kasper Primdal; Balogh, Zoltan Imre

    2017-01-01

    that correlations between the opening and subsequent closing traces may indicate structural memory effects in atomic-sized metallic and molecular junctions. Applying these methods on measured and simulated gold metallic contacts as a test system, we show that the surface diffusion induced flattening of the broken......-molecule junctions, we demonstrate pronounced contact memory effects and recovery of the molecule for junctions breaking before atomic chains are formed. However, if chains are pulled the random relaxation of the chain and molecule after rupture prevents opening-closing correlations....

  18. Double-well potential in annular Josephson junction

    Shaju, P.D.; Kuriakose, V.C.

    2004-01-01

    A double-well potential suitable for quantum-coherent vortex tunnelling can be created in an annular Josephson junction by inserting a microshort in the junction and by applying an in-plane dc magnetic field. Analysis shows that the intensity of the magnetic field determines the depth of the potential well and the strength of the microshort controls the potential barrier height while a dc bias across the junction tilts the potential well. At milli-Kelvin temperatures, the system is expected to behave as a quantum two-level system and may be useful in designing vortex qubits

  19. Steady-state properties of Josephson junctions with direct conductivity

    Zubkov, A.A.; Kupriyanov, M.Y.; Semenov, V.K.

    1981-01-01

    A new criterion for determining the kinetic inductance of Josephson junctions is introduced. The effects of temperature T, the critical temperatures of the superconducting electrodes T/sub c/1 and T/sub c/2, and the weak-link length on the kinetic inductance of ''dirty'' junctions with direct conductivity are analyzed within the framework of the Usadel equations. Numerical calculations show that both a large characteristic voltage and a nearly harmonic dependence of the current on the phase difference of the superconducting-electrode wave functions cannot be obtained by varying the junction parameters

  20. Amorphous molecular junctions produced by ion irradiation on carbon nanotubes

    Wang Zhenxia; Yu Liping; Zhang Wei; Ding Yinfeng; Li Yulan; Han Jiaguang; Zhu Zhiyuan; Xu Hongjie; He Guowei; Chen Yi; Hu Gang

    2004-01-01

    Experiments and molecular dynamics have demonstrated that electron irradiation could create molecular junctions between crossed single-wall carbon nanotubes. Recently molecular dynamics computation predicted that ion irradiation could also join single-walled carbon nanotubes. Employing carbon ion irradiation on multi-walled carbon nanotubes, we find that these nanotubes evolve into amorphous carbon nanowires, more importantly, during the process of which various molecular junctions of amorphous nanowires are formed by welding from crossed carbon nanotubes. It demonstrates that ion-beam irradiation could be an effective way not only for the welding of nanotubes but also for the formation of nanowire junctions