WorldWideScience

Sample records for by-product double-stranded rna

  1. Why double-stranded RNA resists condensation

    Energy Technology Data Exchange (ETDEWEB)

    Tolokh, Igor S.; Pabit, Suzette; Katz, Andrea M.; Chen, Yujie; Drozdetski, Aleksander; Baker, Nathan A.; Pollack, Lois; Onufriev, Alexey

    2014-09-15

    The addition of small amounts of multivalent cations to solutions containing double-stranded DNA leads to attraction between the negatively charged helices and eventually to condensation. Surprisingly, this effect is suppressed in double-stranded RNA, which carries the same charge as the DNA, but assumes a different double helical form. However, additional characterization of short (25 base-pairs) nucleic acid (NA) duplex structures by circular dichroism shows that measured differences in condensation are not solely determined by duplex helical geometry. Here we combine experiment, theory, and atomistic simulations to propose a mechanism that connects the observed variations in condensation of short NA duplexes with the spatial variation of cobalt hexammine (CoHex) binding at the NA duplex surface. The atomistic picture that emerged showed that CoHex distributions around the NA reveals two major NA-CoHex binding modes -- internal and external -- distinguished by the proximity of bound CoHex to the helical axis. Decreasing trends in experimentally observed condensation propensity of the four studied NA duplexes (from B-like form of homopolymeric DNA, to mixed sequence DNA, to DNA:RNA hybrid, to A-like RNA) are explained by the progressive decrease of a single quantity: the fraction of CoHex ions in the external binding mode. Thus, while NA condensation depends on a complex interplay between various structural and sequence features, our coupled experimental and theoretical results suggest a new model in which a single parameter connects the NA condensation propensity with geometry and sequence dependence of CoHex binding.

  2. High resolution atomic force microscopy of double-stranded RNA

    Science.gov (United States)

    Ares, Pablo; Fuentes-Perez, Maria Eugenia; Herrero-Galán, Elías; Valpuesta, José M.; Gil, Adriana; Gomez-Herrero, Julio; Moreno-Herrero, Fernando

    2016-06-01

    Double-stranded (ds) RNA mediates the suppression of specific gene expression, it is the genetic material of a number of viruses, and a key activator of the innate immune response against viral infections. The ever increasing list of roles played by dsRNA in the cell and its potential biotechnological applications over the last decade has raised an interest for the characterization of its mechanical properties and structure, and that includes approaches using Atomic Force Microscopy (AFM) and other single-molecule techniques. Recent reports have resolved the structure of dsDNA with AFM at unprecedented resolution. However, an equivalent study with dsRNA is still lacking. Here, we have visualized the double helix of dsRNA under near-physiological conditions and at sufficient resolution to resolve the A-form sub-helical pitch periodicity. We have employed different high-sensitive force-detection methods and obtained images with similar spatial resolution. Therefore, we show here that the limiting factors for high-resolution AFM imaging of soft materials in liquid medium are, rather than the imaging mode, the force between the tip and the sample and the sharpness of the tip apex.Double-stranded (ds) RNA mediates the suppression of specific gene expression, it is the genetic material of a number of viruses, and a key activator of the innate immune response against viral infections. The ever increasing list of roles played by dsRNA in the cell and its potential biotechnological applications over the last decade has raised an interest for the characterization of its mechanical properties and structure, and that includes approaches using Atomic Force Microscopy (AFM) and other single-molecule techniques. Recent reports have resolved the structure of dsDNA with AFM at unprecedented resolution. However, an equivalent study with dsRNA is still lacking. Here, we have visualized the double helix of dsRNA under near-physiological conditions and at sufficient resolution to

  3. DOUBLE-STRANDED-RNA MYCOVIRUSES IN MYCELIUM OF PLEUROTUS-OSTREATUS

    NARCIS (Netherlands)

    VANDERLENDE, TR; HARMSEN, MC; GO, SJ

    1995-01-01

    Mycelium of Pleurotus ostreatus var. florida with a decreased growth rate contained seven double-stranded RNA segments and isometrical virus particles with diameters of 24 and 30 nm. Mycelium with a normal growth rate lacked dsRNA. Protoclones from virus-containing mycelium contained one to seven of

  4. Baculovirus-mediated gene silencing in insect cells using intracellularly produced long double-stranded RNA

    NARCIS (Netherlands)

    Huang, Yi; Deng, F.; Hu, Z.H.; Vlak, J.M.; Wang, H.

    2007-01-01

    Double-stranded RNA-mediated interference (RNAi) has recently emerged as a powerful reverse genetics tool to silence gene expression in multiple organisms, including plants, nematodes and insects. In this study, DNA vectors capable of promoting the synthesis of long hairpin dsRNAs in vivo from a DNA

  5. Gene silencing: Double-stranded RNA mediated mRNA degradation and gene inactivation

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    The recent development of gene transfer approaches in plants and animals has revealed that transgene can undergo silencing after integration in the genome. Host genes can also be silenced as a consequence of the presence of a homologous transgene. More and more investigations have demonstrated that doublestranded RNA can silence genes by triggering degradation of homologous RNA in the cytoplasm and by directing methylation of homologous nuclear DNA sequences. Analyses of Arabidopsis mutants and plant viral suppressors of silencing are unraveling RNA-silencing mechanisms and are assessing the role of methylation in transcriptional and posttranscriptional gene silencing. This review will focus on double-stranded RNA mediated mRNA degradation and gene inactivation in plants.

  6. Understanding the similarity in thermophoresis between single- and double-stranded DNA or RNA.

    Science.gov (United States)

    Reichl, Maren; Herzog, Mario; Greiss, Ferdinand; Wolff, Manuel; Braun, Dieter

    2015-06-01

    Thermophoresis is the movement of molecules in a temperature gradient. For aqueous solutions its microscopic basis is debated. Understanding thermophoresis for this case is, however, important since it proved very useful to detect the binding affinity of biomolecules and since thermophoresis could have played an important role in early molecular evolution. Here we discuss why the thermophoresis of single- and double-stranded oligonucleotides - DNA and RNA - is surprisingly similar. This finding is understood by comparing the spherical capacitor model for single-stranded species with the case of a rod-shaped model for double-stranded oligonucleotides. The approach describes thermophoresis of DNA and RNA with fitted effective charges consistent with electrophoresis measurements and explains the similarity between single- and double-stranded species. We could not confirm the sign change for the thermophoresis of single- versus double-stranded DNA in crowded solutions containing polyethylene glycol [Y. T. Maeda, T. Tlusty, and A. Libchaber, Proc. Natl. Acad. Sci. USA 109, 17972 (2012)], but find a salt-independent offset while the Debye length dependence still satisfies the capacitor model. Overall, the analysis documents the continuous progress in the microscopic understanding of thermophoresis.

  7. Environmental fate of double-stranded RNA in agricultural soils.

    Directory of Open Access Journals (Sweden)

    Samuel Dubelman

    Full Text Available A laboratory soil degradation study was conducted to determine the biodegradation potential of a DvSnf7 dsRNA transcript derived from a Monsanto genetically modified (GM maize product that confers resistance to corn rootworm (CRW; Diabrotica spp.. This study provides new information to improve the environmental assessment of dsRNAs that become pesticidal through an RNAi process. Three agricultural soils differing in their physicochemical characteristics were obtained from the U.S., Illinois (IL; silt loam, Missouri (MO; loamy sand and North Dakota (ND; clay loam, and exposed to the target dsRNA by incorporating insect-protected maize biomass and purified (in vitro-transcribed DvSnf7 RNA into soil. The GM and control (non-GM maize materials were added to each soil and incubated at ca. 22 °C for 48 hours (h. Samples were collected at 12 time intervals during the incubation period, extracted, and analyzed using QuantiGene molecular analysis and insect bioassay methods. The DT50 (half-life values for DvSnf7 RNA in IL, MO, and ND soils were 19, 28, and 15 h based on QuantiGene, and 18, 29, and 14 h based on insect bioassay, respectively. Furthermore, the DT90 (time to 90% degradation values for DvSnf7 RNA in all three soils were <35 h. These results indicate that DvSnf7 RNA was degraded and biological activity was undetectable within approximately 2 days after application to soil, regardless of texture, pH, clay content and other soil differences. Furthermore, soil-incorporated DvSnf7 RNA was non-detectable in soil after 48 h, as measured by QuantiGene, at levels ranging more than two orders of magnitude (0.3, 1.5, 7.5 and 37.5 µg RNA/g soil. Results from this study indicate that the DvSnf7 dsRNA is unlikely to persist or accumulate in the environment. Furthermore, the rapid degradation of DvSnf7 dsRNA provides a basis to define relevant exposure scenarios for future RNA-based agricultural products.

  8. Sequence selective recognition of double-stranded RNA at physiologically relevant conditions using PNA-peptide conjugates.

    Science.gov (United States)

    Muse, Oluwatoyosi; Zengeya, Thomas; Mwaura, Juddy; Hnedzko, Dziyana; McGee, Dennis W; Grewer, Christof T; Rozners, Eriks

    2013-08-16

    Conjugation of short peptide nucleic acids (PNA) with tetralysine peptides strongly enhanced triple helical binding to RNA at physiologically relevant conditions. The PNA hexamers and heptamers carrying cationic nucleobase and tetralysine modifications displayed high binding affinity for complementary double-stranded RNA without compromising sequence selectivity. The PNA-peptide conjugates had unique preference for binding double-stranded RNA, while having little, if any, affinity for double-stranded DNA. The cationic PNAs were efficiently taken up by HEK293 cells, whereas little uptake was observed for unmodified PNA.

  9. Gene Silencing in Adult Aedes aegypti Mosquitoes Through Oral Delivery of Double-Stranded RNA

    Science.gov (United States)

    2012-01-01

    OR I GI N AL C ONTR I BUTI O N Gene silencing in adult Aedes aegypti mosquitoes through oral delivery of double-stranded RNA M. R. Coy1, N. D...we tested whether such an approach could be used in the yellow fever mosquito, Aedes aegypti . Using a non-specific dsRNA construct, we found that...adult Ae. aegypti ingested dsRNA through this method and that the ingested dsRNA can be recovered from the mosquitoes post-feeding. Through the feeding of

  10. Isolation and characterization of extrachromosomal double-stranded RNA elements in Xanthophyllomyces dendrorhous.

    Science.gov (United States)

    Baeza, Marcelo; Fernández-Lobato, María; Cifuentes, Víctor

    2012-01-01

    Double-stranded RNA (dsRNA) molecules are widely found in yeasts and filamentous fungi. It has been suggested that may play important roles in the evolution of eukaryote genomes and may be a valuable tool in yeast typing. The characterization of these extrachromosomal genetic elements is usually a laborious process, especially when trying to analyze a large number of samples. In this chapter, we describe a simple method to isolate dsRNA elements from yeasts using low amounts of starting material, and their application to different Xanthophyllomyces dendrorhous strains. Furthermore, the methodologies for enzymatic and hybridization characterizations, and quantification of relative dsRNA abundance are detailed.

  11. Detection of double-stranded RNA molecules and virus-like particles in different Mucor species.

    Science.gov (United States)

    Vágvölgyi, C; Magyar, K; Papp, T; Vastag, M; Ferenczy, L; Hornok, L; Fekete, C

    1998-02-01

    The presence of double-stranded RNA elements was examined in 123 strains representing 18 Mucor species. These genetic elements were found to be present in 6 strains: 1 M. aligarensis, 1 M. hiemalis, 2 M. corticolus, 1 M. mucedo and 1 M. ramannianus. Electrophoretic separation of the nucleic acids revealed 4 different RNA patterns, with 1 to 5 discrete dsRNA bands. The molecular weights corresponding to these bands were 1.42-4.15 x 10(6) D. Using electronmicroscopy, for the first time the presence of virus like particles in Mucor species has been revealed.

  12. Complete Genome Sequence of a Double-Stranded RNA Virus from Avocado

    Science.gov (United States)

    Villanueva, Francisco; Sabanadzovic, Sead; Valverde, Rodrigo A.

    2012-01-01

    A number of avocado (Persea americana) cultivars are known to contain high-molecular-weight double-stranded RNA (dsRNA) molecules for which a viral nature has been suggested, although sequence data are not available. Here we report the cloning and complete sequencing of a 13.5-kbp dsRNA virus isolated from avocado and show that it corresponds to the genome of a new species of the genus Endornavirus (family Endornaviridae), tentatively named Persea americana endornavirus (PaEV). PMID:22205720

  13. DMPD: TLR3: interferon induction by double-stranded RNA including poly(I:C). [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 18262679 TLR3: interferon induction by double-stranded RNA including poly(I:C). Mat...sumoto M, Seya T. Adv Drug Deliv Rev. 2008 Apr 29;60(7):805-12. Epub 2008 Jan 2. (.png) (.svg) (.html) (.csml) Show TLR3: inter...feron induction by double-stranded RNA including poly(I:C). PubmedID 18262679 Title TLR3: inter

  14. A non-phenol-chloroform extraction of double-stranded RNA from plant and fungal tissues.

    Science.gov (United States)

    Balijja, Alitukiriza; Kvarnheden, Anders; Turchetti, Tullio

    2008-09-01

    Double-stranded RNA (dsRNA) molecules of viruses are found in nature at a very high frequency. Their detection in plants and fungi has been carried out with difficulty due to the complicated dsRNA extraction techniques used commonly which includes phenol-chloroform extractions. In this study, an extraction method for isolation of dsRNA is described that is free of phenol and chloroform. A lysis buffer, containing beta-mercaptoethanol and polyvinylpolypyrrolidone (PVPP-40), was added to homogenised tissues and the subsequent supernatant was filtered through a cellulose CF-11 mini-column. DsRNA molecules were separated based on the differing affinity of nucleic acids for the cellulose CF-11 resin in 20% ethanol buffer. This easy, rapid and cheap technique has been successfully tested on fungi and plants containing different dsRNA virus molecules, indicating the possibility of a wide use of the method.

  15. Binding by TRBP-dsRBD2 Does Not Induce Bending of Double-Stranded RNA.

    Science.gov (United States)

    Acevedo, Roderico; Evans, Declan; Penrod, Katheryn A; Showalter, Scott A

    2016-06-21

    Protein-nucleic acid interactions are central to a variety of biological processes, many of which involve large-scale conformational changes that lead to bending of the nucleic acid helix. Here, we focus on the nonsequence-specific protein TRBP, whose double-stranded RNA-binding domains (dsRBDs) interact with the A-form geometry of double-stranded RNA (dsRNA). Crystal structures of dsRBD-dsRNA interactions suggest that the dsRNA helix must bend in such a way that its major groove expands to conform to the dsRBD's binding surface. We show through isothermal titration calorimetry experiments that dsRBD2 of TRBP binds dsRNA with a temperature-independent observed binding affinity (KD ∼500 nM). Furthermore, a near-zero observed heat capacity change (ΔCp = 70 ± 40 cal·mol(-1)·K(-1)) suggests that large-scale conformational changes do not occur upon binding. This result is bolstered by molecular-dynamics simulations in which dsRBD-dsRNA interactions generate only modest bending of the RNA along its helical axis. Overall, these results suggest that this particular dsRBD-dsRNA interaction produces little to no change in the A-form geometry of dsRNA in solution. These results further support our previous hypothesis, based on extensive gel-shift assays, that TRBP preferentially binds to sites of nearly ideal A-form structure while being excluded from sites of local deformation in the RNA helical structure. The implications of this mechanism for efficient micro-RNA processing will be discussed.

  16. Double-Stranded-RNA-Activated Protein Kinase PKR Enhances Transcriptional Activation by Tumor Suppressor p53

    OpenAIRE

    1999-01-01

    The tumor suppressor p53 plays a key role in inducing G1 arrest and apoptosis following DNA damage. The double-stranded-RNA-activated protein PKR is a serine/threonine interferon (IFN)-inducible kinase which plays an important role in regulation of gene expression at both transcriptional and translational levels. Since a cross talk between IFN-inducible proteins and p53 had already been established, we investigated whether and how p53 function was modulated by PKR. We analyzed p53 function in...

  17. Rapid isolation of mycoviral double-stranded RNA from Botrytis cinerea and Saccharomyces cerevisiae

    Directory of Open Access Journals (Sweden)

    Sepúlveda Felipe

    2011-01-01

    Full Text Available Abstract Background In most of the infected fungi, the mycoviruses are latent or cryptic, the infected fungus does not show disease symptoms, and it is phenotypically identical to a non-infected strain of the same species. Because of these properties, the initial stage in the search for fungi infected with mycoviruses is the detection of their viral genome, which in most of the described cases corresponds to double-stranded RNA (dsRNA. So to analyze a large number of fungal isolates it is necessary to have a simple and rapid method to detect dsRNA. Results A rapid method to isolate dsRNA from a virus-infected filamentous fungus, Botrytis cinerea, and from a killer strain of Saccharomyces cerevisiae using commercial minicolumns packed with CF11 cellulose was developed. In addition to being a rapid method, it allows to use small quantities of yeasts or mycelium as starting material, being obtained sufficient dsRNA quantity that can later be analyzed by agarose gel electrophoresis, treated with enzymes for its partial characterization, amplified by RT-PCR and cloned in appropriate vectors for further sequencing. Conclusions The method yields high quality dsRNA, free from DNA and ssRNA. The use of nucleases to degrade the DNA or the ssRNA is not required, and it can be used to isolate dsRNA from any type of fungi or any biological sample that contains dsRNA.

  18. Codelivery of zoledronic acid and double-stranded RNA from core-shell nanoparticles

    Directory of Open Access Journals (Sweden)

    Yan W

    2013-01-01

    Full Text Available Li Chen,1 Yunfei Ding,2 Yongzhong Wang,3 Xingrong Liu,2 RJ Babu,1 WR Ravis,1 Weili Yan21Department of Pharmaceutical Sciences, Harrison School of Pharmacy, Auburn University, Auburn, AL, USA; 2Department of Pharmaceutical Sciences, College of Life Sciences and Engineering, Southwest Jiaotong University, Chengdu, China; 3School of Life Sciences, Anhui University, Hefei, ChinaBackground: Zoledronic acid, an inhibitor of osteoclast-mediated bone resorption, has been shown to have both direct and indirect antitumor activity. However, its use in extraskeletal malignancy is limited due to rapid uptake and accumulation within bone. Polyinosinic acid-polycytidylic acid [poly (I:C] is a synthetic double-stranded RNA with direct antitumor cytotoxicity if it can be delivered to tumor cells intracellularly.Methods: Cationic lipid-coated calcium phosphate nanoparticles (LCP were developed to enable intracellular codelivery of zoledronic acid and poly (I:C. LCP codelivering zoledronic acid and poly (I:C were prepared using an ethanol injection method. Briefly, the ethanol solution of lipids was rapidly injected into newly formed calcium phosphate crystals containing poly (I:C and zoledronic acid, and the mixture was then sonicated briefly to form LCP. The LCP were fully characterized for mean diameter size and zeta potential, efficiency in loading zoledronic acid, cytotoxic effect in a B16BL6 melanoma cell line in vitro, and antitumor effect in B16BL6 melanoma-bearing mice.Results: LCP with a mean diameter around 200 nm and a narrow size distribution (polydispersity index 0.17 and high zoledronic acid encapsulation efficiency (94% were achieved. LCP loaded with zoledronic acid and poly (I:C had significantly greater antitumor activity than the free drugs in the B16BL6 melanoma cell line (P < 0.05. Furthermore, codelivery of zoledronic acid and poly (I:C by LCP had higher cytotoxicity than delivering poly (I:C alone by LCP (P < 0.05, indicating a synergism

  19. MDA5 Detects the Double-Stranded RNA Replicative Form in Picornavirus-Infected Cells

    Directory of Open Access Journals (Sweden)

    Qian Feng

    2012-11-01

    Full Text Available RIG-I and MDA5 are cytosolic RNA sensors that play a critical role in innate antiviral responses. Major advances have been made in identifying RIG-I ligands, but our knowledge of the ligands for MDA5 remains restricted to data from transfection experiments mostly using poly(I:C, a synthetic dsRNA mimic. Here, we dissected the IFN-α/β-stimulatory activity of different viral RNA species produced during picornavirus infection, both by RNA transfection and in infected cells in which specific steps of viral RNA replication were inhibited. Our results show that the incoming genomic plus-strand RNA does not activate MDA5, but minus-strand RNA synthesis and production of the 7.5 kbp replicative form trigger a strong IFN-α/β response. IFN-α/β production does not rely on plus-strand RNA synthesis and thus generation of the partially double-stranded replicative intermediate. This study reports MDA5 activation by a natural RNA ligand under physiological conditions.

  20. Double-stranded RNA-activated protein kinase PKR of fishes and amphibians: Varying the number of double-stranded RNA binding domains and lineage-specific duplications

    Directory of Open Access Journals (Sweden)

    Dever Thomas E

    2008-03-01

    Full Text Available Abstract Background Double-stranded (ds RNA, generated during viral infection, binds and activates the mammalian anti-viral protein kinase PKR, which phosphorylates the translation initiation factor eIF2α leading to the general inhibition of protein synthesis. Although PKR-like activity has been described in fish cells, the responsible enzymes eluded molecular characterization until the recent discovery of goldfish and zebrafish PKZ, which contain Z-DNA-binding domains instead of dsRNA-binding domains (dsRBDs. Fish and amphibian PKR genes have not been described so far. Results Here we report the cloning and identification of 13 PKR genes from 8 teleost fish and amphibian species, including zebrafish, demonstrating the coexistence of PKR and PKZ in this latter species. Analyses of their genomic organization revealed up to three tandemly arrayed PKR genes, which are arranged in head-to-tail orientation. At least five duplications occurred independently in fish and amphibian lineages. Phylogenetic analyses reveal that the kinase domains of fish PKR genes are more closely related to those of fish PKZ than to the PKR kinase domains of other vertebrate species. The duplication leading to fish PKR and PKZ genes occurred early during teleost fish evolution after the divergence of the tetrapod lineage. While two dsRBDs are found in mammalian and amphibian PKR, one, two or three dsRBDs are present in fish PKR. In zebrafish, both PKR and PKZ were strongly upregulated after immunostimulation with some tissue-specific expression differences. Using genetic and biochemical assays we demonstrate that both zebrafish PKR and PKZ can phosphorylate eIF2α in yeast. Conclusion Considering the important role for PKR in host defense against viruses, the independent duplication and fixation of PKR genes in different lineages probably provided selective advantages by leading to the recognition of an extended spectrum of viral nucleic acid structures, including both dsRNA

  1. Synthesis of double-stranded RNA in a virus-enriched fraction from Agaricus bisporus

    Energy Technology Data Exchange (ETDEWEB)

    Sriskantha, A.; Wach, P.; Schlagnhaufer, B.; Romaine, C.P.

    1986-03-01

    Partially purified virus preparations from sporophores of Agaricus bisporus affected with LaFrance disease had up to a 15-fold-higher RNA-dependent RNA polymerase activity than did comparable preparations from health sporophores. Enzyme activity was dependent upon the presence of Mg/sup 2 +/ and the four nucleoside triphosphates and was insensitive to actinomycin D, ..cap alpha..-amanitin, and rifampin. The /sup 3/H-labeled enzyme reaction products were double-stranded RNA (dsRNA) as indicated by CF-11 cellulose column chromatography and by their ionic-strength-dependent sensitivity to hydrolysis by RNase A. The principal dsRNA products had estimated molecular weights of 4.3 /times/ 10/sup 6/ and 1.4 /times/ 10/sup 6/. Cs/sub 2/SO/sub 4/ equilibrium centrifugation of the virus preparation resolved a single peak of RNA polymerase activity that banded with a 35-nm spherical virus particle containing dsRNAs with molecular weights of 4.3 /times/ 10/sup 6/ and 1.4 /times/ 10/sup 6/. The data suggest that the RNA-dependent RNA polymerase associated with the 35-nm spherical virus is a replicase which catalyzes the synthesis of the genomic dsRNAs.

  2. Direct transfer of synthetic double-stranded RNA into protoplasts of Arabidopsis thaliana.

    Science.gov (United States)

    Jung, Ha-Il; Zhai, Zhiyang; Vatamaniuk, Olena K

    2011-01-01

    Double-stranded (ds) RNA interference (RNAi) is widely used as a reverse genetic approach for functional analysis of plant genes. Constitutive or transient RNAi effects in plants have been achieved via generating stable transformants expressing dsRNAs or artificial microRNAs (amiRNAs) in planta or by viral-induced gene silencing (VIGS). Although these tools provide outstanding resources for functional genomics, they require generation of vectors expressing dsRNAs or amiRNAs against targeted genes, transformation and propagation of transformed plants, or maintenance of multiple VIGS lines and thus impose time, labor, and space requirements. As we showed recently, these limitations can be circumvented by inducing RNAi effects in protoplasts via transfecting them with in vitro-synthesized dsRNAs. In this chapter we detail the procedure for transient gene silencing in protoplasts using synthetic dsRNAs and provide examples of approaches for subsequent functional analyses.

  3. Gene silencing in tick cell lines using small interfering or long double-stranded RNA.

    Science.gov (United States)

    Barry, Gerald; Alberdi, Pilar; Schnettler, Esther; Weisheit, Sabine; Kohl, Alain; Fazakerley, John K; Bell-Sakyi, Lesley

    2013-03-01

    Gene silencing by RNA interference (RNAi) is an important research tool in many areas of biology. To effectively harness the power of this technique in order to explore tick functional genomics and tick-microorganism interactions, optimised parameters for RNAi-mediated gene silencing in tick cells need to be established. Ten cell lines from four economically important ixodid tick genera (Amblyomma, Hyalomma, Ixodes and Rhipicephalus including the sub-species Boophilus) were used to examine key parameters including small interfering RNA (siRNA), double stranded RNA (dsRNA), transfection reagent and incubation time for silencing virus reporter and endogenous tick genes. Transfection reagents were essential for the uptake of siRNA whereas long dsRNA alone was taken up by most tick cell lines. Significant virus reporter protein knockdown was achieved using either siRNA or dsRNA in all the cell lines tested. Optimum conditions varied according to the cell line. Consistency between replicates and duration of incubation with dsRNA were addressed for two Ixodes scapularis cell lines; IDE8 supported more consistent and effective silencing of the endogenous gene subolesin than ISE6, and highly significant knockdown of the endogenous gene 2I1F6 in IDE8 cells was achieved within 48 h incubation with dsRNA. In summary, this study shows that gene silencing by RNAi in tick cell lines is generally more efficient with dsRNA than with siRNA but results vary between cell lines and optimal parameters need to be determined for each experimental system.

  4. Use of microalgae Chlamydomonas reinhardtii for production of double-stranded RNA against shrimp virus

    Directory of Open Access Journals (Sweden)

    Parinyachat Somchai

    2016-05-01

    Full Text Available RNA interference has been proposed to be a promising tool for combating shrimp viruses. Antiviral double-stranded (dsRNA has been mostly produced in Escherichia coli-expression system because of its high efficiency and inexpensive operations. However, overusing the bacteria may raise concerns regarding public health and environmental contamination, and seeking for a new dsRNA production platform would be alternative for future molecular farming. In this study, we exploited the green microalgae Chlamydomonas reinhardtii to produce dsRNA targeting the lethal shrimp yellow head virus (YHV. The expression plasmid pSL18 for C. reinhardtii was constructed to contain YHV-specific hairpin RNA expression cassette, and the successful assembly of pSL18-YHV was confirmed by PCR and enzymatic digestions. Glass bead method was employed for transformation of C. reinhardtii nuclear genome with pSL18-YHV. Microalgal expression of dsRNA-YHV, approximately 45 ng from 100-mL culture, was detected by qRT-PCR. Oral feeding experiment on postlarval shrimp revealed that the formulated feed with C. reinhardtii expressing dsRNA-YHV, at the ratio of 1 × 108 transformants per gram feed, improved 22% survival rate after YHV challenge. The present study suggests that C. reinhardtii can be bioengineered to produce viral-specific dsRNA for shrimp viral disease control, and the developed qRT-PCR could detect microalgal dsRNA with detection limit of subpicogram.

  5. Double strand RNA delivery system for plant-sap-feeding insects.

    Science.gov (United States)

    Ghosh, Saikat Kumar B; Hunter, Wayne B; Park, Alexis L; Gundersen-Rindal, Dawn E

    2017-01-01

    Double-stranded RNA (dsRNA)-mediated gene silencing, also known as RNA interference (RNAi), has been a breakthrough technology for functional genomic studies and represents a potential tool for the management of insect pests. Since the inception of RNAi numerous studies documented successful introduction of exogenously synthesized dsRNA or siRNA into an organism triggering highly efficient gene silencing through the degradation of endogenous RNA homologous to the presented siRNA. Managing hemipteran insect pests, especially Halyomorpha halys (Stål) (Heteroptera: Pentatomidae), the brown marmorated stink bug (BMSB), is critical to food productivity. BMSB was recently introduced into North America where it is both an invasive agricultural pest of high value specialty, row, and staple crops, as well as an indoor nuisance pest. RNAi technology may serve as a viable tool to manage this voracious pest, but delivery of dsRNA to piercing-sucking insects has posed a tremendous challenge. Effective and practical use of RNAi as molecular biopesticides for biocontrol of insects like BMSB in the environment requires that dsRNAs be delivered in vivo through ingestion. Therefore, the key challenge for molecular biologists in developing insect-specific molecular biopesticides is to find effective and reliable methods for practical delivery of stable dsRNAs such as through oral ingestion. Here demonstrated is a reliable delivery system of effective insect-specific dsRNAs through oral feeding through a new delivery system to induce a significant decrease in expression of targeted genes such as JHAMT and Vg. This state-of-the-art delivery method overcomes environmental delivery challenges so that RNAi is induced through insect-specific dsRNAs orally delivered to hemipteran and other insect pests.

  6. Two Novel Relative Double-Stranded RNA Mycoviruses Infecting Fusarium poae Strain SX63

    Directory of Open Access Journals (Sweden)

    Luan Wang

    2016-04-01

    Full Text Available Two novel double-stranded RNA (dsRNA mycoviruses, termed Fusarium poae dsRNA virus 2 (FpV2 and Fusarium poae dsRNA virus 3 (FpV3, were isolated from the plant pathogenic fungus, Fusarium poae strain SX63, and molecularly characterized. FpV2 and FpV3, with respective genome sequences of 9518 and 9419 base pairs (bps, are both predicted to contain two discontinuous open reading frames (ORFs, ORF1 and ORF2. A hypothetical polypeptide (P1 and a RNA-dependent RNA polymerase (RdRp are encoded by ORF1 and ORF2, respectively. Phytoreo_S7 domain (pfam07236 homologs were detected downstream of the RdRp domain (RdRp_4; pfam02123 of the ORF2-coded proteins of both FpV2 and FpV3. The same shifty heptamers (GGAAAAC were both found immediately before the stop codon UAG of ORF1 in FpV2 and FpV3, which could mediate programmed –1 ribosomal frameshifting (–1 PRF. Phylogenetic analysis based on RdRp sequences clearly place FpV2 and FpV3 in a taxonomically unassigned dsRNA mycovirus group. Together, with a comparison of genome organization, a new taxonomic family termed Fusagraviridae is proposed to be created to include FpV2- and FpV3-related dsRNA mycoviruses, within which FpV2 and FpV3 would represent two distinct virus species.

  7. Double strand RNA delivery system for plant-sap-feeding insects

    Science.gov (United States)

    Ghosh, Saikat Kumar B.; Hunter, Wayne B.; Park, Alexis L.; Gundersen-Rindal, Dawn E.

    2017-01-01

    Double-stranded RNA (dsRNA)-mediated gene silencing, also known as RNA interference (RNAi), has been a breakthrough technology for functional genomic studies and represents a potential tool for the management of insect pests. Since the inception of RNAi numerous studies documented successful introduction of exogenously synthesized dsRNA or siRNA into an organism triggering highly efficient gene silencing through the degradation of endogenous RNA homologous to the presented siRNA. Managing hemipteran insect pests, especially Halyomorpha halys (Stål) (Heteroptera: Pentatomidae), the brown marmorated stink bug (BMSB), is critical to food productivity. BMSB was recently introduced into North America where it is both an invasive agricultural pest of high value specialty, row, and staple crops, as well as an indoor nuisance pest. RNAi technology may serve as a viable tool to manage this voracious pest, but delivery of dsRNA to piercing-sucking insects has posed a tremendous challenge. Effective and practical use of RNAi as molecular biopesticides for biocontrol of insects like BMSB in the environment requires that dsRNAs be delivered in vivo through ingestion. Therefore, the key challenge for molecular biologists in developing insect-specific molecular biopesticides is to find effective and reliable methods for practical delivery of stable dsRNAs such as through oral ingestion. Here demonstrated is a reliable delivery system of effective insect-specific dsRNAs through oral feeding through a new delivery system to induce a significant decrease in expression of targeted genes such as JHAMT and Vg. This state-of-the-art delivery method overcomes environmental delivery challenges so that RNAi is induced through insect-specific dsRNAs orally delivered to hemipteran and other insect pests. PMID:28182760

  8. Double-stranded RNA transcribed from vector-based oligodeoxynucleotide acts as transcription factor decoy

    Energy Technology Data Exchange (ETDEWEB)

    Xiao, Xiao [State Key Laboratory of Cancer Biology and Xijing Hospital of Digestive Diseases, Xijing Hospital, Fourth Military Medical University, Xi’an 710032, Shaanxi Province (China); Gang, Yi [State Key Laboratory of Cancer Biology and Xijing Hospital of Digestive Diseases, Xijing Hospital, Fourth Military Medical University, Xi’an 710032, Shaanxi Province (China); Department of Infectious Diseases, Tangdu Hospital, Fourth Military Medical University, Xi’an 710038, Shaanxi Province (China); Wang, Honghong [No. 518 Hospital of Chinese People’s Liberation Army, Xi’an 710043, Shaanxi Province (China); Wang, Jiayin [The Genome Institute, Washington University in St. Louis, St. Louis, MO 63108 (United States); Zhao, Lina [Department of Radiation Oncology, Xijing Hospital, Fourth Military Medical University, Xi’an 710032, Shaanxi Province (China); Xu, Li, E-mail: lxuhelen@163.com [State Key Laboratory of Cancer Biology and Xijing Hospital of Digestive Diseases, Xijing Hospital, Fourth Military Medical University, Xi’an 710032, Shaanxi Province (China); Liu, Zhiguo, E-mail: liuzhiguo@fmmu.edu.cn [State Key Laboratory of Cancer Biology and Xijing Hospital of Digestive Diseases, Xijing Hospital, Fourth Military Medical University, Xi’an 710032, Shaanxi Province (China)

    2015-02-06

    Highlights: • A shRNA vector based transcription factor decoy, VB-ODN, was designed. • VB-ODN for NF-κB inhibited cell viability in HEK293 cells. • VB-ODN inhibited expression of downstream genes of target transcription factors. • VB-ODN may enhance nuclear entry ratio for its feasibility of virus production. - Abstract: In this study, we designed a short hairpin RNA vector-based oligodeoxynucleotide (VB-ODN) carrying transcription factor (TF) consensus sequence which could function as a decoy to block TF activity. Specifically, VB-ODN for Nuclear factor-κB (NF-κB) could inhibit cell viability and decrease downstream gene expression in HEK293 cells without affecting expression of NF-κB itself. The specific binding between VB-ODN produced double-stranded RNA and NF-κB was evidenced by electrophoretic mobility shift assay. Moreover, similar VB-ODNs designed for three other TFs also inhibit their downstream gene expression but not that of themselves. Our study provides a new design of decoy for blocking TF activity.

  9. Detection and characterisation of bisegmented double-stranded RNA viruses (picobirnaviruses) in human faecal specimens.

    Science.gov (United States)

    Gallimore, C I; Appleton, H; Lewis, D; Green, J; Brown, D W

    1995-02-01

    The prevalence of picobirnaviruses (PBVs) in human stools was investigated by polyacrylamide gel electrophoresis (PAGE) analysis of 832 faecal specimens collected between 1982 and 1993 from patients in various clinical groups. Similar prevalences (9-13%) were detected in patients with or without gastroenteritis and throughout the age range of 3 to > 65 years. Two methods for the extraction of nucleic acid, a phenol/chloroform method and a guanidinium thiocynate (GTC)/silica method, were compared. Detection of PBVs by PAGE was three times more sensitive following RNA extraction by the GTC/silica method. Characterisation of three strains was carried out. Segment sizes ranged from 1.625 to 1.95 kilo base pairs (Kbp) and 2.2 to 2.5 Kbp for the fast and slow migrating bands, respectively. The nuclic acid was shown to be double-stranded RNA (dsRNA) by nuclease digestion. PBV-like particles were detected by electron microscopy in two PAGE-positive stools. Virion diameters ranged from 35 to 41 nm and a buoyant density of 1.38-1.4 g/ml in caesium chloride (CsCl) was demonstrated. These findings suggest that PBVs are widespread in humans in the United Kingdom. However, no disease association could be demonstrated.

  10. Effects of exogenous double-stranded RNA on the basonuclin gene expression in mouse oocytes

    Institute of Scientific and Technical Information of China (English)

    马峻; 周红林; 苏雷; 季维智

    2002-01-01

    In plants and less-advanced animal species, such as C.elegans, introduction of exogenous double-stranded RNA (dsRNA) into cells would trigger degradation of the mRNA with homologous sequence and interfere with the endogenous gene expression. It might represent an ancient anti-virus response which could prevent the mutation in the genome that was caused by virus infection or mobile DNA elements insertion. This phenomenon was named RNA interference, or RNAi. In this study, RNAi was used to investigate the function of basonuclin gene during oogenesis. Microinjection of dsRNA directed towards basonuclin into mouse germinal-vesicle-in- tact (GV) oocytes brought down the abundance of the cognate mRNA effectively in a time- and concentration-dependent manner. This reduction effect was sequence-specific and showed no negative effect on other non-homologous gene expression in oocytes, which indicated that dsRNA can recognize and cause the degradation of the transcriptional products of endogenous basonuclin gene in a sequence-specific manner. Immunofluorescence results showed that RNAi could reduce the concentration of basonuclin protein to some extent, but the effect was less efficient than the dsRNA targeting towards tPA and cMos which was also expressed in oocytes. This result might be due to the long half life of basonuclin protein in oocytes and the short reaction time which was posed by the limited life span of GV oocytes cultured in vitro. In summary, dsRNA could inhibit the expression of the cognate gene in oocytes at both mRNA and protein levels. The effect was similar to Knock-out technique which was based on homologous recombination. Furthermore, hairpin-style dsRNA targeting basonuclin gene could be produced by transcription from a recombinant plasmid and worked efficiently to deplete the cognate mRNA in oocytes. This finding offered a new way to study the function of basonuclin in the early stage of oogenesis by infection of primordial oocytes with the plasmid

  11. Visualizing double-stranded RNA distribution and dynamics in living cells by dsRNA binding-dependent fluorescence complementation

    Energy Technology Data Exchange (ETDEWEB)

    Cheng, Xiaofei [Southern Crop Protection and Food Research Centre, Agriculture and Agri-Food Canada, London, Ontario N5V 4T3 (Canada); College of Life and Environmental Sciences, Hangzhou Normal University, Hangzhou, Zhejiang 310036 (China); Deng, Ping; Cui, Hongguang [Southern Crop Protection and Food Research Centre, Agriculture and Agri-Food Canada, London, Ontario N5V 4T3 (Canada); Wang, Aiming, E-mail: aiming.wang@agr.gc.ca [Southern Crop Protection and Food Research Centre, Agriculture and Agri-Food Canada, London, Ontario N5V 4T3 (Canada)

    2015-11-15

    Double-stranded RNA (dsRNA) is an important type of RNA that plays essential roles in diverse cellular processes in eukaryotic organisms and a hallmark in infections by positive-sense RNA viruses. Currently, no in vivo technology has been developed for visualizing dsRNA in living cells. Here, we report a dsRNA binding-dependent fluorescence complementation (dRBFC) assay that can be used to efficiently monitor dsRNA distribution and dynamics in vivo. The system consists of two dsRNA-binding proteins, which are fused to the N- and C-terminal halves of the yellow fluorescent protein (YFP). Binding of the two fusion proteins to a common dsRNA brings the split YFP halves in close proximity, leading to the reconstitution of the fluorescence-competent structure and restoration of fluorescence. Using this technique, we were able to visualize the distribution and trafficking of the replicative RNA intermediates of positive-sense RNA viruses in living cells. - Highlights: • A live-cell imaging system was developed for visualizing dsRNA in vivo. • It uses dsRNA binding proteins fused with two halves of a fluorescent protein. • Binding to a common dsRNA enables the reporter to become fluorescent. • The system can efficiently monitor viral RNA replication in living cells.

  12. Coupling of double-stranded RNA synthesis and siRNA generation in fission yeast RNAi.

    Science.gov (United States)

    Colmenares, Serafin U; Buker, Shane M; Buhler, Marc; Dlakić, Mensur; Moazed, Danesh

    2007-08-03

    The fission yeast centromeric repeats are transcribed and ultimately processed into small interfering RNAs (siRNAs) that are required for heterochromatin formation. siRNA generation requires dsRNA synthesis by the RNA-directed RNA polymerase complex (RDRC) and processing by the Dicer ribonuclease. Here we show that Dcr1, the fission yeast Dicer, is physically associated with RDRC. Dcr1 generates siRNAs in an ATP-dependent manner that requires its conserved N-terminal helicase domain. Furthermore, C-terminal truncations of Dcr1 that abolish its interaction with RDRC, but can generate siRNA in vitro, abolish siRNA generation and heterochromatic gene silencing in vivo. Finally, reconstitution experiments show that the association of Dcr1 with RDRC strongly stimulates the dsRNA synthesis activity of RDRC. Our results suggest that heterochromatic dsRNA synthesis and siRNA generation are physically coupled processes. This coupling has implications for cis-restriction of siRNA-mediated heterochromatin assembly and for mechanisms that give rise to siRNA strand polarity.

  13. RNA interference in the light brown apple moth, Epiphyas postvittana (Walker) induced by double-stranded RNA feeding.

    Science.gov (United States)

    Turner, C T; Davy, M W; MacDiarmid, R M; Plummer, K M; Birch, N P; Newcomb, R D

    2006-06-01

    RNA interference (RNAi) or gene silencing is typically induced in insects by the injection of double-stranded RNAs (dsRNAs), short interfering RNAs, or through the use of hairpin constructs in transgenic insects. Here we demonstrate in the horticultural pest, Epiphyas postvittana (Lepidoptera: Tortricidae), that RNAi can be triggered by oral delivery of dsRNA to larvae. Transcript levels of a larval gut carboxylesterase gene (EposCXE1) were reduced to less than half that of controls within 2 days of being fed EposCXE1 dsRNA. Transcript levels of the pheromone binding protein gene (EposPBP1) were reduced in adult antennae by feeding larvae EposPBP1 dsRNA. Knockdown of EposPBP1 transcripts was observed for the first 2 days after adult eclosion but recovered to wild-type levels at 4 days posteclosion. The potential mechanisms involved in the initiation, movement and amplification of the silencing signal are discussed.

  14. Using Triple Helix Forming Peptide Nucleic Acids for Sequence-selective Recognition of Double-stranded RNA

    Science.gov (United States)

    Hnedzko, Dziyana; Cheruiyot, Samwel K.; Rozners, Eriks

    2014-01-01

    Non-coding RNAs play important roles in regulation of gene expression. Specific recognition and inhibition of these biologically important RNAs that form complex double-helical structures will be highly useful for fundamental studies in biology and practical applications in medicine. This protocol describes a strategy developed in our laboratory for sequence-selective recognition of double-stranded RNA (dsRNA) using triple helix forming peptide nucleic acids (PNAs) that bind in the major grove of RNA helix. The strategy developed uses chemically modified nucleobases, such as 2-aminopyridine (M) that enables strong triple helical binding at physiologically relevant conditions, and 2-pyrimidinone (P) and 3-oxo-2,3-dihydropyridazine (E) that enable recognition of isolated pyrimidines in the purine rich strand of the RNA duplex. Detailed protocols for preparation of modified PNA monomers, solid-phase synthesis and HPLC purification of PNA oligomers, and measuring dsRNA binding affinity using isothermal titration calorimetry are included. PMID:25199637

  15. Inhibition of Plasmodium falciparum proliferation in vitro by double-stranded RNA nanoparticle against malaria topoisomerase II.

    Science.gov (United States)

    Attasart, Pongsopee; Boonma, Siriwan; Sunintaboon, Panya; Tanwilai, Dolpawan; Pothikasikorn, Jinrapa; Noonpakdee, Wilai Tienrungroj

    2016-05-01

    The need to develop new effective antimalarial agents is urgent due to the rapid emergence of drug resistance to all current drugs by the most virulent human malaria parasite, Plasmodium falciparum. A promising avenue is in the development of antimalarials based on RNA interference targeting expression of malaria parasite vital genes, viz. DNA topoisomerase II gene (PfTOP2). Biodegradable chitosan nanoparticle system has proven to be effective in delivering DNA and small double-stranded interfering RNA to target cells. We have employed a long double-stranded (dsRNA) targeting the coding region of PfTOP2 that is complexed with chitosan nanoparticles in order to interfere with the cognate mRNA expression and examined its effect on P. falciparum growth in culture. Exposure of ring stage-infected erythrocytes to 10 μg/ml PfTOP2 chitosan/dsRNA nanoparticles for 48 h resulted in 71% growth inhibition as determined by [(3)H] hypoxanthine incorporation and microscopic assays, compared with 41% inhibition using an equivalent amount of free PfTOP2 dsRNA or 12% with unrelated chitosan/dsRNA nanoparticles. This inhibition was shown to occur during maturation of trophozoite to schizont stages. RT-PCR analysis indicated 56% and 38% decrease in PfTOP2 transcript levels in P. falciparum trophozoites treated with PfTOP2 dsRNA nanoparticles and free PfTOP2 dsRNA respectively. These results suggest that chitosan-based nanoparticles might be a useful tool for delivering dsRNA into malaria parasites.

  16. Using double-stranded RNA to prevent in vitro and in vivo viral infections by recombinant baculovirus.

    Science.gov (United States)

    Valdes, Victor Julian; Sampieri, Alicia; Sepulveda, Jorge; Vaca, Luis

    2003-05-23

    Introduction of double-stranded RNA (dsRNA) into a wide variety of cells and organisms results in post-transcriptional depletion of the homologue endogenous mRNA. This well-preserved phenomenon known as RNA interference (RNAi) is present in evolutionarily diverse organisms such as plants, fungi, insects, metazoans, and mammals. Because the identification of the targeted mRNA by the RNAi machinery depends upon Watson-Crick base-pairing interactions, RNAi can be exquisitely specific. We took advantage of this powerful and flexible technique to demonstrate that selective silencing of genes essential for viral propagation prevents in vitro and in vivo viral infection. Using the baculovirus Autographa californica, a rapidly replicating and highly cytolytic double-stranded DNA virus that infects many different insect species, we show for the first time that introduction of dsRNA from gp64 and ie1, two genes essential for baculovirus propagation, results in prevention of viral infection in vitro and in vivo. This is the first report demonstrating the use of RNAi to inhibit a viral infection in animals. This inhibition was specific, because dsRNA from the polyhedrin promoter (used as control) or unrelated dsRNAs did not affect the time course of viral infection. The most relevant consequences from the present study are: 1) RNAi offers a rapid and efficient way to interfere with viral genes to assess the role of specific proteins in viral function and 2) using RNAi to interfere with viral genes essential for cell infection may provide a powerful therapeutic tool for the treatment of viral infections.

  17. MKT1, a nonessential Saccharomyces cerevisiae gene with a temperature-dependent effect on replication of M2 double-stranded RNA.

    OpenAIRE

    Wickner, R B

    1987-01-01

    The MKT1 gene was defined by recessive alleles present in many laboratory strains of Saccharomyces cerevisiae that result in loss of M2 double-stranded RNA at temperatures above 30 degrees C if L-A-HN double-stranded RNA is present but not if L-A-H is present. I mapped MKT1 near TOP2 and isolated the gene by chromosome walking from TOP2. The gene location was defined by deletions, and a 2.8-kilobase transcript corresponding to the gene was detected. The recessive natural-variant mutations are...

  18. Primer-Dependent and Primer-Independent Initiation of Double Stranded RNA Synthesis by Purified Arabidopsis RNA-Dependent RNA Polymerases RDR2 and RDR6

    Science.gov (United States)

    Devert, Anthony; Fabre, Nicolas; Floris, Maïna; Canard, Bruno; Robaglia, Christophe; Crété, Patrice

    2015-01-01

    Cellular RNA-dependent RNA polymerases (RDRs) are fundamental components of RNA silencing in plants and many other eukaryotes. In Arabidopsis thaliana genetic studies have demonstrated that RDR2 and RDR6 are involved in the synthesis of double stranded RNA (dsRNA) from single stranded RNA (ssRNA) targeted by RNA silencing. The dsRNA is subsequently cleaved by the ribonuclease DICER-like into secondary small interfering RNAs (siRNAs) that reinforce and/or maintain the silenced state of the target RNA. Models of RNA silencing propose that RDRs could use primer-independent and primer-dependent initiation to generate dsRNA from a transcript targeted by primary siRNA or microRNA (miRNA). However, the biochemical activities of RDR proteins are still partly understood. Here, we obtained active recombinant RDR2 and RDR6 in a purified form. We demonstrate that RDR2 and RDR6 have primer-independent and primer-dependent RNA polymerase activities with different efficiencies. We further show that RDR2 and RDR6 can initiate dsRNA synthesis either by elongation of 21- to 24- nucleotides RNAs hybridized to complementary RNA template or by elongation of self-primed RNA template. These findings provide new insights into our understanding of the molecular mechanisms of RNA silencing in plants. PMID:25793874

  19. Protection of Macrobrachium rosenbergii against white tail disease by oral administration of bacterial expressed and encapsulated double-stranded RNA.

    Science.gov (United States)

    Naveen Kumar, Singaiah; Karunasagar, Indrani; Karunasagar, Iddya

    2013-09-01

    White tail disease (WTD) of cultured Macrobrachium rosenbergii is caused by M. rosenbergii nodavirus (MrNV) and an extra small virus (XSV), both present together, and the mortality rate can be as high as 100% within 2 or 3 days of infection. Possible protection of M. rosenbergii against WTD by oral administration of bacterial expressed and encapsulated double-stranded RNA (dsRNA) was studied. Juvenile M. rosenbergii were fed with the feed coated with inactivated bacteria encapsulated dsRNA of MrNV and XSV genes individually and in combination for 7 days followed by challenge with WTD causing agents at 24 h and 72 h post-feeding. Test animals fed with a combination of dsRNA of MrNV and XSV capsid genes showed the highest relative percent survival (RPS) when compared to other treatments with RPS of 80% and 75% at 24 and 72 h respectively. One hundred percent mortality was observed in test animals fed with control dsRNA coated feed. Although in the literature, injection is the most common method used to deliver dsRNA, this study shows that oral administration is effective, feasible and economical.

  20. Using triple-helix-forming Peptide nucleic acids for sequence-selective recognition of double-stranded RNA.

    Science.gov (United States)

    Hnedzko, Dziyana; Cheruiyot, Samwel K; Rozners, Eriks

    2014-09-08

    Non-coding RNAs play important roles in regulation of gene expression. Specific recognition and inhibition of these biologically important RNAs that form complex double-helical structures will be highly useful for fundamental studies in biology and practical applications in medicine. This protocol describes a strategy developed in our laboratory for sequence-selective recognition of double-stranded RNA (dsRNA) using triple-helix-forming peptide nucleic acids (PNAs) that bind in the major grove of the RNA helix. The strategy developed uses chemically modified nucleobases, such as 2-aminopyridine (M), which enables strong triple-helical binding under physiologically relevant conditions, and 2-pyrimidinone (P) and 3-oxo-2,3-dihydropyridazine (E), which enable recognition of isolated pyrimidines in the purine-rich strand of the RNA duplex. Detailed protocols for preparation of modified PNA monomers, solid-phase synthesis, HPLC purification of PNA oligomers, and measuring dsRNA binding affinity using isothermal titration calorimetry are included.

  1. Mutations Abrogating VP35 Interaction with Double-Stranded RNA Render Ebola Virus Avirulent in Guinea Pigs

    Energy Technology Data Exchange (ETDEWEB)

    Prins, Kathleen C.; Delpeut, Sebastien; Leung, Daisy W.; Reynard, Olivier; Volchkova, Valentina A.; Reid, St. Patrick; Ramanan, Parameshwaran; Cárdenas, Washington B.; Amarasinghe, Gaya K.; Volchkov, Viktor E.; Basler, Christopher F. (CNRS-INSERM); (Mount Sinai Hospital); (LB-Ecuador); (Iowa State)

    2010-10-11

    Ebola virus (EBOV) protein VP35 is a double-stranded RNA (dsRNA) binding inhibitor of host interferon (IFN)-{alpha}/{beta} responses that also functions as a viral polymerase cofactor. Recent structural studies identified key features, including a central basic patch, required for VP35 dsRNA binding activity. To address the functional significance of these VP35 structural features for EBOV replication and pathogenesis, two point mutations, K319A/R322A, that abrogate VP35 dsRNA binding activity and severely impair its suppression of IFN-{alpha}/{beta} production were identified. Solution nuclear magnetic resonance (NMR) spectroscopy and X-ray crystallography reveal minimal structural perturbations in the K319A/R322A VP35 double mutant and suggest that loss of basic charge leads to altered function. Recombinant EBOVs encoding the mutant VP35 exhibit, relative to wild-type VP35 viruses, minimal growth attenuation in IFN-defective Vero cells but severe impairment in IFN-competent cells. In guinea pigs, the VP35 mutant virus revealed a complete loss of virulence. Strikingly, the VP35 mutant virus effectively immunized animals against subsequent wild-type EBOV challenge. These in vivo studies, using recombinant EBOV viruses, combined with the accompanying biochemical and structural analyses directly correlate VP35 dsRNA binding and IFN inhibition functions with viral pathogenesis. Moreover, these studies provide a framework for the development of antivirals targeting this critical EBOV virulence factor.

  2. Therapeutic effect of Artemia enriched with Escherichia coli expressing double-stranded RNA in the black tiger shrimp Penaeus monodon.

    Science.gov (United States)

    Thammasorn, Thitiporn; Somchai, Parinyachat; Laosutthipong, Chaowanee; Jitrakorn, Sarocha; Wongtripop, Somjai; Thitamadee, Siripong; Withyachumnarnkul, Boonsirm; Saksmerprome, Vanvimon

    2013-10-01

    We exploited Artemia as a double-stranded (ds)RNA-delivery system to combat viral diseases in shrimp. First, the transformed Escherichia coli (E. coli) expressing red fluorescent protein (RFP) was tested in the Artemia enrichment process. RFP signals detectable in the gut of Artemia under confocal microscope were evident for the successful encapsulation. Second, the Artemia enrichment process was performed using E. coli producing Laem-Singh virus (LSNV)-specific dsRNA, which has been previously shown to inhibit the viral infection in the black tiger shrimp Penaeus monodon by intramuscular injection and oral administration. The enriched Artemia nauplii were confirmed to contain dsRNA-LSNV by RT-PCR, and were subjected to the feeding test with P. monodon postlarvae. Quantitative RT-PCR indicated that a number of LSNV copies in most of the treated shrimp were, at least, 1000-fold lower than the untreated controls. During 11-17weeks after feeding, average body weight of the treated group was markedly increased relative to the control group. A smaller differential growth rate of the treated group as compared to the control was also noticed. These results suggested that feeding shrimp with the dsRNA-enriched Artemia can eliminate LSNV infection, which is the cause of retarded growth in P. monodon. The present study reveals for the first time the therapeutic effect of dsRNA-enriched Artemia for shrimp disease control.

  3. Double-Stranded RNA-Binding Protein 4 Is Required for Resistance Signaling against Viral and Bacterial Pathogens

    Directory of Open Access Journals (Sweden)

    Shifeng Zhu

    2013-09-01

    Full Text Available Plant viruses often encode suppressors of host RNA silencing machinery, which occasionally function as avirulence factors that are recognized by host resistance (R proteins. For example, the Arabidopsis R protein, hypersensitive response to TCV (HRT, recognizes the turnip crinkle virus (TCV coat protein (CP. HRT-mediated resistance requires the RNA-silencing component double-stranded RNA-binding protein 4 (DRB4 even though it neither is associated with the accumulation of TCV-specific small RNA nor requires the RNA silencing suppressor function of CP. HRT interacts with the cytosolic fraction of DRB4. Interestingly, TCV infection both increases the cytosolic DRB4 pool and inhibits the HRT-DRB4 interaction. The virulent R8A CP derivative, which induces a subset of HRT-derived responses, also disrupts this interaction. The differential localization of DRB4 in the presence of wild-type and R8A CP implies the importance of subcellular compartmentalization of DRB4. The requirement of DRB4 in resistance to bacterial infection suggests a universal role in R-mediated defense signaling.

  4. The dengue virus conceals double-stranded RNA in the intracellular membrane to escape from an interferon response

    Science.gov (United States)

    Uchida, Leo; Espada-Murao, Lyre Anni; Takamatsu, Yuki; Okamoto, Kenta; Hayasaka, Daisuke; Yu, Fuxun; Nabeshima, Takeshi; Buerano, Corazon C.; Morita, Kouichi

    2014-01-01

    The dengue virus (DENV) circulates between humans and mosquitoes and requires no other mammals or birds for its maintenance in nature. The virus is well-adapted to humans, as reflected by high-level viraemia in patients. To investigate its high adaptability, the DENV induction of host type-I interferon (IFN) was assessed in vitro in human-derived HeLa cells and compared with that induced by the Japanese encephalitis virus (JEV), a closely related arbovirus that generally exhibits low viraemia in humans. A sustained viral spread with a poor IFN induction was observed in the DENV-infected cells, whereas the JEV infection resulted in a self-limiting and abortive infection with a high IFN induction. There was no difference between DENV and JEV double-stranded RNA (dsRNA) as IFN inducers. Instead, the dsRNA was poorly exposed in the cytosol as late as 48 h post-infection (p.i.), despite the high level of DENV replication in the infected cells. In contrast, the JEV-derived dsRNA appeared in the cytosol as early as 24 h p.i. Our results provided evidence for the first time in DENV, that concealing dsRNA in the intracellular membrane diminishes the effect of the host defence mechanism, a strategy that differs from an active suppression of IFN activity. PMID:25491663

  5. Enhanced whitefly resistance in transgenic tobacco plants expressing double stranded RNA of v-ATPase A gene.

    Directory of Open Access Journals (Sweden)

    Nidhi Thakur

    Full Text Available BACKGROUND: Expression of double strand RNA (dsRNA designed against important insect genes in transgenic plants have been shown to give protection against pests through RNA interference (RNAi, thus opening the way for a new generation of insect-resistant crops. We have earlier compared the efficacy of dsRNAs/siRNAs, against a number of target genes, for interference in growth of whitefly (Bemisia tabaci upon oral feeding. The v-ATPase subunit A (v-ATPaseA coding gene was identified as a crucial target. We now report the effectiveness of transgenic tobacco plants expressing siRNA to silence v-ATPaseA gene expression for the control of whitefly infestation. METHODOLOGY/PRINCIPAL FINDINGS: Transgenic tobacco lines were developed for the expression of long dsRNA precursor to make siRNA and knock down the v-ATPaseA mRNA in whitefly. Molecular analysis and insecticidal properties of the transgenic plants established the formation of siRNA targeting the whitefly v-ATPaseA, in the leaves. The transcript level of v-ATPaseA in whiteflies was reduced up to 62% after feeding on the transgenic plants. Heavy infestation of whiteflies on the control plants caused significant loss of sugar content which led to the drooping of leaves. The transgenic plants did not show drooping effect. CONCLUSIONS/SIGNIFICANCE: Host plant derived pest resistance was achieved against whiteflies by genetic transformation of tobacco which generated siRNA against the whitefly v-ATPaseA gene. Transgenic tobacco lines expressing dsRNA of v-ATPaseA, delivered sufficient siRNA to whiteflies feeding on them, mounting a significant silencing response, leading to their mortality. The transcript level of the target gene was reduced in whiteflies feeding on transgenic plants. The strategy can be taken up for genetic engineering of plants to control whiteflies in field crops.

  6. Structure-spectrophotometric selectivity relationship in interactions of quercetin related flavonoids with double stranded and single stranded RNA

    Science.gov (United States)

    Piantanida, Ivo; Mašić, Lozika; Rusak, Gordana

    2009-04-01

    Interactions of five flavonoids with dsRNA and single stranded ssRNA were studied by UV/vis titrations. The results obtained supported the intercalative binding mode as a dominant interaction of studied flavonoids with dsRNA as well as major interaction with ssRNA. Furthermore, changes of the UV/vis spectra of flavonoids induced by addition of poly G or poly C, respectively, are significantly stronger than changes induced by double stranded poly G-poly C, pointing to essential role of the free poly G or poly C sequence (not hydrogen bonded in double helix). Exclusively poly G caused significant batochromic shift of the UV/vis maxima of all studied flavonoids, whereby the intensity of batochromic shift is nicely correlated to the number of OH groups of flavonoid. Unlikely to poly G, addition of poly A and poly U induced measurable changes only in the UV/vis spectra of flavonoids characterised by no OH (galangin) or three OH groups (myricetin) on the phenyl part of the molecule. Consequently, flavonoids with one- or two-OH groups on the phenyl part of the molecule (luteolin, fisetin, kaempferol) specifically differentiate between poly A, poly U (negligible changes in the UV/Vis spectra) and poly G (strong changes in the UV/Vis spectra) as well as poly C (moderate changes in the UV/Vis spectra).

  7. Separation and quantification of viral double-stranded RNA fragments by capillary electrophoresis in hydroxyethylcellulose polymer solutions.

    Science.gov (United States)

    Shambaugh, C L; Bodmer, J L; Hsu, D; Ranucci, C S

    2004-10-01

    Capillary electrophoresis (CE) is an analytical technique widely utilized to resolve complex mixtures of nucleic acids. CE uses a variety of polymers in solution that act as a molecular sieve to separate nucleic acid fragments according to size. It has been shown previously that purified dsDNA can be resolved efficiently by solutions of hydroxyethylcellulose (HEC) polymer, providing a rapid and high resolution method of separation. We have applied this separation technique to viral double-stranded (ds) RNA segments derived from rotavirus process samples. HEC polymers of various molecular masses and concentrations were identified and compared for their ability to separate dsRNA based on the extent of expected polymer network formation. The HEC polymer exhibiting the most desirable separation characteristics was then used for subsequent optimization of various method parameters, such as, injection time, electric field strength, dye concentration and capillary equilibration. The optimized method was then applied to the quantification of genome concentration based on a representative segment of the rotavirus genome. This study demonstrated that purified viral dsRNA material of known concentration could be used to generate an external standard curve relating concentration to peak area. This standard curve was used to determine the concentration of unknown samples by interpolation. This novel RNA quantification assay is likely to be applicable to other types of virus, including those containing dsDNA.

  8. RNAi-mediated Mortality of the Whitefly through Transgenic Expression of Double-stranded RNA Homologous to Acetylcholinesterase and Ecdysone Receptor in Tobacco Plants

    Science.gov (United States)

    The whitefly Bemisia tabaci (Genn.) is a pest and vector of plant viruses affecting plants worldwide. Using RNA interference (RNAi) to downregulate whitefly genes by expressing their homologous double stranded RNAs in plants has great potential for management of whiteflies to reduce plant virus dise...

  9. Regulation of CXCL-8 (Interleukin-8) Induction by Double-Stranded RNA Signaling Pathways during Hepatitis C Virus Infection▿

    Science.gov (United States)

    Wagoner, Jessica; Austin, Michael; Green, Jamison; Imaizumi, Tadaatsu; Casola, Antonella; Brasier, Allan; Khabar, Khalid S. A.; Wakita, Takaji; Gale, Michael; Polyak, Stephen J.

    2007-01-01

    Hepatitis C virus (HCV) infection induces the α-chemokine interleukin-8 (CXCL-8), which is regulated at the levels of transcription and mRNA stability. In the current study, CXCL-8 regulation by double-stranded (ds)RNA pathways was analyzed in the context of HCV infection. A constitutively active mutant of the retinoic acid-inducible gene I (RIG-I), RIG-N, activated CXCL-8 transcription. Promoter mutagenesis experiments indicated that NF-κB and interferon (IFN)-stimulated response element (ISRE) binding sites were required for the RIG-N induction of CXCL-8 transcription. IFN-β promoter stimulator 1 (IPS-1) expression also activated CXCL-8 transcription, and mutations of the ISRE and NF-κB binding sites reduced and abrogated CXCL-8 transcription, respectively. In the presence of wild-type RIG-I, transfection of JFH-1 RNA or JFH-1 virus infection of Huh7.5.1 cells activated the CXCL-8 promoter. Expression of IFN regulatory factor 3 (IRF-3) stimulated transcription from both full-length and ISRE-driven CXCL-8 promoters. Chromatin immunoprecipitation assays demonstrated that IRF-3 and NF-κB bound directly to the CXCL-8 promoter in response to virus infection and dsRNA transfection. RIG-N stabilized CXCL-8 mRNA via the AU-rich element in the 3′ untranslated region of CXCL-8 mRNA, leading to an increase in its half-life following tumor necrosis factor alpha induction. The data indicate that HCV infection triggers dsRNA signaling pathways that induce CXCL-8 via transcriptional activation and mRNA stabilization and define a regulatory link between innate antiviral and inflammatory cellular responses to virus infection. PMID:17035306

  10. Impact of folic acid supplementation on single- and double-stranded RNA degradation in human colostrum and mature milk.

    Science.gov (United States)

    Kocic, Gordana; Bjelakovic, Ljiljana; Bjelakovic, Bojko; Jevtoci-Stoimenov, Tatjana; Sokolovic, Dusan; Cvetkovic, Tatjana; Kocic, Hristina; Stojanovic, Svetlana; Langerholc, Tomaz; Jonovic, Marina

    2014-07-01

    Sufficient intake of folic acid is necessary for normal embryogenesis, fetal, and neonatal development. Folic acid facilitates nucleic acid internalization, and protects cellular DNA from nuclease degradation. Human milk contains enzymes, antimicrobial proteins, and antibodies, along with macrophages, that protect against infections and allergies. However, little to no information is available on the effects of folic acid supplementation on degradation of nucleic acids in human milk. In the present study, we aimed to determine the RNase activity (free and inhibitor-bound) in colostrum and mature milk, following folic acid supplementation. The study design included a total of 59 women, 27 of whom received 400 μg of folic acid daily periconceptionally and after. Folic acid supplementation increased the free RNase and polyadenylase activity following lactation. However, the increased RNase activity was not due to de novo enzyme synthesis, as the inhibitor-bound (latent) RNase activity was significantly lower and disappeared after one month. Folic acid reduced RNase activity by using double-stranded RNA as substrate. Data suggests that folic acid supplementation may improve viral RNAs degradation and mRNA degradation, but not dsRNA degradation, preserving in this way the antiviral defense.

  11. Sequence selective recognition of double-stranded RNA using triple helix-forming peptide nucleic acids.

    Science.gov (United States)

    Zengeya, Thomas; Gupta, Pankaj; Rozners, Eriks

    2014-01-01

    Noncoding RNAs are attractive targets for molecular recognition because of the central role they play in gene expression. Since most noncoding RNAs are in a double-helical conformation, recognition of such structures is a formidable problem. Herein, we describe a method for sequence-selective recognition of biologically relevant double-helical RNA (illustrated on ribosomal A-site RNA) using peptide nucleic acids (PNA) that form a triple helix in the major grove of RNA under physiologically relevant conditions. Protocols for PNA preparation and binding studies using isothermal titration calorimetry are described in detail.

  12. The wide distribution of endornaviruses, large double-stranded RNA replicons with plasmid-like properties.

    Science.gov (United States)

    Fukuhara, T; Koga, R; Aoki, N; Yuki, C; Yamamoto, N; Oyama, N; Udagawa, T; Horiuchi, H; Miyazaki, S; Higashi, Y; Takeshita, M; Ikeda, K; Arakawa, M; Matsumoto, N; Moriyama, H

    2006-05-01

    The International Committee on Taxonomy of Viruses (ICTV) recently accepted Endornavirus as a new genus of plant dsRNA virus. We have determined the partial nucleotide sequences of the RNA-dependent RNA polymerase regions from the large dsRNAs (about 14 kbp) isolated from barley (Hordeum vulgare), kidney bean (Phaseolus vulgaris), melon (Cucumis melo), bottle gourd (Lagenaria siceraria), Malabar spinach (Basella alba), seagrass (Zostera marina), and the fungus Helicobasidium mompa. Phylogenetic analyses of these seven dsRNAs indicate that these dsRNAs are new members of the genus Endornavirus that are widely distributed over the plant and fungal kingdoms.

  13. RNA/aTNA Chimeras: RNAi Effects and Nucleases Resistance of Single and Double Stranded RNAs

    Directory of Open Access Journals (Sweden)

    Adele Alagia

    2014-11-01

    Full Text Available The RNA interference pathway (RNAi is a specific and powerful biological process, triggered by small non-coding RNA molecules and involved in gene expression regulation. In this work, we explored the possibility of increasing the biological stability of these RNA molecules by replacing their natural ribose ring with an acyclic L-threoninol backbone. In particular, this modification has been incorporated at certain positions of the oligonucleotide strands and its effects on the biological properties of the siRNA have been evaluated. In vitro cellular RNAi assays have demonstrated that the L-threoninol backbone is well tolerated by the RNAi machinery in both double and single-stranded fashion, with activities significantly higher than those evinced by the unmodified RNAs and comparable to the well-known phosphorothioate modification. Additionally, this modification conferred extremely strong resistance to serum and 3′/5′-exonucleases. In view of these results, we applied this modification to the knockdown of a therapeutically relevant human gene such as apolipoprotein B (ApoB. Further studies on the activation of the innate immune system showed that L-threoninol-modified RNAs are slightly less stimulatory than unmodified RNAs.

  14. Topically Applied AaeIAP1 Double-Stranded RNA Kills Female Adults of Aedes aegypti

    Science.gov (United States)

    2008-05-01

    successfully used to transfect cell cultures with siRNA (Grayson et al. 2006). It is a proprietary cationic polymer/ lipid for- mulation supplied in ethanol...S.C.Diniz, J.C.Oliveira,E.Moreira, M. P. Miagostovich, E. V. Costa, et al. 2002. Outbreak of jaundice andhemorrhagic fever in the Southeast ofBrazil

  15. Functional role of glutamine 28 and arginine 39 in double stranded RNA cleavage by human pancreatic ribonuclease.

    Directory of Open Access Journals (Sweden)

    Md Tabish Rehman

    Full Text Available Human pancreatic ribonuclease (HPR, a member of RNase A superfamily, has a high activity on double stranded (ds RNA. By virtue of this activity HPR appears to be involved in the host-defense against pathogenic viruses. To delineate the mechanism of dsRNA cleavage by HPR, we have investigated the role of glutamine 28 and arginine 39 of HPR in its activity on dsRNA. A non-basic residue glycine 38, earlier shown to be important for dsRNA cleavage by HPR was also included in the study in the context of glutamine 28 and arginine 39. Nine variants of HPR respectively containing Q28A, Q28L, R39A, G38D, Q28A/R39A, Q28L/R39A, Q28A/G38D, R39A/G38D and Q28A/G38D/R39A mutations were generated and functionally characterized. The far-UV CD-spectral analysis revealed all variants, except R39A, to have structures similar to that of HPR. The catalytic activity of all HPR variants on single stranded RNA substrate was similar to that of HPR, whereas on dsRNA, the catalytic efficiency of all single residue variants, except for the Q28L, was significantly reduced. The dsRNA cleavage activity of R39A/G38D and Q28A/G38D/R39A variants was most drastically reduced to 4% of that of HPR. The variants having reduced dsRNA cleavage activity also had reduction in their dsDNA melting activity and thermal stability. Our results indicate that in HPR both glutamine 28 and arginine 39 are important for the cleavage of dsRNA. Although these residues are not directly involved in catalysis, both arginine 39 and glutamine 28 appear to be facilitating a productive substrate-enzyme interaction during the dsRNA cleavage by HPR.

  16. Functional role of glutamine 28 and arginine 39 in double stranded RNA cleavage by human pancreatic ribonuclease.

    Science.gov (United States)

    Rehman, Md Tabish; Dey, Punyatirtha; Hassan, Md Imtaiyaz; Ahmad, Faizan; Batra, Janendra K

    2011-03-08

    Human pancreatic ribonuclease (HPR), a member of RNase A superfamily, has a high activity on double stranded (ds) RNA. By virtue of this activity HPR appears to be involved in the host-defense against pathogenic viruses. To delineate the mechanism of dsRNA cleavage by HPR, we have investigated the role of glutamine 28 and arginine 39 of HPR in its activity on dsRNA. A non-basic residue glycine 38, earlier shown to be important for dsRNA cleavage by HPR was also included in the study in the context of glutamine 28 and arginine 39. Nine variants of HPR respectively containing Q28A, Q28L, R39A, G38D, Q28A/R39A, Q28L/R39A, Q28A/G38D, R39A/G38D and Q28A/G38D/R39A mutations were generated and functionally characterized. The far-UV CD-spectral analysis revealed all variants, except R39A, to have structures similar to that of HPR. The catalytic activity of all HPR variants on single stranded RNA substrate was similar to that of HPR, whereas on dsRNA, the catalytic efficiency of all single residue variants, except for the Q28L, was significantly reduced. The dsRNA cleavage activity of R39A/G38D and Q28A/G38D/R39A variants was most drastically reduced to 4% of that of HPR. The variants having reduced dsRNA cleavage activity also had reduction in their dsDNA melting activity and thermal stability. Our results indicate that in HPR both glutamine 28 and arginine 39 are important for the cleavage of dsRNA. Although these residues are not directly involved in catalysis, both arginine 39 and glutamine 28 appear to be facilitating a productive substrate-enzyme interaction during the dsRNA cleavage by HPR.

  17. Nature of the polypeptide encoded by each of the 10 double-stranded RNA segments of reovirus type 3

    Energy Technology Data Exchange (ETDEWEB)

    McCrae, M.A.; Joklik, W.K.

    1978-09-01

    Under suitable conditions of denaturation, the double-stranded (ds) RNA segments of reovirus can be translated in cell-free protein synthesizing systems. Since all 10 segments of reovirus ds RNA can be isolated in virtually pure form, this provides a means for determining the nature of the polypeptide encoded by each individual segment. The complete coding assignment set was determined for the Dearing strain of reovirus serotype 3. Polypeptide identification was made not only on the basis of electrophoretic migration rates in both the phosphate- and Tri-glycine (Laemmli)-based polyacrylamide gel systems, but also on the basis of comparing peptide profiles of in vitro translation products and authentic reovirus polypeptides after digestion with staphylococcal V8 protease. The latter method provides absolute identification. The assignment set is (using the commonly accepted designation for the ds RNA segments, but a newly proposed nomenclature for the polypeptides); segment L1 codes for the minor virion components lambda 3, and segments L2 and L3 code for the two major virion core components lambda 2 and lambda 1, respectively; segment M1 codes for a minor virion component ..mu..2, segment M2 codes for the polypeptide that is present in virions both in the form of the minor component ..mu..1 and as the major component ..mu..1C which is derived from it by cleavage, and segment M3 codes for the nonstructural polypeptide ..mu..NS; and segment S1 codes for the minor outer capsid shell component sigma 1, segment S2 codes for the core component sigma 2, segment S3 codes for the nonstructural polypeptide sigma NS, and segment S4 codes for the major outer capsid shell component sigma 3.

  18. Transfection of the Giardia lamblia double-stranded RNA virus into giardia lamblia by electroporation of a single-stranded RNA copy of the viral genome.

    OpenAIRE

    1990-01-01

    The development of a genetic vector for protozoan parasites is a major hurdle yet to be crossed in the study of the molecular and cellular biology of these parasites. We have identified and isolated a double-stranded RNA virus (G. lamblia virus [GLV]) from certain strains of the intestinal parasitic protozoan Giardia lamblia (A. L. Wang and C. C. Wang, Mol. Biochem. Parasitol. 21:269-276, 1986), which is capable of infecting other virus-free strains of G. lamblia (R. L. Miller, A. L. Wang, an...

  19. Comparative sequence analysis of double stranded RNA binding protein encoding gene of parapoxviruses from Indian camels

    Directory of Open Access Journals (Sweden)

    G. Nagarajan

    2014-03-01

    Full Text Available The dsRNA binding protein (RBP encoding gene of parapoxviruses (PPVs from the Dromedary camels, inhabitating different geographical region of Rajasthan, India were amplified by polymerase chain reaction using the primers of pseudocowpoxvirus (PCPV from Finnish reindeer and cloned into pGEM-T for sequence analysis. Analysis of RBP encoding gene revealed that PPV DNA from Bikaner shared 98.3% and 76.6% sequence identity at the amino acid level, with Pali and Udaipur PPV DNA, respectively. Reference strains of Bovine papular stomatitis virus (BPSV and PCPV (reindeer PCPV and human PCPV shared 52.8% and 86.9% amino acid identity with RBP gene of camel PPVs from Bikaner, respectively. But different strains of orf virus (ORFV from different geographical areas of the world shared 69.5–71.7% amino acid identity with RBP gene of camel PPVs from Bikaner. These findings indicate that the camel PPVs described are closely related to bovine PPV (PCPV in comparison to caprine and ovine PPV (ORFV.

  20. Double-stranded RNA evokes exacerbation in a mouse model of corticosteroid refractory asthma.

    Science.gov (United States)

    De Alba, Jorge; Otal, Raquel; Calama, Elena; Domenech, Anna; Prats, Neus; Gozzard, Neil; Miralpeix, Montserrat

    2015-12-01

    RNA viruses are a major cause of respiratory infections and are known to exacerbate asthma and other respiratory diseases. Our aim was to test the ability of poly(I:C) (polyinosinic:polycytidylic acid), a viral surrogate, to elicit exacerbation in a model of severe asthma driven by HDM (house dust mite) in FCA (Freund's complete adjuvant). Poly(I:C) was administered intranasally around the HDM challenge in FCA-HDM-sensitized animals. Changes in AHR (airway hyperresponsiveness), BALF (bronchoalveolar lavage fluid) inflammatory infiltrate, HDM-specific immunoglobulins and cytokine/chemokine release were evaluated at different points after the challenge. The effect of oral dexamethasone was also assessed. Exacerbation was achieved when poly(I:C) was administered 24 h before the HDM challenge and was characterized by enhanced AHR and an increase in the numbers of neutrophils, macrophages and lymphocytes in the BALF. Th1, Th2 and Th17 cytokines were also elevated at different time points after the challenge. Peribronchial and alveolar inflammation in lung tissue were also augmented. AHR and inflammatory infiltration showed reduced sensitivity to dexamethasone treatment. We have set up a model that mimics key aspects of viral exacerbation in a corticosteroid-refractory asthmatic phenotype which could be used to evaluate new therapies for this condition.

  1. Evolutionary history of double-stranded RNA binding proteins in plants: identification of new cofactors involved in easiRNA biogenesis.

    Science.gov (United States)

    Clavel, Marion; Pélissier, Thierry; Montavon, Thomas; Tschopp, Marie-Aude; Pouch-Pélissier, Marie-Noëlle; Descombin, Julie; Jean, Viviane; Dunoyer, Patrice; Bousquet-Antonelli, Cécile; Deragon, Jean-Marc

    2016-05-01

    In this work, we retrace the evolutionary history of plant double-stranded RNA binding proteins (DRBs), a group of non-catalytic factors containing one or more double-stranded RNA binding motif (dsRBM) that play important roles in small RNA biogenesis and functions. Using a phylogenetic approach, we show that multiple dsRBM DRBs are systematically composed of two different types of dsRBMs evolving under different constraints and likely fulfilling complementary functions. In vascular plants, four distinct clades of multiple dsRBM DRBs are always present with the exception of Brassicaceae species, that do not possess member of the newly identified clade we named DRB6. We also identified a second new and highly conserved DRB family (we named DRB7) whose members possess a single dsRBM that shows concerted evolution with the most C-terminal dsRBM domain of the Dicer-like 4 (DCL4) proteins. Using a BiFC approach, we observed that Arabidopsis thaliana DRB7.2 (AtDRB7.2) can directly interact with AtDRB4 but not with AtDCL4 and we provide evidence that both AtDRB7.2 and AtDRB4 participate in the epigenetically activated siRNAs pathway.

  2. Molecular characterization of double-stranded RNA virus in Trichomonas vaginalis Egyptian isolates and its association with pathogenicity.

    Science.gov (United States)

    El-Gayar, Eman K; Mokhtar, Amira B; Hassan, Wael A

    2016-10-01

    Trichomoniasis is a common human sexually transmitted infection caused by Trichomonas vaginalis. The parasite can be infected with double-stranded RNA viruses (TVV). This viral infection may have important implications on trichomonal virulence and disease pathogenesis. This study aimed to determine the prevalence of T. vaginalis virus among isolates obtained from infected (symptomatic and asymptomatic) women in Ismailia City, Egypt, and to correlate the virus-infected isolates with the clinical manifestations of patients. In addition, the pathogenicity of TVV infected isolates on mice was also evaluated. T. vaginalis isolates were obtained from symptomatic and asymptomatic female patients followed by axenic cultivation in Diamond's TYM medium. The presence of T. vaginalis virus was determined from total extraction of nucleic acids (DNA-RNA) followed by reverse transcriptase-PCR. Representative samples were inoculated intraperitoneally in female albino/BALB mice to assess the pathogenicity of different isolates. A total of 110 women were examined; 40 (36.3 %) samples were positive for T. vaginalis infection. Of these 40 isolates, 8 (20 %) were infected by TVV. Five isolates contained TVV-2 virus species, and the remaining three isolates were infected withTVV-4 variant. A significant association was found between the presence of TVV and particular clinical manifestations of trichomoniasis. Experimental mice infection showed varying degrees of pathogenicity. This is the first report on T. vaginalis infection by TVV in Egypt. The strong association detected between TVV and particular clinical features of trichomoniasis and also the degree of pathogenicity in experimentally infected mice may indicate a possible clinical significance of TVV infection of T. vaginalis isolates.

  3. Non-Target Effects of Green Fluorescent Protein (GFP-Derived Double-Stranded RNA (dsRNA-GFP Used in Honey Bee RNA Interference (RNAi Assays

    Directory of Open Access Journals (Sweden)

    Francis M. F. Nunes

    2013-01-01

    Full Text Available RNA interference has been frequently applied to modulate gene function in organisms where the production and maintenance of mutants is challenging, as in our model of study, the honey bee, Apis mellifera. A green fluorescent protein (GFP-derived double-stranded RNA (dsRNA-GFP is currently commonly used as control in honey bee RNAi experiments, since its gene does not exist in the A. mellifera genome. Although dsRNA-GFP is not expected to trigger RNAi responses in treated bees, undesirable effects on gene expression, pigmentation or developmental timing are often observed. Here, we performed three independent experiments using microarrays to examine the effect of dsRNA-GFP treatment (introduced by feeding on global gene expression patterns in developing worker bees. Our data revealed that the expression of nearly 1,400 genes was altered in response to dsRNA-GFP, representing around 10% of known honey bee genes. Expression changes appear to be the result of both direct off-target effects and indirect downstream secondary effects; indeed, there were several instances of sequence similarity between putative siRNAs generated from the dsRNA-GFP construct and genes whose expression levels were altered. In general, the affected genes are involved in important developmental and metabolic processes associated with RNA processing and transport, hormone metabolism, immunity, response to external stimulus and to stress. These results suggest that multiple dsRNA controls should be employed in RNAi studies in honey bees. Furthermore, any RNAi studies involving these genes affected by dsRNA-GFP in our studies should use a different dsRNA control.

  4. Activation of double-stranded RNA-dependent protein kinase inhibits proliferation of pancreatic β-cells

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Shan-Shan [Key Laboratory of Human Functional Genomics of Jiangsu Province, Nanjing Medical University, Nanjing (China); Department of Biochemistry and Molecular Biology, Nanjing Medical University, Nanjing (China); Jiang, Teng [Department of Neurology, Qingdao Municipal Hospital, Nanjing Medical University, Nanjing (China); Wang, Yi; Gu, Li-Ze [Key Laboratory of Human Functional Genomics of Jiangsu Province, Nanjing Medical University, Nanjing (China); Department of Biochemistry and Molecular Biology, Nanjing Medical University, Nanjing (China); Wu, Hui-Wen [Laboratory Center for Basic Medical Sciences, Nanjing Medical University, Nanjing (China); Tan, Lan [Department of Neurology, Qingdao Municipal Hospital, Nanjing Medical University, Nanjing (China); Guo, Jun, E-mail: Guoj@njmu.edu.cn [Key Laboratory of Human Functional Genomics of Jiangsu Province, Nanjing Medical University, Nanjing (China); Department of Biochemistry and Molecular Biology, Nanjing Medical University, Nanjing (China)

    2014-01-17

    Highlights: •PKR can be activated by glucolipitoxicity and pro-inflammatory cytokines in β-cells. •Activated PKR inhibited β-cell proliferation by arresting cell cycle at G1 phase. •Activated PKR fully abrogated the pro-proliferative effects of IGF-I on β-cells. -- Abstract: Double-stranded RNA-dependent protein kinase (PKR) is revealed to participate in the development of insulin resistance in peripheral tissues in type 2 diabetes (T2DM). Meanwhile, PKR is also characterized as a critical regulator of cell proliferation. To date, no study has focused on the impact of PKR on the proliferation of pancreatic β-cells. Here, we adopted insulinoma cell lines and mice islet β-cells to investigate: (1) the effects of glucolipotoxicity and pro-inflammatory cytokines on PKR activation; (2) the effects of PKR on proliferation of pancreatic β-cells and its underlying mechanisms; (3) the actions of PKR on pro-proliferative effects of IGF-I and its underlying pathway. Our results provided the first evidence that PKR can be activated by glucolipitoxicity and pro-inflammatory cytokines in pancreatic β-cells, and activated PKR significantly inhibited cell proliferation by arresting cell cycle at G1 phase. Reductions in cyclin D1 and D2 as well as increases in p27 and p53 were associated with the anti-proliferative effects of PKR, and proteasome-dependent degradation took part in the reduction of cyclin D1 and D2. Besides, PKR activation abrogated the pro-proliferative effects of IGF-I by activating JNK and disrupting IRS1/PI3K/Akt signaling pathway. These findings indicate that the anti-proliferative actions of PKR on pancreatic β-cells may contribute to the pathogenesis of T2DM.

  5. Persistence of double-stranded RNA in insect hemolymph as a potential determiner of RNA interference success: evidence from Manduca sexta and Blattella germanica.

    Science.gov (United States)

    Garbutt, Jennie S; Bellés, Xavier; Richards, Elaine H; Reynolds, Stuart E

    2013-02-01

    RNA interference (RNAi) is a specific gene silencing mechanism mediated by double-stranded RNA (dsRNA), which has been harnessed as a useful reverse genetics tool in insects. Unfortunately, however, this technology has been limited by the variable sensitivity of insect species to RNAi. We propose that rapid degradation of dsRNA in insect hemolymph could impede gene silencing by RNAi and experimentally investigate the dynamics of dsRNA persistence in two insects, the tobacco hornworm, Manduca sexta, a species in which experimental difficulty has been experienced with RNAi protocols and the German cockroach, Blattella germanica, which is known to be highly susceptible to experimental RNAi. An ex vivo assay revealed that dsRNA was rapidly degraded by an enzyme in M. sexta hemolymph plasma, whilst dsRNA persisted much longer in B. germanica plasma. A quantitative reverse transcription PCR-based assay revealed that dsRNA, accordingly, disappeared rapidly from M. sexta hemolymph in vivo. The M. sexta dsRNAse is inactivated by exposure to high temperature and is inhibited by EDTA. These findings lead us to propose that the rate of persistence of dsRNA in insect hemolymph (mediated by the action of one or more nucleases) could be an important factor in determining the susceptibility of insect species to RNAi.

  6. Sequence-selective recognition of double-stranded RNA and enhanced cellular uptake of cationic nucleobase and backbone-modified peptide nucleic acids.

    Science.gov (United States)

    Hnedzko, Dziyana; McGee, Dennis W; Karamitas, Yannis A; Rozners, Eriks

    2017-01-01

    Sequence-selective recognition of complex RNAs in live cells could find broad applications in biology, biomedical research, and biotechnology. However, specific recognition of structured RNA is challenging, and generally applicable and effective methods are lacking. Recently, we found that peptide nucleic acids (PNAs) were unusually well-suited ligands for recognition of double-stranded RNAs. Herein, we report that 2-aminopyridine (M) modified PNAs and their conjugates with lysine and arginine tripeptides form strong (Ka = 9.4 to 17 × 10(7) M(-1)) and sequence-selective triple helices with RNA hairpins at physiological pH and salt concentration. The affinity of PNA-peptide conjugates for the matched RNA hairpins was unusually high compared to the much lower affinity for DNA hairpins of the same sequence (Ka = 0.05 to 1.1 × 10(7) M(-1)). The binding of double-stranded RNA by M-modified PNA-peptide conjugates was a relatively fast process (kon = 2.9 × 10(4) M(-1) sec(-1)) compared to the notoriously slow triple helix formation by oligodeoxynucleotides (kon ∼ 10(3) M(-1) sec(-1)). M-modified PNA-peptide conjugates were not cytotoxic and were efficiently delivered in the cytosol of HEK293 cells at 10 µM. Surprisingly, M-modified PNAs without peptide conjugation were also taken up by HEK293 cells, which, to the best of our knowledge, is the first example of heterocyclic base modification that enhances the cellular uptake of PNA. Our results suggest that M-modified PNA-peptide conjugates are promising probes for sequence-selective recognition of double-stranded RNA in live cells and other biological systems.

  7. Deformability in the cleavage site of primary microRNA is not sensed by the double-stranded RNA binding domains in the microprocessor component DGCR8.

    Science.gov (United States)

    Quarles, Kaycee A; Chadalavada, Durga; Showalter, Scott A

    2015-06-01

    The prevalence of double-stranded RNA (dsRNA) in eukaryotic cells has only recently been appreciated. Of interest here, RNA silencing begins with dsRNA substrates that are bound by the dsRNA-binding domains (dsRBDs) of their processing proteins. Specifically, processing of microRNA (miRNA) in the nucleus minimally requires the enzyme Drosha and its dsRBD-containing cofactor protein, DGCR8. The smallest recombinant construct of DGCR8 that is sufficient for in vitro dsRNA binding, referred to as DGCR8-Core, consists of its two dsRBDs and a C-terminal tail. As dsRBDs rarely recognize the nucleotide sequence of dsRNA, it is reasonable to hypothesize that DGCR8 function is dependent on the recognition of specific structural features in the miRNA precursor. Previously, we demonstrated that noncanonical structural elements that promote RNA flexibility within the stem of miRNA precursors are necessary for efficient in vitro cleavage by reconstituted Microprocessor complexes. Here, we combine gel shift assays with in vitro processing assays to demonstrate that neither the N-terminal dsRBD of DGCR8 in isolation nor the DGCR8-Core construct is sensitive to the presence of noncanonical structural elements within the stem of miRNA precursors, or to single-stranded segments flanking the stem. Extending DGCR8-Core to include an N-terminal heme-binding region does not change our conclusions. Thus, our data suggest that although the DGCR8-Core region is necessary for dsRNA binding and recruitment to the Microprocessor, it is not sufficient to establish the previously observed connection between RNA flexibility and processing efficiency.

  8. Screening for plant viruses by next generation sequencing using a modified double strand RNA extraction protocol with an internal amplification control.

    Science.gov (United States)

    Kesanakurti, Prasad; Belton, Mark; Saeed, Hanaa; Rast, Heidi; Boyes, Ian; Rott, Michael

    2016-10-01

    The majority of plant viruses contain RNA genomes. Detection of viral RNA genomes in infected plant material by next generation sequencing (NGS) is possible through the extraction and sequencing of total RNA, total RNA devoid of ribosomal RNA, small RNA interference (RNAi) molecules, or double stranded RNA (dsRNA). Plants do not typically produce high molecular weight dsRNA, therefore the presence of dsRNA makes it an attractive target for plant virus diagnostics. The sensitivity of NGS as a diagnostic method demands an effective dsRNA protocol that is both representative of the sample and minimizes sample cross contamination. We have developed a modified dsRNA extraction protocol that is more efficient compared to traditional protocols, requiring reduced amounts of starting material, that is less prone to sample cross contamination. This was accomplished by using bead based homogenization of plant material in closed, disposable 50ml tubes. To assess the quality of extraction, we also developed an internal control by designing a real-time (quantitative) PCR (qPCR) assay that targets endornaviruses present in Phaseolus vulgaris cultivar Black Turtle Soup (BTS).

  9. Diphosphates at the 5' end of the positive strand of yeast L-A double-stranded RNA virus as a molecular self-identity tag.

    Science.gov (United States)

    Fujimura, Tsutomu; Esteban, Rosa

    2016-10-01

    The 5'end of RNA conveys important information on self-identity. In mammalian cells, double-stranded RNA (dsRNA) with 5'di- or triphosphates generated during virus infection is recognized as foreign and elicits the host innate immune response. Here, we analyze the 5' ends of the dsRNA genome of the yeast L-A virus. The positive strand has largely diphosphates with a minor amount of triphosphates, while the negative strand has only diphosphates. Although the virus can produce capped transcripts by cap snatching, neither strand carried a cap structure, suggesting that only non-capped transcripts serve as genomic RNA for encapsidation. We also found that the 5' diphosphates of the positive but not the negative strand within the dsRNA genome are crucial for transcription in vitro. Furthermore, the presence of a cap structure in the dsRNA abrogated its template activity. Given that the 5' diphosphates of the transcripts are also essential for cap acquisition and that host cytosolic RNAs (mRNA, rRNA, and tRNA) are uniformly devoid of 5' pp-structures, the L-A virus takes advantage of its 5' terminal diphosphates, using them as a self-identity tag to propagate in the host cytoplasm.

  10. Folate deficiency facilitates recruitment of upstream binding factor to hot spots of DNA double-strand breaks of rRNA genes and promotes its transcription.

    Science.gov (United States)

    Xie, Qiu; Li, Caihua; Song, Xiaozhen; Wu, Lihua; Jiang, Qian; Qiu, Zhiyong; Cao, Haiyan; Yu, Kaihui; Wan, Chunlei; Li, Jianting; Yang, Feng; Huang, Zebing; Niu, Bo; Jiang, Zhengwen; Zhang, Ting

    2016-12-06

    The biogenesis of ribosomes in vivo is an essential process for cellular functions. Transcription of ribosomal RNA (rRNA) genes is the rate-limiting step in ribosome biogenesis controlled by environmental conditions. Here, we investigated the role of folate antagonist on changes of DNA double-strand breaks (DSBs) landscape in mouse embryonic stem cells. A significant DSB enhancement was detected in the genome of these cells and a large majority of these DSBs were found in rRNA genes. Furthermore, spontaneous DSBs in cells under folate deficiency conditions were located exclusively within the rRNA gene units, representing a H3K4me1 hallmark. Enrichment H3K4me1 at the hot spots of DSB regions enhanced the recruitment of upstream binding factor (UBF) to rRNA genes, resulting in the increment of rRNA genes transcription. Supplement of folate resulted in a restored UBF binding across DNA breakage sites of rRNA genes, and normal rRNA gene transcription. In samples from neural tube defects (NTDs) with low folate level, up-regulation of rRNA gene transcription was observed, along with aberrant UBF level. Our results present a new view by which alterations in folate levels affects DNA breakage through epigenetic control leading to the regulation of rRNA gene transcription during the early stage of development.

  11. Effects of double-stranded RNA on virulence of Paecilomyces fumosoroseus (Deuteromycotina: Hyphomycetes against the silverleaf whitefly, Bemisia tabaci strain B (Homoptera: Aleyrodidae

    Directory of Open Access Journals (Sweden)

    Andréia Cristiane Souza Azevedo

    2000-03-01

    Full Text Available Bands of double-stranded RNA (dsRNA were detected in three out of twelve isolates of Paecilomyces fumosoroseus. Identity of these bands was confirmed by RNAse, DNAse and S1 nuclease treatments. The cure of dsRNA for one isolate (P92 was successfully carried out for a single conidium subculture. Isogenic strains, with or without dsRNA, were submitted to virulence tests against the whitefly Bemisia tabaci strain B. In contrast to findings for some phytopathogenic fungi, these dsRNA fragments did not cause hypovirulence in P. fumosoroseus.Bandas de dsRNA foram detectadas em três dos doze isolados de Paecilomyces fumosoroseus. A identidade destas bandas foi provada através de tratamentos com RNAse, DNAse e S1 nuclease. A cura do dsRNA para um dos isolados (P92 foi obtida através do isolamento de colônias monospóricas. Linhagens isogênicas, com e sem dsRNA, foram submetidas ao teste de virulência contra a mosca branca Bemisia tabaci biotipo B. Ao contrário do que ocorre para vários fungos fitopatogênicos, os fragmentos de dsRNA não causaram hipovirulência em P. fumosoroseus.

  12. Transient silencing mediated by in vitro synthesized double-stranded RNA indicates that PsCdc14 is required for sporangial development in a soybean root rot pathogen.

    Science.gov (United States)

    Zhao, Wei; Yang, Xinyu; Dong, Suomeng; Sheng, Yuting; Wang, Yuanchao; Zheng, Xiaobo

    2011-12-01

    In many eukaryotic organisms, Cdc14 phosphatase regulates multiple biological events during anaphase and is essential for mitosis. It has been shown that Cdc14 is required for sporulation in the potato blight pathogen Phytophthora infestans; however, the role that the Cdc14 homolog (PsCdc14) plays in the soil-borne soybean root rot pathogen P. sojae remains ambiguous. PsCdc14 is highly expressed in sporulation, zoospore, and cyst life stages, but not in vegetative mycelia and infection stages, suggesting that it contributes to asexual reproduction and thus the spread of the disease. Double-stranded RNA (dsRNA) mediates gene silencing, a post-transcriptional and highly conserved process in eukaryotes, involving specific gene silencing through degradation of target mRNA. We combined in vitro dsRNA synthesis and a polyethylene glycol-mediated transformation system to construct a dsRNA-mediated transient gene silencing system; and then performed a functional analysis of PsCdc14 in P. sojae. PsCdc14 mRNA was dramatically reduced in transformants after protoplasts were exposed in in vitro synthesized PsCdc14 dsRNA, resulting in low sporangial production and abnormal development in P. sojae silencing lines. Furthermore, dsRNA-mediated transient gene silencing could enable elucidation of P. sojae rapid gene function, facilitating our understanding of the development and pathogenicity mechanisms of this oomycete fungus.

  13. PI3K-delta mediates double-stranded RNA-induced upregulation of B7-H1 in BEAS-2B airway epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Kan-o, Keiko [Research Institute for Diseases of the Chest, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582 (Japan); Matsumoto, Koichiro, E-mail: koichi@kokyu.med.kyushu-u.ac.jp [Research Institute for Diseases of the Chest, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582 (Japan); Asai-Tajiri, Yukari; Fukuyama, Satoru; Hamano, Saaka; Seki, Nanae; Nakanishi, Yoichi [Research Institute for Diseases of the Chest, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582 (Japan); Inoue, Hiromasa [Research Institute for Diseases of the Chest, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582 (Japan); Department of Pulmonary Medicine, Graduate School of Medical and Dental Sciences, Kagoshima University, 8-35-1 Sakuragaoka, Kagoshima 890-8520 (Japan)

    2013-05-31

    Highlights: •Double-stranded RNA upregulates B7-H1 on BEAS-2B airway epithelial cells. •The upregulation of B7-H1 is attenuated by inhibition of PI3Kδ isoform. •PI3Kδ-mediated upregulation of B7-H1 is independent of NF-κB activation. •Inhibition of PI3Kδ may prevent persistent viral infection induced by B7-H1. -- Abstract: Airway viral infection disturbs the health-related quality of life. B7-H1 (also known as PD-L1) is a coinhibitory molecule associated with the escape of viruses from the mucosal immunity, leading to persistent infection. Most respiratory viruses generate double-stranded (ds) RNA during replication. The stimulation of cultured airway epithelial cells with an analog of viral dsRNA, polyinosinic-polycytidylic acid (poly IC) upregulates the expression of B7-H1 via activation of the nuclear factor κB(NF-κB). The mechanism of upregulation was investigated in association with phosphatidylinositol 3-kinases (PI3Ks). Poly IC-induced upregulation of B7-H1 was profoundly suppressed by a pan-PI3K inhibitor and partially by an inhibitor or a small interfering (si)RNA for PI3Kδ in BEAS-2B cells. Similar results were observed in the respiratory syncytial virus-infected cells. The expression of p110δ was detected by Western blot and suppressed by pretreatment with PI3Kδ siRNA. The activation of PI3Kδ is typically induced by oxidative stress. The generation of reactive oxygen species was increased by poly IC. Poly IC-induced upregulation of B7-H1 was attenuated by N-acetyl-L-cysteine, an antioxidant, or by oxypurinol, an inhibitor of xanthine oxidase. Poly IC-induced activation of NF-κB was suppressed by a pan-PI3K inhibitor but not by a PI3Kδ inhibitor. These results suggest that PI3Kδ mediates dsRNA-induced upregulation of B7-H1 without affecting the activation of NF-κB.

  14. Double strand break repair by capture of retrotransposon sequences and reverse-transcribed spliced mRNA sequences in mouse zygotes.

    Science.gov (United States)

    Ono, Ryuichi; Ishii, Masayuki; Fujihara, Yoshitaka; Kitazawa, Moe; Usami, Takako; Kaneko-Ishino, Tomoko; Kanno, Jun; Ikawa, Masahito; Ishino, Fumitoshi

    2015-07-28

    The CRISPR/Cas system efficiently introduces double strand breaks (DSBs) at a genomic locus specified by a single guide RNA (sgRNA). The DSBs are subsequently repaired through non-homologous end joining (NHEJ) or homologous recombination (HR). Here, we demonstrate that DSBs introduced into mouse zygotes by the CRISPR/Cas system are repaired by the capture of DNA sequences deriving from retrotransposons, genomic DNA, mRNA and sgRNA. Among 93 mice analysed, 57 carried mutant alleles and 22 of them had long de novo insertion(s) at DSB-introduced sites; two were spliced mRNAs of Pcnt and Inadl without introns, indicating the involvement of reverse transcription (RT). Fifteen alleles included retrotransposons, mRNAs, and other sequences without evidence of RT. Two others were sgRNAs with one containing T7 promoter-derived sequence suggestive of a PCR product as its origin. In conclusion, RT-product-mediated DSB repair (RMDR) and non-RMDR repair were identified in the mouse zygote. We also confirmed that both RMDR and non-RMDR take place in CRISPR/Cas transfected NIH-3T3 cells. Finally, as two de novo MuERV-L insertions in C57BL/6 mice were shown to have characteristic features of RMDR in natural conditions, we hypothesize that RMDR contributes to the emergence of novel DNA sequences in the course of evolution.

  15. Modeling of the catalytic core of Arabidopsis thaliana Dicer-like 4 protein and its complex with double-stranded RNA.

    Science.gov (United States)

    Mickiewicz, Agnieszka; Sarzyńska, Joanna; Miłostan, Maciej; Kurzyńska-Kokorniak, Anna; Rybarczyk, Agnieszka; Łukasiak, Piotr; Kuliński, Tadeusz; Figlerowicz, Marek; Błażewicz, Jacek

    2017-02-01

    Plant Dicer-like proteins (DCLs) belong to the Ribonuclease III (RNase III) enzyme family. They are involved in the regulation of gene expression and antiviral defense through RNA interference pathways. A model plant, Arabidopsis thaliana encodes four DCL proteins (AtDCL1-4) that produce different classes of small regulatory RNAs. Our studies focus on AtDCL4 that processes double-stranded RNAs (dsRNAs) into 21 nucleotide trans-acting small interfering RNAs. So far, little is known about the structures of plant DCLs and the complexes they form with dsRNA. In this work, we present models of the catalytic core of AtDCL4 and AtDCL4-dsRNA complex constructed by computational methods. We built a homology model of the catalytic core of AtDCL4 comprising Platform, PAZ, Connector helix and two RNase III domains. To assemble the AtDCL4-dsRNA complex two modeling approaches were used. In the first method, to establish conformations that allow building a consistent model of the complex, we used Normal Mode Analysis for both dsRNA and AtDCL4. The second strategy involved template-based approach for positioning of the PAZ domain and manual arrangement of the Connector helix. Our results suggest that the spatial orientation of the Connector helix, Platform and PAZ relative to the RNase III domains is crucial for measuring dsRNA of defined length. The modeled complexes provide information about interactions that may contribute to the relative orientations of these domains and to dsRNA binding. All these information can be helpful for understanding the mechanism of AtDCL4-mediated dsRNA recognition and binding, to produce small RNA of specific size.

  16. Double-stranded RNA induces biphasic STAT1 phosphorylation by both type I interferon (IFN)-dependent and type I IFN-independent pathways.

    Science.gov (United States)

    Dempoya, Junichi; Matsumiya, Tomoh; Imaizumi, Tadaatsu; Hayakari, Ryo; Xing, Fei; Yoshida, Hidemi; Okumura, Ken; Satoh, Kei

    2012-12-01

    Upon viral infection, pattern recognition receptors sense viral nucleic acids, leading to the production of type I interferons (IFNs), which initiate antiviral activities. Type I IFNs bind to their cognate receptor, IFNAR, resulting in the activation of signal-transducing activators of transcription 1 (STAT1). Thus, it has long been thought that double-stranded RNA (dsRNA)-induced STAT1 phosphorylation is mediated by the transactivation of type I IFN signaling. Foreign RNA, such as viral RNA, in cells is sensed by the cytoplasmic sensors retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA-5). In this study, we explored the molecular mechanism responsible for STAT1 phosphorylation in response to the sensing of dsRNA by cytosolic RNA sensors. Polyinosinic-poly(C) [poly(I:C)], a synthetic dsRNA that is sensed by both RIG-I and MDA-5, induces STAT1 phosphorylation. We found that the poly(I:C)-induced initial phosphorylation of STAT1 is dependent on the RIG-I pathway and that MDA-5 is not involved in STAT1 phosphorylation. Furthermore, pretreatment of the cells with neutralizing antibody targeting the IFN receptor suppressed the initial STAT1 phosphorylation in response to poly(I:C), suggesting that this initial phosphorylation event is predominantly type I IFN dependent. In contrast, neither the known RIG-I pathway nor type I IFN is involved in the late phosphorylation of STAT1. In addition, poly(I:C) stimulated STAT1 phosphorylation in type I IFN receptor-deficient U5A cells with delayed kinetics. Collectively, our study provides evidence of a comprehensive regulatory mechanism in which dsRNA induces STAT1 phosphorylation, indicating the importance of STAT1 in maintaining very tight regulation of the innate immune system.

  17. Clathrin-dependent endocytosis plays a predominant role in cellular uptake of double-stranded RNA in the red flour beetle.

    Science.gov (United States)

    Xiao, Da; Gao, Xiwu; Xu, Jiaping; Liang, Xiao; Li, Qingqing; Yao, Jianxiu; Zhu, Kun Yan

    2015-05-01

    RNA interference (RNAi) is a highly conserved gene regulatory mechanism in eukaryotic organisms; however, an understanding of mechanisms of cellular uptake of double-stranded RNA (dsRNA) in different organisms remains elusive. By using pharmacological inhibitors of different endocytic pathways in conjunction with RNAi of a marker gene (lethal giant larvae, TcLgl) in the red flour beetle (Tribolium castaneum), we demonstrated that two inhibitors (chlorpromazine and bafilomycin-A1) of clathrin-dependent endocytosis can nearly abolish or significantly diminish RNAi of TcLgl, whereas methyl-β-cyclodextrin and cytochalasin-D, known to inhibit other endocytic pathways, showed no effect on RNAi of TcLgl. By using Cy3-labeled TcLgl dsRNA, we observed significantly reduced cellular uptake of TcLgl dsRNA in midgut cells after larvae were injected with each of the two clathrin-dependent endocytosis inhibitors. By using an "RNAi of RNAi" strategy, we further demonstrated that suppression of each transcript of the four key genes encoding clathrin heavy chain (TcChc), clathrin coat assembly protein AP50 (TcAP50), vacuolar (H(+))-ATPase subunit H (TcVhaSFD) and a ras-related protein (TcRab7) in clathrin-dependent endocytosis by RNAi can significantly impair RNAi of TcLgl. These results support our conclusion that clathrin-dependent endocytosis is a major mechanism in cellular uptake of dsRNA in T. castaneum. Our study also provides new insights into improving RNAi efficiency by enhancing dsRNA endosomal release.

  18. Interferons specifically suppress the translation from the internal ribosome entry site of hepatitis C virus through a double-stranded RNA-activated protein kinase-independent pathway.

    Science.gov (United States)

    Kato, Jun; Kato, Naoya; Moriyama, Masaru; Goto, Tadashi; Taniguchi, Hiroyoshi; Shiratori, Yasushi; Omata, Masao

    2002-07-15

    Interferon (IFN) therapy is used worldwide as the best available treatment for hepatitis C virus (HCV) infection; however, little is known about how IFN or other drugs work against liver diseases. The effect of 6 drugs (glycyrrhizin, ursodeoxycholic acid, ribavirin, methylprednisolone, IFN-alpha, and IFN-beta) on HCV RNA translation from the HCV internal ribosome entry site (IRES) was investigated, using a bicistronic reporter containing the HCV IRES. IFNs suppressed both cap-dependent and HCV IRES-dependent translation, with HCV IRES-dependent translation being more significantly suppressed. In contrast to HCV IRES, IFN did not suppress either foot-and-mouth disease virus IRES-dependent or encephalomyocarditis virus IRES-dependent translation more than it suppressed cap-dependent translation. Moreover, dominant inhibition of HCV IRES-dependent over cap-dependent translation depended neither on the double-stranded RNA-activated protein kinase activation nor on La protein function. These results indicate a novel antiviral effect of IFNs against HCV.

  19. Double-stranded RNA induces inflammation via the NF-κB pathway and inflammasome activation in the outer root sheath cells of hair follicles

    Science.gov (United States)

    Shin, Jung-Min; Choi, Dae-Kyoung; Sohn, Kyung-Cheol; Kim, Soo-Yeon; Min Ha, Jeong; Ho Lee, Young; Im, Myung; Seo, Young-Joon; Deok Kim, Chang; Lee, Jeung-Hoon; Lee, Young

    2017-01-01

    Alopecia areata (AA), a chronic, relapsing, hair-loss disorder, is considered to be a T cell-mediated autoimmune disease. It affects approximately 1.7% of the population, but its precise pathogenesis remains to be elucidated. Despite the recent attention focused on the roles of inflammasomes in the pathogenesis of autoinflammatory diseases, little is known about inflammasome activation in AA. Thus, in this study, we investigated the pattern of NLRP3 inflammasome activation in the outer root sheath (ORS) cells of hair follicles. We found that interleukin (IL)-1β and caspase-1 expression was increased in hair follicle remnants and inflammatory cells of AA tissue specimens. After stimulation of ORS cells with the double-stranded (ds)RNA mimic polyinosinic:polycytidylic acid (poly[I:C]), the activation of caspase-1 and secretion of IL-1β were enhanced. Moreover, NLRP3 knockdown decreased this poly(I:C)-induced IL-1β production. Finally, we found that high-mobility group box 1 (HMGB1) translocated from the nucleus to the cytosol and was secreted into the extracellular space by inflammasome activation. Taken together, these findings suggest that ORS cells are important immunocompetent cells that induce NLRP3 inflammasomes. In addition, dsRNA-induced IL-1β and HMGB1 secretion from ORS cells may contribute to clarifying the pathogenesis and therapeutic targets of AA. PMID:28266599

  20. Characterizing the mechanism of action of double-stranded RNA activity against western corn rootworm (Diabrotica virgifera virgifera LeConte.

    Directory of Open Access Journals (Sweden)

    Renata Bolognesi

    Full Text Available RNA interference (RNAi has previously been shown to be effective in western corn rootworm (WCR, Diabrotica virgifera virgifera LeConte larvae via oral delivery of synthetic double-stranded RNA (dsRNA in an artificial diet bioassay, as well as by ingestion of transgenic corn plant tissues engineered to express dsRNA. Although the RNAi machinery components appear to be conserved in Coleopteran insects, the key steps in this process have not been reported for WCR. Here we characterized the sequence of events that result in mortality after ingestion of a dsRNA designed against WCR larvae. We selected the Snf7 ortholog (DvSnf7 as the target mRNA, which encodes an essential protein involved in intracellular trafficking. Our results showed that dsRNAs greater than or equal to approximately 60 base-pairs (bp are required for biological activity in artificial diet bioassays. Additionally, 240 bp dsRNAs containing a single 21 bp match to the target sequence were also efficacious, whereas 21 bp short interfering (si RNAs matching the target sequence were not. This result was further investigated in WCR midgut tissues: uptake of 240 bp dsRNA was evident in WCR midgut cells while a 21 bp siRNA was not, supporting the size-activity relationship established in diet bioassays. DvSnf7 suppression was observed in a time-dependent manner with suppression at the mRNA level preceding suppression at the protein level when a 240 bp dsRNA was fed to WCR larvae. DvSnf7 suppression was shown to spread to tissues beyond the midgut within 24 h after dsRNA ingestion. These events (dsRNA uptake, target mRNA and protein suppression, systemic spreading, growth inhibition and eventual mortality comprise the overall mechanism of action by which DvSnf7 dsRNA affects WCR via oral delivery and provides insights as to how targeted dsRNAs in general are active against insects.

  1. Distinct signaling pathways leading to the induction of human β-defensin 2 by stimulating an electrolyticaly-generated acid functional water and double strand RNA in oral epithelial cells.

    Science.gov (United States)

    Gojoubori, Takahiro; Nishio, Yukina; Asano, Masatake; Nishida, Tetsuya; Komiyama, Kazuo; Ito, Koichi

    2014-04-01

    Defensins, a major family of cationic antimicrobial peptides, play important roles in innate immunity. In the present study, we investigated whether double-stranded RNA (dsRNA), a by-product of RNA virus replication, can induce human β-defensins-2 (hBD-2) expression in oral epithelial cells (OECs). We also examined the hBD-2-inducible activity of acid-electrolyzed functional water (FW). The results indicated that both dsRNA- and FW-induced hBD-2 expression in OECs. The induction efficiency was much higher for FW than for dsRNA. FW-induced production of hBD-2 was clearly observed by immunofluorescence staining. A luciferase assay was performed with 1.2 kb of the 5'-untranslated region (5'-UTR) of the hBD-2 gene. The results indicated that the nuclear factor-kappa B (NF-κB)-binding site proximal to the translation initiation site was indispensable for dsRNA-stimulated hBD-2 expression, but not in the case of FW. Moreover, FW-stimulated hBD-2 expression did not depend on NF-κB activity; instead, FW inhibited NF-κB activity. Pretreatment of the cells with specific inhibitors against NF-κB further confirmed NF-κB-independent hBD-2 induction by FW. In analogy to the results for intestinal epithelial cells (IECs), the dsRNA signal, but not FW, was sensed by toll-like receptor 3 (TLR3) in OECs. These results suggested that hBD-2 expression induced by dsRNA and FW is regulated by distinct mechanisms in OECs.

  2. The SID-1 double-stranded RNA transporter is not required for systemic RNAi in the migratory locust.

    Science.gov (United States)

    Luo, Yuan; Wang, Xianhui; Yu, Dan; Kang, Le

    2012-05-01

    Systemic RNAi, the spreading of RNAi effects to cells and tissues that have not initially encountered a dsRNA trigger, is a common phenomenon in many organisms. However, the underlying mechanisms of systemic RNAi remain largely unknown. Here, we studied the characteristics and possible mechanisms of systemic RNAi in Locusta migratoria. We observed that the locust has pronounced sensitive systemic RNAi in response to dsRNA injection for both broadly-expressed as well as tissue-specific genes. Only a 30 pg (dsRNA / mg tissues) dose could induce significant systemic RNAi effects. Only one SID-1 ortholog (LmSID-1) was identified in the locust genome, which exhibited a progressively increasing expression pattern with development and its expression was enriched in the gonad. To test the role of LmSID-1 in systemic RNAi, we performed in vitro and in vivo experiments. The results from in vivo experiments showed that silencing of LmSID-1 gene dose not influence RNAi effects of other genes. The results from in vitro experiments confirmed that the expression of the LmSID-1 protein in Drosophila S2 cells could not enhance dsRNA uptake. Thus, these findings imply the existence of alternative mechanisms underlying insect systemic RNAi, which may be different from Caenorhabditis elegans or mammals.

  3. Association of the Endobiont Double-Stranded RNA Virus LRV1 With Treatment Failure for Human Leishmaniasis Caused by Leishmania braziliensis in Peru and Bolivia.

    Science.gov (United States)

    Adaui, Vanessa; Lye, Lon-Fye; Akopyants, Natalia S; Zimic, Mirko; Llanos-Cuentas, Alejandro; Garcia, Lineth; Maes, Ilse; De Doncker, Simonne; Dobson, Deborah E; Arevalo, Jorge; Dujardin, Jean-Claude; Beverley, Stephen M

    2016-01-01

    Cutaneous and mucosal leishmaniasis, caused in South America by Leishmania braziliensis, is difficult to cure by chemotherapy (primarily pentavalent antimonials [Sb(V)]). Treatment failure does not correlate well with resistance in vitro, and the factors responsible for treatment failure in patients are not well understood. Many isolates of L. braziliensis (>25%) contain a double-stranded RNA virus named Leishmaniavirus 1 (LRV1), which has also been reported in Leishmania guyanensis, for which an association with increased pathology, metastasis, and parasite replication was found in murine models. Here we probed the relationship of LRV1 to drug treatment success and disease in 97 L. braziliensis-infected patients from Peru and Bolivia. In vitro cultures were established, parasites were typed as L. braziliensis, and the presence of LRV1 was determined by reverse transcription-polymerase chain reaction, followed by sequence analysis. LRV1 was associated significantly with an increased risk of treatment failure (odds ratio, 3.99; P = .04). There was no significant association with intrinsic Sb(V) resistance among parasites, suggesting that treatment failure arises from LRV1-mediated effects on host metabolism and/or parasite survival. The association of LRV1 with clinical drug treatment failure could serve to guide more-effective treatment of tegumentary disease caused by L. braziliensis.

  4. Sustained Action of Ceramide on the Insulin Signaling Pathway in Muscle Cells: IMPLICATION OF THE DOUBLE-STRANDED RNA-ACTIVATED PROTEIN KINASE.

    Science.gov (United States)

    Hage Hassan, Rima; Pacheco de Sousa, Ana Catarina; Mahfouz, Rana; Hainault, Isabelle; Blachnio-Zabielska, Agnieszka; Bourron, Olivier; Koskas, Fabien; Górski, Jan; Ferré, Pascal; Foufelle, Fabienne; Hajduch, Eric

    2016-02-01

    In vivo, ectopic accumulation of fatty acids in muscles leads to alterations in insulin signaling at both the IRS1 and Akt steps. However, in vitro treatments with saturated fatty acids or their derivative ceramide demonstrate an effect only at the Akt step. In this study, we adapted our experimental procedures to mimic the in vivo situation and show that the double-stranded RNA-dependent protein kinase (PKR) is involved in the long-term effects of saturated fatty acids on IRS1. C2C12 or human muscle cells were incubated with palmitate or directly with ceramide for short or long periods, and insulin signaling pathway activity was evaluated. PKR involvement was assessed through pharmacological and genetic studies. Short-term treatments of myotubes with palmitate, a ceramide precursor, or directly with ceramide induce an inhibition of Akt, whereas prolonged periods of treatment show an additive inhibition of insulin signaling through increased IRS1 serine 307 phosphorylation. PKR mRNA, protein, and phosphorylation are increased in insulin-resistant muscles. When PKR activity is reduced (siRNA or a pharmacological inhibitor), serine phosphorylation of IRS1 is reduced, and insulin-induced phosphorylation of Akt is improved. Finally, we show that JNK mediates ceramide-activated PKR inhibitory action on IRS1. Together, in the long term, our results show that ceramide acts at two distinct levels of the insulin signaling pathway (IRS1 and Akt). PKR, which is induced by both inflammation signals and ceramide, could play a major role in the development of insulin resistance in muscle cells.

  5. High rates of double-stranded RNA viruses and Mycoplasma hominis in Trichomonas vaginalis clinical isolates in South Brazil.

    Science.gov (United States)

    da Luz Becker, Débora; dos Santos, Odelta; Frasson, Amanda Piccoli; de Vargas Rigo, Graziela; Macedo, Alexandre José; Tasca, Tiana

    2015-08-01

    Trichomonas vaginalis is the etiological agent of trichomoniasis, the most common non-viral sexually transmitted disease (STD) in world, with 276.4 million new cases each year. T. vaginalis can be naturally infected with Mycoplasma hominis and Trichomonasvirus species. This study aimed to evaluate the prevalence of T. vaginalis infected with four distinct T. vaginalis viruses (TVVs) and M. hominis among isolates from patients in Porto Alegre city, South Brazil. An additional goal of this study was to investigate whether there is association between metronidazole resistance and the presence of M. hominis during TVV infection. The RNA expression level of the pyruvate ferredoxin oxidoreductase (PFOR) gene was also evaluated among metronidazole-resistant and metronidazole-sensitive T. vaginalis isolates. A total of 530 urine samples were evaluated, and 5.7% samples were positive for T. vaginalis infection. Among them, 4.51% were isolated from female patients and 1.12% were from male patients. Remarkably, the prevalence rates of M. hominis and TVV-positive T. vaginalis isolates were 56.7% and 90%, respectively. Most of the T. vaginalis isolates were metronidazole-sensitive (86.7%), and only four isolates (13.3%) were resistant. There is no statistically significant association between infection by M. hominis and infection by TVVs. Our results refute the hypothesis that the presence of the M. hominis and TVVs is enough to confer metronidazole resistance to T. vaginalis isolates. Additionally, the role of PFOR RNA expression levels in metronidazole resistance as the main mechanism of resistance to metronidazole could not be established. This study is the first report of the T. vaginalis infection by M. hominis and TVVs in a large collection of isolates from South Brazil.

  6. Fugu double U6 promoter-driven long double-stranded RNA inhibits proliferation of viral hemorrhagic septicemia virus (VHSV) in fish cell lines.

    Science.gov (United States)

    Kim, Min Sun; Jee, Bo Young; Cho, Mi Young; Kim, Jin Woo; Jeong, Hyun Do; Kim, Ki Hong

    2012-06-01

    A long double-stranded RNA (dsRNA)-producing vector driven by fugu double U6 promotors, in which the two promoters were arranged in a head-to-head fashion, was newly constructed. To determine whether the DNA-vector-based long dsRNAs can induce sequence-specific RNA interference (RNAi), Epithelioma papulosum cyprini (EPC) cells and chinook salmon embryonic (CHSE-214) cells were transfected with the long dsRNA vector targeting the G gene of VHSV, and its effect on expression of the G gene and viral proliferation was investigated. The sequence-specific inhibitory effect was further confirmed by analysis of interferon (IFN)-triggered Mx1 gene expression and cross-protection against infectious hematopoietic necrosis virus (IHNV). The fugu double U6 promoter-driven vector successfully produced long dsRNAs in EPC cells, a system that allows continuous production of long dsRNAs in transfected cells. The plasmid-based long dsRNAs targeting the VHSV G gene effectively suppressed G gene expression, but control dsRNAs targeting the EGFP gene did not. Furthermore, there was no significant difference in Mx gene expression between cells transfected with the long dsRNA-producing vector and those transfected with the control empty vector. These results suggest that G gene expression was suppressed not by type-I-IFN-mediated nonspecific inhibition but in a sequence-specific manner. Both EPC and CHSE-214 cells transfected with plasmids producing long dsRNAs targeting the VHSV G gene were protected against VHSV infection but were not protected against IHNV infection, suggesting sequence-specific RNAi-mediated inhibition of viral proliferation. In conclusion, we show, for the first time, long-dsRNA-mediated RNAi in fish cells. The DNA-vector-based long dsRNAs may provide an efficient tool for analysis of gene function in fish cells without preliminary burdensome work for selection of effective siRNA clones, and it may be applied as an antiviral measure in cultured fish.

  7. Double-stranded RNA-binding protein DRB3 negatively regulates anthocyanin biosynthesis by modulating PAP1 expression in Arabidopsis thaliana.

    Science.gov (United States)

    Sawano, Hikaru; Matsuzaki, Takuma; Usui, Tomoyuki; Tabara, Midori; Fukudome, Akihito; Kanaya, Akihiro; Tanoue, Daichi; Hiraguri, Akihiro; Horiguchi, Gorou; Ohtani, Misato; Demura, Taku; Kozaki, Toshinori; Ishii, Kazuo; Moriyama, Hiromitsu; Fukuhara, Toshiyuki

    2017-01-01

    The model plant Arabidopsis thaliana has five double-stranded RNA-binding proteins (DRB1-DRB5), two of which, DRB1 and DRB4, are well characterized. In contrast, the functions of DRB2, DRB3 and DRB5 have yet to be elucidated. In this study, we tried to uncover their functions using drb mutants and DRB-over-expressed lines. In over-expressed lines of all five DRB genes, the over-expression of DRB2 or DRB3 (DRB2ox or DRB3ox) conferred a downward-curled leaf phenotype, but the expression profiles of ten small RNAs were similar to that of the wild-type (WT) plant. Phenotypes were examined in response to abiotic stresses. Both DRB2ox and DRB3ox plants exhibited salt-tolerance. When these plants were exposed to cold stress, drb2 and drb3 over-accumulated anthocyanin but DRB2ox and DRB3ox did not. Therefore, the over-expression of DRB2 or DRB3 had pleiotropic effects on host plants. Microarray and deep-sequencing analyses indicated that several genes encoding key enzymes for anthocyanin biosynthesis, including chalcone synthase (CHS), dihydroflavonol reductase (DFR) and anthocyanidin synthase (ANS), were down-regulated in DRB3ox plants. When DRB3ox was crossed with the pap1-D line, which is an activation-tagged transgenic line that over-expresses the key transcription factor PAP1 (Production of anthocyanin pigmentation1) for anthocyanin biosynthesis, over-expression of DRB3 suppressed the expression of PAP1, CHS, DFR and ANS genes. DRB3 negatively regulates anthocyanin biosynthesis by modulating the level of PAP1 transcript. Since two different small RNAs regulate PAP1 gene expression, a possible function of DRB3 for small RNA biogenesis is discussed.

  8. EGF receptor-targeted synthetic double-stranded RNA eliminates glioblastoma, breast cancer, and adenocarcinoma tumors in mice.

    Directory of Open Access Journals (Sweden)

    Alexei Shir

    2006-01-01

    Full Text Available BACKGROUND: Glioblastoma multiforme (GBM is the most lethal form of brain cancer. With the available treatments, survival does not exceed 12-14 mo from the time of diagnosis. We describe a novel strategy to selectively induce the death of glioblastoma cells and other cancer cells that over-express the EGF receptor. Using a non-viral delivery vector that homes to the EGF receptor, we target synthetic anti-proliferative dsRNA (polyinosine-cytosine [poly IC], a strong activator of apoptosis, selectively to cancer cells. METHODS AND FINDINGS: Poly IC was delivered by means of a non-viral vector: 25kDa polyethylenimine-polyethyleneglycol-EGF (PEI25-PEG-EGF. EGFR-targeted poly IC induced rapid apoptosis in the target cells in vitro and in vivo. Expression of several cytokines and "bystander killing" of untransfected tumor cells was detected in vitro and in vivo. Intra-tumoral delivery of the EGFR-targeted poly IC induced the complete regression of pre-established intracranial tumors in nude mice, with no obvious adverse toxic effects on normal brain tissue. A year after treatment completion the treated mice remain cancer-free and healthy. Similarly, non-viral delivery of poly IC completely eliminated pre-established breast cancer and adenocarcinoma xenografts derived from EGFR over-expressing cancer cell lines, suggesting that the strategy is applicable to other EGFR-over-expressing tumors. CONCLUSION: The strategy described has yielded an effective treatment of EGFR over-expressing GBM in an animal model. If this strategy is translated successfully to the clinical setting, it may actually offer help to GBM patients. Moreover the elimination of two additional EGFR over-expressing cancers in vivo suggests that in principle this strategy can be applied to treat other tumors that over-express EGFR.

  9. TODRA, a lncRNA at the RAD51 Locus, Is Oppositely Regulated to RAD51, and Enhances RAD51-Dependent DSB (Double Strand Break) Repair.

    Science.gov (United States)

    Gazy, Inbal; Zeevi, David A; Renbaum, Paul; Zeligson, Sharon; Eini, Lital; Bashari, Dana; Smith, Yoav; Lahad, Amnon; Goldberg, Michal; Ginsberg, Doron; Levy-Lahad, Ephrat

    2015-01-01

    Expression of RAD51, a crucial player in homologous recombination (HR) and DNA double-strand break (DSB) repair, is dysregulated in human tumors, and can contribute to genomic instability and tumor progression. To further understand RAD51 regulation we functionally characterized a long non-coding (lnc) RNA, dubbed TODRA (Transcribed in the Opposite Direction of RAD51), transcribed 69bp upstream to RAD51, in the opposite direction. We demonstrate that TODRA is an expressed transcript and that the RAD51 promoter region is bidirectional, supporting TODRA expression (7-fold higher than RAD51 in this assay, p = 0.003). TODRA overexpression in HeLa cells induced expression of TPIP, a member of the TPTE family which includes PTEN. Similar to PTEN, we found that TPIP co-activates E2F1 induction of RAD51. Analysis of E2F1's effect on the bidirectional promoter showed that E2F1 binding to the same site that promotes RAD51 expression, results in downregulation of TODRA. Moreover, TODRA overexpression induces HR in a RAD51-dependent DSB repair assay, and increases formation of DNA damage-induced RAD51-positive foci. Importantly, gene expression in breast tumors supports our finding that E2F1 oppositely regulates RAD51 and TODRA: increased RAD51 expression, which is associated with an aggressive tumor phenotype (e.g. negative correlation with positive ER (r = -0.22, p = 0.02) and positive PR status (r = -0.27, pDSB repair in malignancy. Importantly, gene expression in breast tumors supports our finding that E2F1 oppositely regulates RAD51 and TODRA: increased RAD51 expression, which is associated with an aggressive tumor phenotype (e.g. negative correlation with positive ER (r = -0.22, p = 0.02) and positive PR status (r = -0.27, pDSB repair in malignancy.

  10. Interaction of DAPI with double-stranded ribonucleic acids.

    OpenAIRE

    Manzini, G.; Xodo, L; Barcellona, M L; Quadrifoglio, F

    1985-01-01

    The interaction of DAPI with natural and synthetic double-stranded polyribonucleotides was studied with different optical and calorimetric methods. The results were similar to those obtained previously with double-stranded polydeoxynucleotides, i.e. two interaction modes, the first of which shows high affinity for AU clusters and consequent strong fluorescence enhancement. The results suggest caution in the use of DAPI as selective fluorescent staining agent for DNA in the presence of RNA. A ...

  11. Systemic RNA Interference Deficiency-1 (SID-1) Extracellular Domain Selectively Binds Long Double-stranded RNA and Is Required for RNA Transport by SID-1.

    Science.gov (United States)

    Li, Weiqiang; Koutmou, Kristin S; Leahy, Daniel J; Li, Min

    2015-07-31

    During systemic RNA interference (RNAi) in Caenorhabditis elegans, RNA spreads across different cells and tissues in a process that requires the systemic RNA interference deficient-1 (sid-1) gene, which encodes an integral membrane protein. SID-1 acts cell-autonomously and is required for cellular import of interfering RNAs. Heterologous expression of SID-1 in Drosophila Schneider 2 cells enables passive uptake of dsRNA and subsequent soaking RNAi. Previous studies have suggested that SID-1 may serve as an RNA channel, but its precise molecular role remains unclear. To test the hypothesis that SID-1 mediates a direct biochemical recognition of RNA molecule and subsequent permeation, we expressed the extracellular domain (ECD) of SID-1 and purified it to near homogeneity. Recombinant purified SID-1 ECD selectively binds dsRNA but not dsDNA in a length-dependent and sequence-independent manner. Genetic missense mutations in SID-1 ECD causal for deficient systemic RNAi resulted in significant reduction in its affinity for dsRNA. Furthermore, full-length proteins with these mutations decrease SID-1-mediated RNA transport efficiency, providing evidence that dsRNA binding to SID-1 ECD is related to RNA transport. To examine the functional similarity of mammalian homologs of SID-1 (SIDT1 and SIDT2), we expressed and purified mouse SIDT1 and SIDT2 ECDs. We show that they bind long dsRNA in vitro, supportive of dsRNA recognition. In summary, our study illustrates the functional importance of SID-1 ECD as a dsRNA binding domain that contributes to RNA transport.

  12. Transportable data from non-target arthropod field studies for the environmental risk assessment of genetically modified maize expressing an insecticidal double-stranded RNA.

    Science.gov (United States)

    Ahmad, Aqeel; Negri, Ignacio; Oliveira, Wladecir; Brown, Christopher; Asiimwe, Peter; Sammons, Bernard; Horak, Michael; Jiang, Changjian; Carson, David

    2016-02-01

    As part of an environmental risk assessment, the potential impact of genetically modified (GM) maize MON 87411 on non-target arthropods (NTAs) was evaluated in the field. MON 87411 confers resistance to corn rootworm (CRW; Diabrotica spp.) by expressing an insecticidal double-stranded RNA (dsRNA) transcript and the Cry3Bb1 protein and tolerance to the herbicide glyphosate by producing the CP4 EPSPS protein. Field trials were conducted at 14 sites providing high geographic and environmental diversity within maize production areas from three geographic regions including the U.S., Argentina, and Brazil. MON 87411, the conventional control, and four commercial conventional reference hybrids were evaluated for NTA abundance and damage. Twenty arthropod taxa met minimum abundance criteria for valid statistical analysis. Nine of these taxa occurred in at least two of the three regions and in at least four sites across regions. These nine taxa included: aphid, predatory earwig, lacewing, ladybird beetle, leafhopper, minute pirate bug, parasitic wasp, sap beetle, and spider. In addition to wide regional distribution, these taxa encompass the ecological functions of herbivores, predators and parasitoids in maize agro-ecosystems. Thus, the nine arthropods may serve as representative taxa of maize agro-ecosystems, and thereby support that analysis of relevant data generated in one region can be transportable for the risk assessment of the same or similar GM crop products in another region. Across the 20 taxa analyzed, no statistically significant differences in abundance were detected between MON 87411 and the conventional control for 123 of the 128 individual-site comparisons (96.1%). For the nine widely distributed taxa, no statistically significant differences in abundance were detected between MON 87411 and the conventional control. Furthermore, no statistically significant differences were detected between MON 87411 and the conventional control for 53 out of 56 individual

  13. TODRA, a lncRNA at the RAD51 Locus, Is Oppositely Regulated to RAD51, and Enhances RAD51-Dependent DSB (Double Strand Break) Repair

    Science.gov (United States)

    Renbaum, Paul; Zeligson, Sharon; Eini, Lital; Bashari, Dana; Smith, Yoav; Lahad, Amnon; Goldberg, Michal; Ginsberg, Doron; Levy-Lahad, Ephrat

    2015-01-01

    Expression of RAD51, a crucial player in homologous recombination (HR) and DNA double-strand break (DSB) repair, is dysregulated in human tumors, and can contribute to genomic instability and tumor progression. To further understand RAD51 regulation we functionally characterized a long non-coding (lnc) RNA, dubbed TODRA (Transcribed in the Opposite Direction of RAD51), transcribed 69bp upstream to RAD51, in the opposite direction. We demonstrate that TODRA is an expressed transcript and that the RAD51 promoter region is bidirectional, supporting TODRA expression (7-fold higher than RAD51 in this assay, p = 0.003). TODRA overexpression in HeLa cells induced expression of TPIP, a member of the TPTE family which includes PTEN. Similar to PTEN, we found that TPIP co-activates E2F1 induction of RAD51. Analysis of E2F1's effect on the bidirectional promoter showed that E2F1 binding to the same site that promotes RAD51 expression, results in downregulation of TODRA. Moreover, TODRA overexpression induces HR in a RAD51-dependent DSB repair assay, and increases formation of DNA damage-induced RAD51-positive foci. Importantly, gene expression in breast tumors supports our finding that E2F1 oppositely regulates RAD51 and TODRA: increased RAD51 expression, which is associated with an aggressive tumor phenotype (e.g. negative correlation with positive ER (r = -0.22, p = 0.02) and positive PR status (r = -0.27, p<0.001); positive correlation with ki67 status (r = 0.36, p = 0.005) and HER2 amplification (r = 0.41, p = 0.001)), correlates as expected with lower TODRA and higher E2F1 expression. However, although E2F1 induction resulted in TPIP downregulation in cell lines, we find that TPIP expression in tumors is not reduced despite higher E2F1 expression, perhaps contributing to increased RAD51 expression. Our results identify TPIP as a novel E2F1 co-activator, suggest a similar role for other TPTEs, and indicate that the TODRA lncRNA affects RAD51 dysregulation and RAD51

  14. Phenotypic silencing of cytoplasmic genes using sequence-specific double-stranded short interfering RNA and its application in the reverse genetics of wild type negative-strand RNA viruses

    Directory of Open Access Journals (Sweden)

    Barik Sailen

    2001-12-01

    Full Text Available Abstract Background Post-transcriptional gene silencing (PTGS by short interfering RNA has opened up new directions in the phenotypic mutation of cellular genes. However, its efficacy on non-nuclear genes and its effect on the interferon pathway remain unexplored. Since directed mutation of RNA genomes is not possible through conventional mutagenesis, we have tested sequence-specific 21-nucleotide long double-stranded RNAs (dsRNAs for their ability to silence cytoplasmic RNA genomes. Results Short dsRNAs were generated against specific mRNAs of respiratory syncytial virus, a nonsegmented negative-stranded RNA virus with a cytoplasmic life cycle. At nanomolar concentrations, the dsRNAs specifically abrogated expression of the corresponding viral proteins, and produced the expected mutant phenotype ex vivo. The dsRNAs did not induce an interferon response, and did not inhibit cellular gene expression. The ablation of the viral proteins correlated with the loss of the specific mRNAs. In contrast, viral genomic and antigenomic RNA, which are encapsidated, were not directly affected. Conclusions Synthetic inhibitory dsRNAs are effective in specific silencing of RNA genomes that are exclusively cytoplasmic and transcribed by RNA-dependent RNA polymerases. RNA-directed RNA gene silencing does not require cloning, expression, and mutagenesis of viral cDNA, and thus, will allow the generation of phenotypic null mutants of specific RNA viral genes under normal infection conditions and at any point in the infection cycle. This will, for the first time, permit functional genomic studies, attenuated infections, reverse genetic analysis, and studies of host-virus signaling pathways using a wild type RNA virus, unencumbered by any superinfecting virus.

  15. Molecular characterization of a bipartite double-stranded RNA virus and its satellite-like RNA co-infecting the phytopathogenic fungus Sclerotinia sclerotiorum

    Directory of Open Access Journals (Sweden)

    Lijiang eLiu

    2015-05-01

    Full Text Available A variety of mycoviruses have been found in Sclerotinia sclerotiorum. In this study, we report a novel mycovirus Sclerotinia sclerotiorum botybirnavirus 1 (SsBRV1 that was originally isolated from the hypovirulent strain SCH941 of S. sclerotiorum. SsBRV1 has rigid spherical virions that are ~38 nm in diameter, and three dsRNA segments (dsRNA1, 2 and 3 with lengths of 6.4, 6.0 and 1.7 kbp, respectively were packaged in the virions. dsRNA1 encodes a cap-pol fusion protein, and dsRNA2 encodes a polyprotein with unknown functions but contributes to the formation of virus particles. The dsRNA3 is dispensable and may be a satellite-like RNA (SatlRNA of SsBRV1. Although phylogenetic analysis of the RdRp domain demonstrated that SsBRV1 is related to Botrytis porri RNA virus 1 (BpRV1 and Ustilago maydis dsRNA virus-H1 (UmV-H1, the structure proteins of SsBRV1 do not have any significant sequence similarities with other known viral proteins with the exception of those of BpRV1. SsBRV1 carrying dsRNA3 seems to have no obvious effects on the colony morphology, but can significantly reduce the growth rate and virulence of S. sclerotiorum. Notably, a growth hormone receptor binding domain (GHBP, Pfam12772 is detected in ORF2-encoded protein of SsBRV1, which have not been reported in any other viruses. These findings provide new insights into the virus taxonomy, virus evolution and the interactions between SsBRV1 and the fungal hosts.

  16. Interaction of DAPI with double-stranded ribonucleic acids.

    Science.gov (United States)

    Manzini, G; Xodo, L; Barcellona, M L; Quadrifoglio, F

    1985-01-01

    The interaction of DAPI with natural and synthetic double-stranded polyribonucleotides was studied with different optical and calorimetric methods. The results were similar to those obtained previously with double-stranded polydeoxynucleotides, i.e. two interaction modes, the first of which shows high affinity for AU clusters and consequent strong fluorescence enhancement. The results suggest caution in the use of DAPI as selective fluorescent staining agent for DNA in the presence of RNA. A narrow groove binding model with hydrogen bonds between DAPI and AU pairs is proposed. An intercalation mechanism can be excluded because of the non planarity of DAPI molecule. PMID:4080554

  17. Nucleic Acid Analogue Induced Transcription of Double Stranded DNA

    DEFF Research Database (Denmark)

    1998-01-01

    RNA is transcribed from a double stranded DNA template by forming a complex by hybridizing to the template at a desired transcription initiation site one or more oligonucleic acid analogues of the PNA type capable of forming a transcription initiation site with the DNA and exposing the complex...

  18. CD11b/CD18 (Mac-1) is a novel surface receptor for extracellular double-stranded RNA to mediate cellular inflammatory responses.

    Science.gov (United States)

    Zhou, Hui; Liao, Jieying; Aloor, Jim; Nie, Hui; Wilson, Belinda C; Fessler, Michael B; Gao, Hui-Ming; Hong, Jau-Shyong

    2013-01-01

    During viral infection, extracellular dsRNA is a potent signaling molecule that activates many innate immune cells, including macrophages. TLR3 is a well-known receptor for extracellular dsRNA, and internalization of extracellular dsRNA is required for endosomal TLR3 activation. Preserved inflammatory responses of TLR3-deficient macrophages to extracellular dsRNA strongly support a TLR3-independent mechanism in dsRNA-mediated immune responses. The present study demonstrated that CD11b/CD18 (Mac-1 [macrophage-1 Ag]), a surface integrin receptor, recognized extracellular dsRNA and induced macrophage immune responses. CD11b deficiency reduced inflammatory cytokine induction elicited by polyinosinic:polycytidylic acid (poly I:C; a synthetic dsRNA) in mouse sera and livers, as well as in cultured peritoneal macrophages. dsRNA-binding assay and confocal immunofluorescence showed that Mac-1, especially the CD11b subunit, interacted and colocalized with poly I:C on the surface of macrophages. Further mechanistic studies revealed two distinct signaling events following dsRNA recognition by Mac-1. First, Mac-1 facilitated poly I:C internalization through the activation of PI3K signaling and enhanced TLR3-dependent activation of IRF3 in macrophages. Second, poly I:C induced activation of phagocyte NADPH oxidase in a TLR3-independent, but Mac-1-dependent, manner. Subsequently, phagocyte NADPH oxidase-derived intracellular reactive oxygen species activated MAPK and NF-κB pathways. Our results indicate that extracellular dsRNA activates Mac-1 to enhance TLR3-dependent signaling and to trigger TLR3-independent, but Mac-1-dependent, inflammatory oxidative signaling, identifying a novel mechanistic basis for macrophages to recognize extracellular dsRNA to regulate innate immune responses. This study identifies Mac-1 as a novel surface receptor for extracellular dsRNA and implicates it as a potential therapeutic target for virus-related inflammatory diseases.

  19. Efficacy of double-stranded RNA against white spot syndrome virus (WSSV non-structural (orf89, wsv191 and structural (vp28, vp26 genes in the Pacific white shrimp Litopenaeus vannamei

    Directory of Open Access Journals (Sweden)

    César M. Escobedo-Bonilla

    2015-04-01

    Full Text Available White spot syndrome virus (WSSV is a major pathogen in shrimp aquaculture. RNA interference (RNAi is a promising tool against viral infections. Previous works with RNAi showed different antiviral efficacies depending on the silenced gene. This work evaluated the antiviral efficacy of double-stranded (ds RNA against two non-structural (orf89, wsv191 WSSV genes compared to structural (vp26, vp28 genes to inhibit an experimental WSSV infection. Gene orf89 encodes a putative regulatory protein and gene white spot virus (wsv191 encodes a nonspecific nuclease; whereas genes vp26 and vp28 encode envelope proteins, respectively. Molecules of dsRNA against each of the WSSV genes were intramuscularly injected (4 μg per shrimp into a group of shrimp 48 h before a WSSV challenge. The highest antiviral activity occurred with dsRNA against orf89, vp28 and vp26 (cumulative mortalities 10%, 10% and 21%, respectively. In contrast, the least effective treatment was wsv191 dsRNA (cumulative mortality 83%. All dead animals were WSSV-positive by one-step PCR, whereas reverse-transcription PCR of all surviving shrimp confirmed inhibition of virus replication. This study showed that dsRNA against WSSV genes orf89, vp28 and vp26 were highly effective to inhibit virus replication and suggest an essential role in WSSV infection. Non-structural WSSV genes such as orf89 can be used as novel targets to design therapeutic RNAi molecules against WSSV infection.

  20. Derivation of an endogenous small RNA from double-stranded Sox4 sense and natural antisense transcripts in the mouse brain.

    Science.gov (United States)

    Ling, King-Hwa; Brautigan, Peter J; Moore, Sarah; Fraser, Rachel; Cheah, Pike-See; Raison, Joy M; Babic, Milena; Lee, Young Kyung; Daish, Tasman; Mattiske, Deidre M; Mann, Jeffrey R; Adelson, David L; Thomas, Paul Q; Hahn, Christopher N; Scott, Hamish S

    2016-03-01

    Natural antisense transcripts (NATs) are involved in cellular development and regulatory processes. Multiple NATs at the Sox4 gene locus are spatiotemporally regulated throughout murine cerebral corticogenesis. In the study, we evaluated the potential functional role of Sox4 NATs at Sox4 gene locus. We demonstrated Sox4 sense and NATs formed dsRNA aggregates in the cytoplasm of brain cells. Over expression of Sox4 NATs in NIH/3T3 cells generally did not alter the level of Sox4 mRNA expression or protein translation. Upregulation of a Sox4 NAT known as Sox4ot1 led to the production of a novel small RNA, Sox4_sir3. Its biogenesis is Dicer1-dependent and has characteristics resemble piRNA. Expression of Sox4_sir3 was observed in the marginal and germinative zones of the developing and postnatal brains suggesting a potential role in regulating neurogenesis. We proposed that Sox4 sense-NATs serve as Dicer1-dependent templates to produce a novel endo-siRNA- or piRNA-like Sox4_sir3.

  1. Transgenic accumulation of a defective cucumber mosaic virus (CMV) replicase derived double stranded RNA modulates plant defence against CMV strains O and Y in potato.

    Science.gov (United States)

    Ntui, Valentine Otang; Kynet, Kong; Azadi, Pejman; Khan, Raham Sher; Chin, Dong Poh; Nakamura, Ikuo; Mii, Masahiro

    2013-12-01

    Cucumber mosaic virus is an important plant pathogen with a broad host range encompassing many plant species. This study demonstrates the production of transgenic potato lines exhibiting complete resistance to cucumber mosaic virus strain O and Y by post transcriptional gene silencing. Two constructs were used, one, pEKH2IN2CMVai, contains inverted repeat of 1,138 bp fragment of a defective CMV replicase gene derived from RNA2 of cucumber mosaic virus strain O (CMV-O), while the other, TRV-based VIGS vector (pTRV2CMVai), contains the same fragment of the replicase gene, but without inverted repeat. These constructs were used to produce transgenic potato lines of cultivar 'Danshaku', a susceptible genotype to CMV. Transgenic lines derived from pEKH2IN2CMVai accumulated small interfering RNA (siRNA) before and after virus challenge, whereas those derived from pTRV2CMVai showed siRNA expression after virus challenge. When transgenic lines were challenged with CMV-O or CMV-Y, four lines exhibited complete (100%) resistance to both strains, whereas the other lines had high levels of resistance. Infectivity of CMV-O was lower than that of CMV-Y in the highly resistant plants. There were no significant differences with regard to resistance between plants derived from pEKH2IN2CMVai and those obtained from pTRV2CMVai. The presence of CMV-specific siRNA in the resistant phenotypes indicates that the resistance was acquired through RNA silencing.

  2. Relationships and Evolution of Double-Stranded RNA Totiviruses of Yeasts Inferred from Analysis of L-A-2 and L-BC Variants in Wine Yeast Strain Populations.

    Science.gov (United States)

    Rodríguez-Cousiño, Nieves; Esteban, Rosa

    2017-02-15

    Saccharomyces cerevisiae killer strains secrete a protein toxin active on nonkiller strains of the same (or other) yeast species. Different killer toxins, K1, K2, K28, and Klus, have been described. Each toxin is encoded by a medium-size (1.5- to 2.3-kb) M double-stranded RNA (dsRNA) located in the cytoplasm. M dsRNAs require L-A helper virus for maintenance. L-A belongs to the Totiviridae family, and its dsRNA genome of 4.6 kb codes for the major capsid protein Gag and a minor Gag-Pol protein, which form the virions that separately encapsidate L-A or the M satellites. Different L-A variants exist in nature; on average, 24% of their nucleotides are different. Previously, we reported that L-A-lus was specifically associated with Mlus, suggesting coevolution, and proposed a role of the toxin-encoding M dsRNAs in the appearance of new L-A variants. Here we confirm this by analyzing the helper virus in K2 killer wine strains, which we named L-A-2. L-A-2 is required for M2 maintenance, and neither L-A nor L-A-lus shows helper activity for M2 in the same genetic background. This requirement is overcome when coat proteins are provided in large amounts by a vector or in ski mutants. The genome of another totivirus, L-BC, frequently accompanying L-A in the same cells shows a lower degree of variation than does L-A (about 10% of nucleotides are different). Although L-BC has no helper activity for M dsRNAs, distinct L-BC variants are associated with a particular killer strain. The so-called L-BC-lus (in Klus strains) and L-BC-2 (in K2 strains) are analyzed.

  3. Directional secretory response of double stranded RNA-induced thymic stromal lymphopoetin (TSLP and CCL11/eotaxin-1 in human asthmatic airways.

    Directory of Open Access Journals (Sweden)

    Gustavo Nino

    Full Text Available BACKGROUND: Thymic stromal lymphoproetin (TSLP is a cytokine secreted by the airway epithelium in response to respiratory viruses and it is known to promote allergic Th2 responses in asthma. This study investigated whether virally-induced secretion of TSLP is directional in nature (apical vs. basolateral and/or if there are TSLP-mediated effects occurring at both sides of the bronchial epithelial barrier in the asthmatic state. METHODS: Primary human bronchial epithelial cells (HBEC from control (n = 3 and asthmatic (n = 3 donors were differentiated into polarized respiratory tract epithelium under air-liquid interface (ALI conditions and treated apically with dsRNA (viral surrogate or TSLP. Sub-epithelial effects of TSLP were examined in human airway smooth muscle cells (HASMC from normal (n = 3 and asthmatic (n = 3 donors. Clinical experiments examined nasal airway secretions obtained from asthmatic children during naturally occurring rhinovirus-induced exacerbations (n = 20 vs. non-asthmatic uninfected controls (n = 20. Protein levels of TSLP, CCL11/eotaxin-1, CCL17/TARC, CCL22/MDC, TNF-α and CXCL8 were determined with a multiplex magnetic bead assay. RESULTS: Our data demonstrate that: 1 Asthmatic HBEC exhibit an exaggerated apical, but not basal, secretion of TSLP after dsRNA exposure; 2 TSLP exposure induces unidirectional (apical secretion of CCL11/eotaxin-1 in asthmatic HBEC and enhanced CCL11/eotaxin-1 secretion in asthmatic HASMC; 3 Rhinovirus-induced asthma exacerbations in children are associated with in vivo airway secretion of TSLP and CCL11/eotaxin-1. CONCLUSIONS: There are virally-induced TSLP-driven secretory immune responses at both sides of the bronchial epithelial barrier characterized by enhanced CCL11/eotaxin-1 secretion in asthmatic airways. These results suggest a new model of TSLP-mediated eosinophilic responses in the asthmatic airway during viral-induced exacerbations.

  4. Double-Stranded Peptide Nucleic Acids

    DEFF Research Database (Denmark)

    2001-01-01

    A novel class of compounds, known as peptide nucleic acids, form double-stranded structures with one another and with ssDNA. The peptide nucleic acids generally comprise ligands such as naturally occurring DNA bases attached to a peptide backbone through a suitable linker....

  5. Prevention of the β-amyloid peptide-induced inflammatory process by inhibition of double-stranded RNA-dependent protein kinase in primary murine mixed co-cultures

    Directory of Open Access Journals (Sweden)

    Terro F

    2011-06-01

    Full Text Available Abstract Background Inflammation may be involved in the pathogenesis of Alzheimer's disease (AD. There has been little success with anti-inflammatory drugs in AD, while the promise of anti-inflammatory treatment is more evident in experimental models. A new anti-inflammatory strategy requires a better understanding of molecular mechanisms. Among the plethora of signaling pathways activated by β-amyloid (Aβ peptides, the nuclear factor-kappa B (NF-κB pathway could be an interesting target. In virus-infected cells, double-stranded RNA-dependent protein kinase (PKR controls the NF-κB signaling pathway. It is well-known that PKR is activated in AD. This led us to study the effect of a specific inhibitor of PKR on the Aβ42-induced inflammatory response in primary mixed murine co-cultures, allowing interactions between neurons, astrocytes and microglia. Methods Primary mixed murine co-cultures were prepared in three steps: a primary culture of astrocytes and microglia for 14 days, then a primary culture of neurons and astrocytes which were cultured with microglia purified from the first culture. Before exposure to Aβ neurotoxicity (72 h, co-cultures were treated with compound C16, a specific inhibitor of PKR. Levels of tumor necrosis factor-α (TNFα, interleukin (IL-1β, and IL-6 were assessed by ELISA. Levels of PT451-PKR and activation of IκB, NF-κB and caspase-3 were assessed by western blotting. Apoptosis was also followed using annexin V-FITC immunostaining kit. Subcellular distribution of PT451-PKR was assessed by confocal immunofluorescence and morphological structure of cells by scanning electron microscopy. Data were analysed using one-way ANOVA followed by a Newman-Keuls' post hoc test Results In these co-cultures, PKR inhibition prevented Aβ42-induced activation of IκB and NF-κB, strongly decreased production and release of tumor necrosis factor (TNFα and interleukin (IL-1β, and limited apoptosis. Conclusion In spite of the

  6. Repair of DNA Double-Strand Breaks

    Science.gov (United States)

    Falk, Martin; Lukasova, Emilie; Kozubek, Stanislav

    The genetic information of cells continuously undergoes damage induced by intracellular processes including energy metabolism, DNA replication and transcription, and by environmental factors such as mutagenic chemicals and UV and ionizing radiation. This causes numerous DNA lesions, including double strand breaks (DSBs). Since cells cannot escape this damage or normally function with a damaged genome, several DNA repair mechanisms have evolved. Although most "single-stranded" DNA lesions are rapidly removed from DNA without permanent damage, DSBs completely break the DNA molecule, presenting a real challenge for repair mechanisms, with the highest risk among DNA lesions of incorrect repair. Hence, DSBs can have serious consequences for human health. Therefore, in this chapter, we will refer only to this type of DNA damage. In addition to the biochemical aspects of DSB repair, which have been extensively studied over a long period of time, the spatio-temporal organization of DSB induction and repair, the importance of which was recognized only recently, will be considered in terms of current knowledge and remaining questions.

  7. Nampt is involved in DNA double-strand break repair

    Institute of Scientific and Technical Information of China (English)

    Bingtao Zhu; Xiaoli Deng; Yifan Sun; Lin Bai; Zhikai Xiahou; Yusheng Cong; Xingzhi Xu

    2012-01-01

    DNA double-strand break (DSB) is the most severe form of DNA damage,which is repaired mainly through high-fidelity homologous recombination (HR) or error-prone non-homologous end joining (NHEJ).Defects in the DNA damage response lead to genomic instability and ultimately predispose organs to cancer.Nicotinamide phosphoribosyltransferase (Nampt),which is involved in nicotinamide adenine dinucleotide metabolism,is overexpressed in a variety of tumors.In this report,we found that Nampt physically associated with CtlP and DNA-PKcs/Ku80,which are key factors in HR and NHEJ,respectively.Depletion of Nampt by small interfering RNA (siRNA) led to defective NHEJ-mediated DSB repair and enhanced HR-mediated repair.Furthermore,the inhibition of Nampt expression promoted proliferation of cancer cells and normal human fibroblasts and decreased β-galactosidase staining,indicating a delay in the onset of cellular senescence in normal human fibroblasts.Taken together,our results suggest that Nampt is a suppressor of HR-mediated DSB repair and an enhancer of NHEJ-mediated DSB repair,contributing to the acceleration of cellular senescence.

  8. Strong resistance against Rice grassy stunt virus is induced in transgenic rice plants expressing double-stranded RNA of the viral genes for nucleocapsid or movement proteins as targets for RNA interference.

    Science.gov (United States)

    Shimizu, Takumi; Ogamino, Takumi; Hiraguri, Akihiro; Nakazono-Nagaoka, Eiko; Uehara-Ichiki, Tamaki; Nakajima, Masami; Akutsu, Katsumi; Omura, Toshihiro; Sasaya, Takahide

    2013-05-01

    Rice grassy stunt virus (RGSV), a member of the genus Tenuivirus, causes significant economic losses in rice production in South, Southeast, and East Asian countries. Growing resistant varieties is the most efficient method to control RGSV; however, suitable resistance genes have not yet been found in natural rice resources. One of the most promising methods to confer resistance against RGSV is the use of RNA interference (RNAi). It is important to target viral genes that play important roles in viral infection and proliferation at an early stage of viral replication. Our recent findings obtained from an RNAi experiment with Rice stripe virus (RSV), a tenuivirus, revealed that the genes for nucleocapsid and movement proteins were appropriate targets for RNAi to confer resistance against RSV. In this study, we transformed rice plants by introducing an RNAi construct of the RGSV genes for the nucelocapsid protein pC5 or movement protein pC6. All progenies from self-fertilized transgenic plants had strong resistance against RGSV infection and did not allow the proliferation of RGSV. Thus, our strategy to target genes for nucleocapsid and movement proteins for conferring viral resistance might be applicable to the plant viruses in the genus Tenuivirus.

  9. Translocation of double-stranded DNA through membrane-adapted phi29 motor protein nanopores

    Science.gov (United States)

    Wendell, David; Jing, Peng; Geng, Jia; Subramaniam, Varuni; Lee, Tae Jin; Montemagno, Carlo; Guo, Peixuan

    2009-11-01

    Biological pores have been used to study the transport of DNA and other molecules, but most pores have channels that allow only the movement of small molecules and single-stranded DNA and RNA. The bacteriophage phi29 DNA-packaging motor, which allows double-stranded DNA to enter the virus during maturation and exit during an infection, contains a connector protein with a channel that is between 3.6 and 6 nm wide. Here we show that a modified version of this connector protein, when reconstituted into liposomes and inserted into planar lipid bilayers, allows the translocation of double-stranded DNA. The measured conductance of a single connector channel was 4.8 nS in 1 M KCl. This engineered and membrane-adapted phage connector is expected to have applications in microelectromechanical sensing, microreactors, gene delivery, drug loading and DNA sequencing.

  10. Saliva of TBP digests double stranded RNAs

    Science.gov (United States)

    The tarnished plant bug (TPB), Lygus lineolaris, is a generalist pest of many crops including cotton, strawberries, and alfalfa. While studying gene expression and gene knockdown through RNA interference, my lab was frustrated by an observation. We were unable to replicate knock down effects observe...

  11. Chromatin remodelers in the DNA double strand break response

    NARCIS (Netherlands)

    Smeenk, Godelieve

    2012-01-01

    During my PhD project, I studied the role of several chromatin remodelers in the DNA double strand break (DSB) response. We discovered that both CHD4 and SMARCA5 are required for ubiquitin signaling through the E3 ubiquitin ligases RNF8 and RNF168, which is a central signaling event in the response

  12. Heavy ion induced double strand breaks in bacteria and bacteriophages

    Science.gov (United States)

    Micke, U.; Schäfer, M.; Anton, A.; Horneck, G.; Bücker, H.

    DNA damage induced by heavy ions in bacterial cells and bacteriophages such as Bacillus subtilis, E. coli and Bacteriophage Tl were investigated by analyzing the double strand breaks in the chromosomal DNA. This kind of lesion is considered as one of the main reasons for lethal events. To analyze double strand breaks in long molecules of DNA - up to some Mbp in length - the technique of pulse field agarose gel electrophoresis has been used. This allows the detection of one double strand break per genome. Cell lysis and DNA isolation were performed in small agarose blocks directly. This procedure secured minimum DNA destruction by shearing forces. After running a gel, the DNA was stained with ethidium bromide. The light intensity of ethidium bromide fluorescence for both the outcoming (running) DNA and the remaining intact DNA were measured by scanning. The mean number of double strand breaks was calculated by determining the quotient of these intensities. Strand break induction after heavy ion and X-ray irradiation was compared.

  13. Targeting DNA double-strand breaks with TAL effector nucleases.

    Science.gov (United States)

    Christian, Michelle; Cermak, Tomas; Doyle, Erin L; Schmidt, Clarice; Zhang, Feng; Hummel, Aaron; Bogdanove, Adam J; Voytas, Daniel F

    2010-10-01

    Engineered nucleases that cleave specific DNA sequences in vivo are valuable reagents for targeted mutagenesis. Here we report a new class of sequence-specific nucleases created by fusing transcription activator-like effectors (TALEs) to the catalytic domain of the FokI endonuclease. Both native and custom TALE-nuclease fusions direct DNA double-strand breaks to specific, targeted sites.

  14. Double strand break repair functions of histone H2AX

    Energy Technology Data Exchange (ETDEWEB)

    Scully, Ralph, E-mail: rscully@bidmc.harvard.edu; Xie, Anyong

    2013-10-15

    Chromosomal double strand breaks provoke an extensive reaction in neighboring chromatin, characterized by phosphorylation of histone H2AX on serine 139 of its C-terminal tail (to form “γH2AX”). The γH2AX response contributes to the repair of double strand breaks encountered in a variety of different contexts, including those induced by ionizing radiation, physiologically programmed breaks that characterize normal immune cell development and the pathological exposure of DNA ends triggered by telomere dysfunction. γH2AX also participates in the evolutionarily conserved process of sister chromatid recombination, a homologous recombination pathway involved in the suppression of genomic instability during DNA replication and directly implicated in tumor suppression. At a biochemical level, the γH2AX response provides a compelling example of how the “histone code” is adapted to the regulation of double strand break repair. Here, we review progress in research aimed at understanding how γH2AX contributes to double strand break repair in mammalian cells.

  15. What Governs the Unzipping Process of Double-Stranded DNA

    Institute of Scientific and Technical Information of China (English)

    WANG Xiao-Feng; LEI Xiao-Ling; FANG Hai-Ping

    2006-01-01

    @@ The unzipping process of double-stranded DNA is analysed using a discrete model at the base level [Chin. Phys.Lett. 22 (2005)1540]. The numerical results are consistent with the experimental observations on the force-displacement behaviour including the sequence-dependence. We find that the hydrogen bond interaction in a base pair is crucially important to the force-displacement profile.

  16. 萝卜中一种新dsRNA的全长cDNA克隆及序列分析%Full-length cDNA Clone and Sequence Analysis of A New Double-stranded RNA from Radish

    Institute of Scientific and Technical Information of China (English)

    李露露; 李力强; 乔爱民; 陈亮; 陈集双

    2008-01-01

    从'一点红'萝卜(Raphanus sativus-root'Yidianhong')叶片中提取dsRNA,应用SPAT方法对各dsRNA条带进行cDNA克隆并测序,除获得先前报道的5条序列(RasR 1~RasR 5)以外,还得到一条新的全长序列,将其定名为RasR 6(EU285027).序列分析结果表明:RasR 6全长为1 778 bp,其正义链编码1个由502个氨基酸组成、分子量约为55.1 kD的蛋白质.该序列与前人报道的双分病毒科5个病毒序列具有相似性,且它们均编码双分病毒科病毒的外壳蛋白(capsid protein,CP).核苷酸序列比对结果显示:RasR 6与同时来源于萝卜的RasR 1和RasR 2的5'UTR序列高度同源,且其3'末端具有poly A结构,而与RasR 3、RasR 4和RasR 5的UTR则没有明显的相似性.因此,推测RasR6与RasR1、RasR 2同属于双分病毒RasV 1(Raphanus sativus virus 1),可能与RasR 2共同编码该病毒的CP或作为RasV 1的卫星RNA存在.

  17. dsRNA介导的番木瓜环斑病毒(PRSV)的抗病性研究%Virus-resistance of Papaya Ringspot Virus Mediated by Double-Strand RNA

    Institute of Scientific and Technical Information of China (English)

    张帆; 姜玲

    2011-01-01

    Tobacco ( Nicotiana tabacum) , Arabidopsis thaliana, and host papaya( Carica papaya L. )of PRSV were used as model plant materials to testing whether RNAi could mediate resistance to papaya ringspot virus (PRSV). The agrobacterium was harbored with pHellsgate12-CPIR containing structured inverted sequences of the CP gene of PRSV,where were transformed into the tobacco and Arabidopsis. We also conducted an Agrobacterium-mediated transient expression experiment by infiltration in papaya. The three transgenic plants inoculated with PRSV were tested for resistance to PRSV. After PRSV challenge-inoculation for 3 to 7 days, leaves of the wild type plants displayed symptoms of necrosis, wrinkling and/or chlorosis, and the scale of the abnormal leaves in the transgenic plants was significantly lower than that of the wild type when the PRSV were inoculated in papaya and Arabidopsis separately.However, the difference in symptoms was not significant in transgenic tobacco and the control wild plant. We carried out a RT-PCR experiment on the three kinds of plants. The expression of CP mRNA was detected in the wild type plants infected with PRSV, but not in the transgenic plants. These results suggest that the transgenic plants may induce the RNAi process to resist PRSV.%以模式植物拟南芥(Arabidopsis thaliana)和烟草(Nicotiana tabacum)及PRSV寄主植物番木瓜(Carica papaya L.)作为试验材料,开展了番木瓜环斑病毒外壳蛋白基因dsRNA介导的PRSV病原抗性的研究.利用农杆菌介导法将番木瓜环斑病毒外壳蛋白CP基因反向重复表达载体pHellsgate12-CPIR(简称PHG12-CPIR)分别转化到烟草和拟南芥中,获得阳性植株,并利用渗透法和农杆菌介导的瞬时表达体系将pHG12-CPIR载体导入到番木瓜中.对转基因植株进行攻毒试验并分析了其抗病性.在接种3~7 d内,在拟南芥和番木瓜上转基因植株的发病情况较轻,而野生型植株叶片与转基因植株相比,均表现出不同

  18. 干扰素诱导的双链 RNA 依赖性蛋白激酶体外抗乙型肝炎病毒活性的研究%Study of IFN-inducible double-stranded RNA dependent protein kinase on antiviral activity of HBV in vitro

    Institute of Scientific and Technical Information of China (English)

    王爱华; 管世鹤; 杨凯; 张浩; 孙蓓蓓; 潘颖; 沈继龙

    2015-01-01

    目的:构建表达双链 RNA 依赖性蛋白激酶(PKR)融合绿色增强荧光蛋白(pEGFP-PKR)真核表达质粒,并进一步研究 PKR 蛋白在体外抗乙型肝炎病毒(HBV)活性。方法以 pEGFP-N1为空载体,运用分子克隆技术构建重组质粒pEGFP-PKR,通过双酶切和直接测序两种方法验证重组质粒pEGFP-PKR 是否构建成功。以能分泌完整 HBV 病毒颗粒子的肝胚瘤细胞株 HepG2.2.15细胞为细胞模型,采用重组质粒转染方式处理 HepG2.2.15细胞,运用荧光显微镜观察融合蛋白 pEGFP-PKR 在细胞内的表达,以电化学发光方法和实时荧光定量 PCR 技术分析细胞上清 HBV 抗原表达和细胞病毒复制水平。结果酶切鉴定和序列分析证实成功构建重组质粒 pEGFP-PKR,转染 HepG2.2.15细胞后在荧光显微镜下可见融合蛋白 pEGFP-PKR 表达,同时细胞分泌的HBV 抗原与空载体组相比较明显下降(P <0.05),而细胞外HBV 复制水平未见明显变化。结论在体外肝细胞模型中,PKR 蛋白具有一定的抗 HBV 活性作用。%Aim To construct and express the eukary-otic expression vector of double-stranded RNA-depend-ent protein kinase (PKR)fusion green fluorescent and analyse its antiviral activity of HBV in vitro.Methods The PKR gene was cloned into an empty expression vector pEGFP-N1 using molecular clone technology. After being confirmed by restriction enzyme digestion and sequencing methods,the recombinant plasmid was named as pEGFP-PKR that was subsequently transfect-ed into HepG2.2.15 cells using LipofectamineTM2000. The expression level of PKR in HepG2.2.15 cells was confirmed by using fluorescent microscopy. Mean-while,HBV DNA and HBsAg/HBeAg were detected by real-time PCR and electrochemiluminescence meth-od,respectively.Results Both restriction enyme di-gestion and sequencing assays showed that the recombi-nant vector pEGFP-PKR was successfully constructed in our study

  19. Charge Migration in DNA: A Double Stranded Model

    Institute of Scientific and Technical Information of China (English)

    BAO, Han; LU, Jing; FAN, Kang-Nian

    2006-01-01

    In particular, charge migration phenomena in DNA have attracted much interest because of relevance to the generation of damage and mutations which play important roles in most of life processes. In this paper a theory method was presented in which the DNA chain was treated as a double-stranded system, and the charge migration in DNA based on the donor-bridge-acceptor system was investigated by this model. After having obtained the Hamiltonian, the effects of the surrounding were explained and calculated. The double-strand calculation could lead to good exponential decay curves and this time two different falloff parameters were found respectively before and after 3 or 4 AT base pair bridge lengths as prediction. Lately theoretical study showed this result by addition of more parameter, and sequence effect was then concentrated on. The difference of transfer integral caused the different decay rate of unlike sequences, but bridge length was still proved to be the main factor on the decay rates.

  20. The isothermal amplification detection of double-stranded DNA based on a double-stranded fluorescence probe.

    Science.gov (United States)

    Shi, Chao; Shang, Fanjin; Pan, Mei; Liu, Sen; Ma, Cuiping

    2016-06-15

    Here we have developed a novel method of isothermal amplification detection of double-stranded DNA (dsDNA) based on double-stranded fluorescence probe (ds-probe). Target dsDNA repeatedly generated single-stranded DNA (ssDNA) with polymerase and nicking enzyme. The ds-probe as a primer hybridized with ssDNA and extended to its 5'-end. The displaced ssDNA served as a new detection target to initiate above-described reaction. Meanwhile, the extended ds-probe could dynamically dissociate from ssDNA and self-hybridize, converting into a turn-back structure to initiate another amplification reaction. In particular, the ds-probe played a key role in the entire experimental process, which not only was as a primer but also produced the fluorescent signal by an extension and displacement reaction. Our method could detect the pBluescript II KS(+) plasmid with a detection limit of 2.3 amol, and it was also verified to exhibit a high specificity, even one-base mismatch. Overall, it was a true isothermal dsDNA detection strategy with a strongly anti-jamming capacity and one-pot, only requiring one ds-probe, which greatly reduced the cost and the probability of contamination. With its advantages, the approach of dsDNA detection will offer a promising tool in the field of point-of-care testing (POCT).

  1. Electronic transport in double-strand DNA segments

    Science.gov (United States)

    Albuquerque, E. L.; Mauriz, P. W.; Moreira, D. A.

    2008-03-01

    We report in this work a numerical study of the electronic density of states in π-stacked arrays of DNA double-strand segments made up from the nucleotides guanine G, adenine A, cytosine C and thymine T. In order to reveal the relevance of the underlying correlations in the nucleotides distribution, we compare the results for a genomic DNA sequence, considering a segment of the first sequenced human chromosome 22 (Ch 22), with those of two artificial sequences forming a Rudin-Shapiro (RS) as well as a Fibonacci (FB) polyGC quasiperiodic sequences. Our theoretical method uses an electronic tight-binding Hamiltonian suitable to describe the DNA segments modeled by the quasiperiodic chains.

  2. Current-voltage characteristics of double-strand DNA sequences

    Science.gov (United States)

    Bezerril, L. M.; Moreira, D. A.; Albuquerque, E. L.; Fulco, U. L.; de Oliveira, E. L.; de Sousa, J. S.

    2009-09-01

    We use a tight-binding formulation to investigate the transmissivity and the current-voltage (I-V) characteristics of sequences of double-strand DNA molecules. In order to reveal the relevance of the underlying correlations in the nucleotides distribution, we compare the results for the genomic DNA sequence with those of artificial sequences (the long-range correlated Fibonacci and Rudin-Shapiro one) and a random sequence, which is a kind of prototype of a short-range correlated system. The random sequence is presented here with the same first neighbors pair correlations of the human DNA sequence. We found that the long-range character of the correlations is important to the transmissivity spectra, although the I-V curves seem to be mostly influenced by the short-range correlations.

  3. Phosphorylation: The Molecular Switch of Double-Strand Break Repair

    Directory of Open Access Journals (Sweden)

    K. C. Summers

    2011-01-01

    Full Text Available Repair of double-stranded breaks (DSBs is vital to maintaining genomic stability. In mammalian cells, DSBs are resolved in one of the following complex repair pathways: nonhomologous end-joining (NHEJ, homologous recombination (HR, or the inclusive DNA damage response (DDR. These repair pathways rely on factors that utilize reversible phosphorylation of proteins as molecular switches to regulate DNA repair. Many of these molecular switches overlap and play key roles in multiple pathways. For example, the NHEJ pathway and the DDR both utilize DNA-PK phosphorylation, whereas the HR pathway mediates repair with phosphorylation of RPA2, BRCA1, and BRCA2. Also, the DDR pathway utilizes the kinases ATM and ATR, as well as the phosphorylation of H2AX and MDC1. Together, these molecular switches regulate repair of DSBs by aiding in DSB recognition, pathway initiation, recruitment of repair factors, and the maintenance of repair mechanisms.

  4. Electronic transport in double-strand DNA segments

    Energy Technology Data Exchange (ETDEWEB)

    Albuquerque, E L; Moreira, D A [Departamento de Fisica, Universidade Federal do Rio Grande do Norte 59072-970 Natal-RN (Brazil); Mauriz, P W [Departamento de Ciencias Exatas, Centro Federal de Educacao Tecnologica do Maranhao 65025-001 Sao Luis-MA (Brazil)], E-mail: eudenilson@dfte.ufrn.br

    2008-03-15

    We report in this work a numerical study of the electronic density of states in {pi}-stacked arrays of DNA double-strand segments made up from the nucleotides guanine G, adenine A, cytosine C and thymine T. In order to reveal the relevance of the underlying correlations in the nucleotides distribution, we compare the results for a genomic DNA sequence, considering a segment of the first sequenced human chromosome 22 (Ch 22), with those of two artificial sequences forming a Rudin-Shapiro (RS) as well as a Fibonacci (FB) polyGC quasiperiodic sequences. Our theoretical method uses an electronic tight-binding Hamiltonian suitable to describe the DNA segments modeled by the quasiperiodic chains.

  5. Current-voltage characteristics of double-strand DNA sequences

    Energy Technology Data Exchange (ETDEWEB)

    Bezerril, L.M.; Moreira, D.A. [Departamento de Fisica, Universidade Federal do Rio Grande do Norte, 59072-970, Natal-RN (Brazil); Albuquerque, E.L., E-mail: eudenilson@dfte.ufrn.b [Departamento de Fisica, Universidade Federal do Rio Grande do Norte, 59072-970, Natal-RN (Brazil); Fulco, U.L. [Departamento de Biofisica e Farmacologia, Universidade Federal do Rio Grande do Norte, 59072-970, Natal-RN (Brazil); Oliveira, E.L. de; Sousa, J.S. de [Departamento de Fisica, Universidade Federal do Ceara, 60455-760, Fortaleza-CE (Brazil)

    2009-09-07

    We use a tight-binding formulation to investigate the transmissivity and the current-voltage (I-V) characteristics of sequences of double-strand DNA molecules. In order to reveal the relevance of the underlying correlations in the nucleotides distribution, we compare the results for the genomic DNA sequence with those of artificial sequences (the long-range correlated Fibonacci and Rudin-Shapiro one) and a random sequence, which is a kind of prototype of a short-range correlated system. The random sequence is presented here with the same first neighbors pair correlations of the human DNA sequence. We found that the long-range character of the correlations is important to the transmissivity spectra, although the I-V curves seem to be mostly influenced by the short-range correlations.

  6. Heavy Metal Exposure Influences Double Strand Break DNA Repair Outcomes.

    Directory of Open Access Journals (Sweden)

    Maria E Morales

    Full Text Available Heavy metals such as cadmium, arsenic and nickel are classified as carcinogens. Although the precise mechanism of carcinogenesis is undefined, heavy metal exposure can contribute to genetic damage by inducing double strand breaks (DSBs as well as inhibiting critical proteins from different DNA repair pathways. Here we take advantage of two previously published culture assay systems developed to address mechanistic aspects of DNA repair to evaluate the effects of heavy metal exposures on competing DNA repair outcomes. Our results demonstrate that exposure to heavy metals significantly alters how cells repair double strand breaks. The effects observed are both specific to the particular metal and dose dependent. Low doses of NiCl2 favored resolution of DSBs through homologous recombination (HR and single strand annealing (SSA, which were inhibited by higher NiCl2 doses. In contrast, cells exposed to arsenic trioxide preferentially repaired using the "error prone" non-homologous end joining (alt-NHEJ while inhibiting repair by HR. In addition, we determined that low doses of nickel and cadmium contributed to an increase in mutagenic recombination-mediated by Alu elements, the most numerous family of repetitive elements in humans. Sequence verification confirmed that the majority of the genetic deletions were the result of Alu-mediated non-allelic recombination events that predominantly arose from repair by SSA. All heavy metals showed a shift in the outcomes of alt-NHEJ repair with a significant increase of non-templated sequence insertions at the DSB repair site. Our data suggest that exposure to heavy metals will alter the choice of DNA repair pathway changing the genetic outcome of DSBs repair.

  7. A role for small RNAs in DNA double-strand break repair

    DEFF Research Database (Denmark)

    Wei, W.; Ba, Z.; Wu, Y.;

    2012-01-01

    Eukaryotes have evolved complex mechanisms to repair DNA double-strand breaks (DSBs) through coordinated actions of protein sensors, transducers, and effectors. Here we show that ∼21-nucleotide small RNAs are produced from the sequences in the vicinity of DSB sites in Arabidopsis and in human cells....... We refer to these as diRNAs for DSB-induced small RNAs. In Arabidopsis, the biogenesis of diRNAs requires the PI3 kinase ATR, RNA polymerase IV (Pol IV), and Dicer-like proteins. Mutations in these proteins as well as in Pol V cause significant reduction in DSB repair efficiency. In Arabidopsis, di......RNAs are recruited by Argonaute 2 (AGO2) to mediate DSB repair. Knock down of Dicer or Ago2 in human cells reduces DSB repair. Our findings reveal a conserved function for small RNAs in the DSB repair pathway. We propose that diRNAs may function as guide molecules directing chromatin modifications or the recruitment...

  8. Molecular detection of bacterial pathogens using microparticle enhanced double-stranded DNA probes.

    Science.gov (United States)

    Riahi, Reza; Mach, Kathleen E; Mohan, Ruchika; Liao, Joseph C; Wong, Pak Kin

    2011-08-15

    Rapid, specific, and sensitive detection of bacterial pathogens is essential toward clinical management of infectious diseases. Traditional approaches for pathogen detection, however, often require time-intensive bacterial culture and amplification procedures. Herein, a microparticle enhanced double-stranded DNA probe is demonstrated for rapid species-specific detection of bacterial 16S rRNA. In this molecular assay, the binding of the target sequence to the fluorophore conjugated probe thermodynamically displaces the quencher probe and allows the fluorophore to fluoresce. By incorporation of streptavidin-coated microparticles to localize the biotinylated probes, the sensitivity of the assay can be improved by 3 orders of magnitude. The limit of detection of the assay is as few as eight bacteria without target amplification and is highly specific against other common pathogens. Its applicability toward clinical diagnostics is demonstrated by directly identifying bacterial pathogens in urine samples from patients with urinary tract infections.

  9. Single- and double-stranded viral RNAs in plants infected with the potexviruses papaya mosaic virus and foxtail mosaic virus.

    Science.gov (United States)

    Mackie, G A; Johnston, R; Bancroft, J B

    1988-01-01

    Three classes of viral RNA were recovered from polyribosomes purified from papaya leaves infected with papaya mosaic virus (PapMV) and from barley leaves infected with foxtail mosaic virus (FoMV): full-length viral RNAs [6.8 and 6.2 kilobases (kb), respectively]; less abundant intermediate subgenomic RNAs (2.2 and 1.9 kb), and abundant, small subgenomic RNAs (1 and 0.9 kb). Small amounts of the PapMV-specified 1.0-kb subgenomic RNA were encapsidated, whereas no encapsidated subgenomic RNAs could be found in preparations of FoMV. Immunoprecipitation of the products of in vitro translation of the small subgenomic RNA of both viruses showed that it codes for the corresponding viral coat protein. FoMV genomic RNA isolated from polyribosomes also directed the efficient synthesis of a 37- to 38-kilodalton protein which was immunoprecipitated by an antiserum raised against the coat protein. We presume this product to be a readthrough protein initiated to the 5' side of and in the same reading frame as the coat protein-coding sequences in FoMV RNA. The predominant double-stranded viral-specified RNAs in tissues infected with PapMV, FoMV, and clover yellow mosaic virus were genome sized (6.8, 6.2, and 7.0 kb pairs, respectively). If double-stranded RNAs corresponding to coat protein subgenomic RNAs exist, they must be present in much lower relative abundances.

  10. 利拉鲁肽下调游离脂肪酸作用下βTC3细胞PERK的表达%The Down-regulation Effect of Liraglutide (Lira) on the Expression of Double-stranded RNA-dependent Protein Kinase-like Endoplasmic Reticulum Kinase (PERK) in βTC3 Cells Induced by Free Fatty Acids (FFA)

    Institute of Scientific and Technical Information of China (English)

    彭红艳; 姬秋和; 周洁; 邢影; 高彬; 曹宏伟; 刘涛

    2012-01-01

    Objective: To investigate the expression of PERK in pTC3 cells exposed to different concentrations of FFA and the intervention effect of Lira on the expression of double-stranded RNA-dependent protein kinase-like endoplasmic reticulum kinase (PERK) induced by FFA. Methods: βTC3 cells were treated with different concentrations of FFA (0,0.125,0.25, 0.5 and 1.0 mmol/L). Western Blot analysis was used to determine the expression of PERK in (3TC3 cells after 24 hours. Afterwards, |JTC3 cells were prein-cubated with different concentrations of Lira(0,0,0.5,1 mg/L)for 6 hours, and different concentrations of FFA(0,l, 1,1 mmol/L) were then added and the cells were incubated for another 24 hours. The expression of PERK was detected. Results:1. After the cells were incubated with FFA of different concentrations for 24 hours, compared with the control group, the expression of PERK in pTC3 cells in the group with 1 mmol/L FFA increased (P<0.05). 2.Compared with that in the group with 1 mmol/L FFA, the expression of PERK decreased (P<0.05) in the group with 0.5 mg/L Lira+1 mmol/L FFA and the group with 1 mg/L Lira+1 mmol/L FFA, and there was statistical difference between the two groups (P<0.05). Conclusion: The expression of PERK in βTC3 cells is up-regulated by administration of FFA of certain concentration, while Lira can reverse this response to some extent, partly inhibiting endoplasmic reticulum stress.%目的:探讨游离脂肪酸(FFA)作用下胰岛βTC3细胞双链RNA依赖性蛋白样内质网激酶(PERK)的表达以及利拉鲁肽(Lira)对其表达的干预作用.方法:以βTC3细胞为研究对象,分为对照组和FFA组(0.125,0.25,0.5及1 mmol/L)孵育24h,Westernblot方法检测PERK的表达.然后,分为对照组,FFA组,和FFA+Lira组(0.5 mg/L和1 mg/L),Lira预孵育6h后,1mmol/L FFA 继续孵育24h,Western blot检测PERK的表达.结果:①不同浓度FFA孵育24h后,与对照组相比,1mmol/L FFA组PERK表达增加(P<0.05).②与1 mmo1/L FFA组相比,0

  11. Signalling of double strand breaks and deprotected telomeres in Arabidopsis.

    Directory of Open Access Journals (Sweden)

    Simon eAmiard

    2013-10-01

    Full Text Available Failure to repair DNA double strand breaks (DSB can lead to chromosomal rearrangements and eventually to cancer or cell death. Radiation and environmental pollutants induce DSB and this is of particular relevance to plants due to their sessile life style. DSB also occur naturally in cells during DNA replication and programmed induction of DSB initiates the meiotic recombination essential for gametogenesis in most eukaryotes. The linear nature of most eukaryotic chromosomes means that each chromosome has two "broken" ends. Chromosome ends, or telomeres, are protected by nucleoprotein caps which avoid their recognition as DSB by the cellular DNA repair machinery. Deprotected telomeres are recognized as DSB and become substrates for recombination leading to chromosome fusions, the "bridge-breakage-fusion" cycle, genome rearrangements and cell death. The importance of repair of DSB and the severity of the consequences of their misrepair have led to the presence of multiple, robust mechanisms for their detection and repair. After a brief overview of DSB repair pathways to set the context, we present here an update of current understanding of the detection and signalling of DSB in the plant, Arabidopsis thaliana.

  12. DNA double strand break repair, aging and the chromatin connection.

    Science.gov (United States)

    Gorbunova, Vera; Seluanov, Andrei

    2016-06-01

    Are DNA damage and mutations possible causes or consequences of aging? This question has been hotly debated by biogerontologists for decades. The importance of DNA damage as a possible driver of the aging process went from being widely recognized to then forgotten, and is now slowly making a comeback. DNA double strand breaks (DSBs) are particularly relevant to aging because of their toxicity, increased frequency with age and the association of defects in their repair with premature aging. Recent studies expand the potential impact of DNA damage and mutations on aging by linking DNA DSB repair and age-related chromatin changes. There is overwhelming evidence that increased DNA damage and mutations accelerate aging. However, an ultimate proof of causality would be to show that enhanced genome and epigenome stability delays aging. This is not an easy task, as improving such complex biological processes is infinitely more difficult than disabling it. We will discuss the possibility that animal models with enhanced DNA repair and epigenome maintenance will be generated in the near future.

  13. Euler buckling and nonlinear kinking of double-stranded DNA.

    Science.gov (United States)

    Fields, Alexander P; Meyer, Elisabeth A; Cohen, Adam E

    2013-11-01

    The bending stiffness of double-stranded DNA (dsDNA) at high curvatures is fundamental to its biological activity, yet this regime has been difficult to probe experimentally, and literature results have not been consistent. We created a 'molecular vise' in which base-pairing interactions generated a compressive force on sub-persistence length segments of dsDNA. Short dsDNA strands (Euler buckling'. We monitored the buckling transition via Förster Resonance Energy Transfer (FRET) between appended fluorophores. For low-to-moderate concentrations of monovalent salt (up to ∼150 mM), our results are in quantitative agreement with the worm-like chain (WLC) model of DNA elasticity, without the need to invoke any 'kinked' states. Greater concentrations of monovalent salts or 1 mM Mg(2+) induced an apparent softening of the dsDNA, which was best accounted for by a kink in the region of highest curvature. We tested the effects of all single-nucleotide mismatches on the DNA bending. Remarkably, the propensity to kink correlated with the thermodynamic destabilization of the mismatched DNA relative the perfectly complementary strand, suggesting that the kinked state is locally melted. The molecular vise is exquisitely sensitive to the sequence-dependent linear and nonlinear elastic properties of dsDNA.

  14. An effective mesoscopic model of double-stranded DNA.

    Science.gov (United States)

    Jeon, Jae-Hyung; Sung, Wokyung

    2014-01-01

    Watson and Crick's epochal presentation of the double helix structure in 1953 has paved the way to intense exploration of DNA's vital functions in cells. Also, recent advances of single molecule techniques have made it possible to probe structures and mechanics of constrained DNA at length scales ranging from nanometers to microns. There have been a number of atomistic scale quantum chemical calculations or molecular level simulations, but they are too computationally demanding or analytically unfeasible to describe the DNA conformation and mechanics at mesoscopic levels. At micron scales, on the other hand, the wormlike chain model has been very instrumental in describing analytically the DNA mechanics but lacks certain molecular details that are essential in describing the hybridization, nano-scale confinement, and local denaturation. To fill this fundamental gap, we present a workable and predictive mesoscopic model of double-stranded DNA where the nucleotides beads constitute the basic degrees of freedom. With the inter-strand stacking given by an interaction between diagonally opposed monomers, the model explains with analytical simplicity the helix formation and produces a generalized wormlike chain model with the concomitant large bending modulus given in terms of the helical structure and stiffness. It also explains how the helical conformation undergoes overstretch transition to the ladder-like conformation at a force plateau, in agreement with the experiment.

  15. Double-strand break repair-adox: Restoration of suppressed double-strand break repair during mitosis induces genomic instability.

    Science.gov (United States)

    Terasawa, Masahiro; Shinohara, Akira; Shinohara, Miki

    2014-12-01

    Double-strand breaks (DSBs) are one of the severest types of DNA damage. Unrepaired DSBs easily induce cell death and chromosome aberrations. To maintain genomic stability, cells have checkpoint and DSB repair systems to respond to DNA damage throughout most of the cell cycle. The failure of this process often results in apoptosis or genomic instability, such as aneuploidy, deletion, or translocation. Therefore, DSB repair is essential for maintenance of genomic stability. During mitosis, however, cells seem to suppress the DNA damage response and proceed to the next G1 phase, even if there are unrepaired DSBs. The biological significance of this suppression is not known. In this review, we summarize recent studies of mitotic DSB repair and discuss the mechanisms of suppression of DSB repair during mitosis. DSB repair, which maintains genomic integrity in other phases of the cell cycle, is rather toxic to cells during mitosis, often resulting in chromosome missegregation and aberration. Cells have multiple safeguards to prevent genomic instability during mitosis: inhibition of 53BP1 or BRCA1 localization to DSB sites, which is important to promote non-homologous end joining or homologous recombination, respectively, and also modulation of the non-homologous end joining core complex to inhibit DSB repair. We discuss how DSBs during mitosis are toxic and the multiple safeguard systems that suppress genomic instability.

  16. Double-stranded Let-7 mimics, potential candidates for cancer gene therapy.

    Science.gov (United States)

    Wang, Qi-zhao; Lv, Ying-hui; Gong, Yu-hua; Li, Zhao-fa; Xu, William; Diao, Yong; Xu, Ruian

    2012-03-01

    MicroRNAs (miRNAs), a class of small, single-stranded endogenous RNAs, act as post-transcriptional regulators of gene expression. The ability of one single miRNA regulating multiple functionally related mRNAs makes it a new potential candidate for cancer gene therapy. Let-7s miRNAs have been demonstrated as tumor-suppressor genes in various types of cancers, providing one choice of gene therapy by replenishing this miRNA. In the present studies, we demonstrate that the chemically synthesized, double-stranded Let-7 mimics can inhibit the growth and migration and induce the cell cycle arrest of lung cancer cell lines in vitro. Let-7 mimics silence gene expression by binding to the 3' UTR of targeting mRNAs. Mutation of seed sequence significantly depresses the gene silencing activity of Let-7 mimics. Our results also demonstrate that it is possible to increase the activity of Let-7s through mutating the sequence within the 3'end of the antisense strand. Directly, co-transfection Let-7 mimics with active siRNAs impairs the anti-cancer activities of Let-7 mimics. However, a 3-h interval between the introduction of Let-7 mimics and a kind of siRNA avoids the competition and enhances the anti-cancer activities of Let-7 mimics. Taken together, these results have revealed that Let-7s mimics are potential candidates for cancer gene therapy.

  17. Regulation of DNA double-strand break repair pathway choice

    Institute of Scientific and Technical Information of China (English)

    Meena Shrivastav; Leyma P De Haro; Jac A Nickoloff

    2008-01-01

    DNA double-strand breaks (DSBs) are critical lesions that can result in cell death or a wide variety of genetic alterations including large- or small-scale deletions, loss of heterozygosity, translocations, and chromosome loss. DSBs are repaired by non-homologous end-joining (NHEJ) and homologous recombination (HR), and defects in these pathways cause genome instability and promote tumorigenesis. DSBs arise from endogenous sources includ-ing reactive oxygen species generated during cellular metabolism, collapsed replication forks, and nucleases, and from exogenous sources including ionizing radiation and chemicals that directly or indirectly damage DNA and are commonly used in cancer therapy. The DSB repair pathways appear to compete for DSBs, but the balance between them differs widely among species, between different cell types of a single species, and during different cell cycle phases of a single cell type. Here we review the regulatory factors that regulate DSB repair by NHEJ and HR in yeast and higher eukaryotes. These factors include regulated expression and phosphorylation of repair proteins, chromatin modulation of repair factor accessibility, and the availability of homologous repair templates. While most DSB repair proteins appear to function exclusively in NHEJ or HR, a number of proteins influence both pathways, including the MRE11/RAD50/NBS1 (XRS2) complex, BRCA1, histone H2AX, PARP-1, RAD18, DNA-dependent protein kinase catalytic subunit (DNA-PKcs), and ATM. DNA-PKcs plays a role in mammalian NHEJ, but it also influences HR through a complex regulatory network that may involve crosstalk with ATM, and the regulation of at least 12 proteins involved in HR that are phosphorylated by DNA-PKcs and/or ATM.

  18. Protein kinase CK2 localizes to sites of DNA double-strand break regulating the cellular response to DNA damage

    Directory of Open Access Journals (Sweden)

    Olsen Birgitte B

    2012-03-01

    Full Text Available Abstract Background The DNA-dependent protein kinase (DNA-PK is a nuclear complex composed of a large catalytic subunit (DNA-PKcs and a heterodimeric DNA-targeting subunit Ku. DNA-PK is a major component of the non-homologous end-joining (NHEJ repair mechanism, which is activated in the presence of DNA double-strand breaks induced by ionizing radiation, reactive oxygen species and radiomimetic drugs. We have recently reported that down-regulation of protein kinase CK2 by siRNA interference results in enhanced cell death specifically in DNA-PKcs-proficient human glioblastoma cells, and this event is accompanied by decreased autophosphorylation of DNA-PKcs at S2056 and delayed repair of DNA double-strand breaks. Results In the present study, we show that CK2 co-localizes with phosphorylated histone H2AX to sites of DNA damage and while CK2 gene knockdown is associated with delayed DNA damage repair, its overexpression accelerates this process. We report for the first time evidence that lack of CK2 destabilizes the interaction of DNA-PKcs with DNA and with Ku80 at sites of genetic lesions. Furthermore, we show that CK2 regulates the phosphorylation levels of DNA-PKcs only in response to direct induction of DNA double-strand breaks. Conclusions Taken together, these results strongly indicate that CK2 plays a prominent role in NHEJ by facilitating and/or stabilizing the binding of DNA-PKcs and, possibly other repair proteins, to the DNA ends contributing to efficient DNA damage repair in mammalian cells.

  19. A requirement for polymerized actin in DNA double-strand break repair.

    Science.gov (United States)

    Andrin, Christi; McDonald, Darin; Attwood, Kathleen M; Rodrigue, Amélie; Ghosh, Sunita; Mirzayans, Razmik; Masson, Jean-Yves; Dellaire, Graham; Hendzel, Michael J

    2012-07-01

    Nuclear actin is involved in several nuclear processes from chromatin remodeling to transcription. Here we examined the requirement for actin polymerization in DNA double-strand break repair. Double-strand breaks are considered the most dangerous type of DNA lesion. Double-strand break repair consists of a complex set of events that are tightly regulated. Failure at any step can have catastrophic consequences such as genomic instability, oncogenesis or cell death. Many proteins involved in this repair process have been identified and their roles characterized. We discovered that some DNA double-strand break repair factors are capable of associating with polymeric actin in vitro and specifically, that purified Ku70/80 interacts with polymerized actin under these conditions. We find that the disruption of polymeric actin inhibits DNA double strand break repair both in vitro and in vivo. Introduction of nuclear targeted mutant actin that cannot polymerize, or the depolymerization of endogenous actin filaments by the addition of cytochalasin D, alters the retention of Ku80 at sites of DNA damage in live cells. Our results suggest that polymeric actin is required for proper DNA double-strand break repair and may function through the stabilization of the Ku heterodimer at the DNA damage site.

  20. Regulated restriction endonuclease expression: A novel, radiomimetic model of DNA double strand break induction

    Energy Technology Data Exchange (ETDEWEB)

    Radany, E.H.; Pu, A.T. [Univ. of Michigan School of Medicine, Ann Arbor, MI (United States)

    1997-10-01

    Exposure of mammalian cells to ionizing radiations (IR) produces a plethora of damages in DNA and non-DNA targets. Although DNA double strand breaks (DSB) are thought to be the critical lesion generated by IR with respect to conventional cytotoxicity, it is clear that signaling events regulating cellular responses to IR arise from multiple other lesions in addition to these. The authors are interested in identifying cellular signaling events that derive from DSB specifically, as well as the distal effects (e.g., repair, apoptosis, cell cycle delay) of such signaling. Although electroporation of restriction enzymes might afford an approach to such studies, serious concerns would be raised by the non-uniformity of enzyme transfer and general disruption of the intracellular environment (with the possibility of associated signaling processes) when using this method. The authors have established a radiomimetic model for DSB induction, based upon expression of a hybrid steroid hormone receptor: this system is subject to tight, rapid postranslational regulation of endonuclease activity via addition or withdrawl of the cognate hormone ligand. In preliminary experiments, The authors have demonstrated ligand dose and exposure time-dependent cytotoxicity and DSB induction (the latter assayed by PFGE). Cytogenetic characterization of this system, as well as studies of the interaction between enzyme- and IR-generated DSB are in progress. RNA differential display and subtractive enrichment cloning approaches will ultimately be used to identify genes whose expression changes as a consequence of isolated DSB induction.

  1. Sequence-specific double strand breaks trigger P-TEFb-dependent Rpb1-CTD hyperphosphorylation

    Energy Technology Data Exchange (ETDEWEB)

    Napolitano, Giuliana; Amente, Stefano; Lavadera, Miriam Lubrano; Di Palo, Giacomo; Ambrosio, Susanna; Lania, Luigi [Department of Biology, University of Naples ‘Federico II’, Naples (Italy); Dellino, Gaetano Ivan; Pelicci, Pier Giuseppe [Department of Experimental Oncology, European Institute of Oncology, Milan (Italy); Majello, Barbara, E-mail: majello@unina.it [Department of Biology, University of Naples ‘Federico II’, Naples (Italy)

    2013-09-15

    Highlights: • Using an inducible restriction enzyme, hundreds of site-specific DSBs are generated across the genome. • Site-specific DSBs trigger activation of P-TEFb and consequent Rpb1-CTD hyperphosphorylation. • Site-specific DSBs induce activation of p53-transcriptional axis. - Abstract: Double strand DNA breaks (DSBs) are one of the most challenging forms of DNA damage which, if left unrepaired, can trigger cellular death and can contribute to cancer. A number of studies have been focused on DNA-damage response (DDR) mechanisms, and most of them rely on the induction of DSBs triggered by chemical compounds or radiations. However, genotoxic drugs and radiation treatments of cultured cell lines induce random DSBs throughout the genome, thus heterogeneously across the cell population, leading to variability of the cellular response. To overcome this aspect, we used here a recently described cell-based DSBs system whereby, upon induction of an inducible restriction enzyme, hundreds of site-specific DSBs are generated across the genome. We show here that sequence-specific DSBs are sufficient to activate the positive transcription elongation factor b (P-TEFb), to trigger hyperphosphorylation of the largest RNA polymerase II carboxyl-terminal-domain (Rpb1-CTD) and to induce activation of p53-transcriptional axis resulting in cell cycle arrest.

  2. Evolution of susceptibility to ingested double-stranded RNAs in Caenorhabditis nematodes.

    Directory of Open Access Journals (Sweden)

    Isabelle Nuez

    Full Text Available BACKGROUND: The nematode Caenorhabditis elegans is able to take up external double-stranded RNAs (dsRNAs and mount an RNA interference response, leading to the inactivation of specific gene expression. The uptake of ingested dsRNAs into intestinal cells has been shown to require the SID-2 transmembrane protein in C. elegans. By contrast, C. briggsae was shown to be naturally insensitive to ingested dsRNAs, yet could be rendered sensitive by transgenesis with the C. elegans sid-2 gene. Here we aimed to elucidate the evolution of the susceptibility to external RNAi in the Caenorhabditis genus. PRINCIPAL FINDINGS: We study the sensitivity of many new species of Caenorhabditis to ingested dsRNAs matching a conserved actin gene sequence from the nematode Oscheius tipulae. We find ample variation in the Caenorhabditis genus in the ability to mount an RNAi response. We map this sensitivity onto a phylogenetic tree, and show that sensitivity or insensitivity have evolved convergently several times. We uncover several evolutionary losses in sensitivity, which may have occurred through distinct mechanisms. We could render C. remanei and C. briggsae sensitive to ingested dsRNAs by transgenesis of the Cel-sid-2 gene. We thus provide tools for RNA interference studies in these species. We also show that transgenesis by injection is possible in many Caenorhabditis species. CONCLUSIONS: The ability of animals to take up dsRNAs or to respond to them by gene inactivation is under rapid evolution in the Caenorhabditis genus. This study provides a framework and tools to use RNA interference and transgenesis in various Caenorhabditis species for further comparative and evolutionary studies.

  3. Sequence and phylogenetic analyses of novel totivirus-like double-stranded RNAs from field-collected powdery mildew fungi.

    Science.gov (United States)

    Kondo, Hideki; Hisano, Sakae; Chiba, Sotaro; Maruyama, Kazuyuki; Andika, Ida Bagus; Toyoda, Kazuhiro; Fujimori, Fumihiro; Suzuki, Nobuhiro

    2016-02-02

    The identification of mycoviruses contributes greatly to understanding of the diversity and evolutionary aspects of viruses. Powdery mildew fungi are important and widely studied obligate phytopathogenic agents, but there has been no report on mycoviruses infecting these fungi. In this study, we used a deep sequencing approach to analyze the double-stranded RNA (dsRNA) segments isolated from field-collected samples of powdery mildew fungus-infected red clover plants in Japan. Database searches identified the presence of at least ten totivirus (genus Totivirus)-like sequences, termed red clover powdery mildew-associated totiviruses (RPaTVs). The majority of these sequences shared moderate amino acid sequence identity with each other (powdery mildew fungus populations infecting red clover plants in the field.

  4. The enzyme and the cDNA sequence of a thermolabile and double-strand specific DNase from Northern shrimps (Pandalus borealis.

    Directory of Open Access Journals (Sweden)

    Inge W Nilsen

    Full Text Available BACKGROUND: We have previously isolated a thermolabile nuclease specific for double-stranded DNA from industrial processing water of Northern shrimps (Pandalus borealis and developed an application of the enzyme in removal of contaminating DNA in PCR-related technologies. METHODOLOGY/PRINCIPAL FINDINGS: A 43 kDa nuclease with a high specific activity of hydrolysing linear as well as circular forms of DNA was purified from hepatopancreas of Northern shrimp (Pandalus borealis. The enzyme displayed a substrate preference that was shifted from exclusively double-stranded DNA in the presence of magnesium to also encompass significant activity against single-stranded DNA when calcium was added. No activity against RNA was detected. Although originating from a cold-environment animal, the shrimp DNase has only minor low-temperature activity. Still, the enzyme was irreversibly inactivated by moderate heating with a half-life of 1 min at 65 degrees C. The purified protein was partly sequenced and derived oligonucleotides were used to prime amplification of the encoding cDNA. This cDNA sequence revealed an open reading frame encoding a 404 amino acid protein containing a signal peptide. By sequence similarity the enzyme is predicted to belong to a family of DNA/RNA non-specific nucleases even though this shrimp DNase lacks RNase activity and is highly double-strand specific in some respects. These features are in agreement with those previously established for endonucleases classified as similar to the Kamchatka crab duplex-specific nuclease (Par_DSN. Sequence comparisons and phylogenetic analyses confirmed that the Northern shrimp nuclease resembles the Par_DSN-like nucleases and displays a more distant relationship to the Serratia family of nucleases. CONCLUSIONS/SIGNIFICANCE: The shrimp nuclease contains enzyme activity that may be controlled by temperature or buffer compositions. The double-stranded DNA specificity, as well as the thermolabile feature

  5. Facile synthesis of Graphene Oxide/Double-stranded DNA composite liquid crystals and Hydrogels

    Indian Academy of Sciences (India)

    Rajendra Kurapati; Ashok M Raichur; U Venkateswara Reddy; N Suryaprakash

    2016-03-01

    Investigation of the interactions between graphene oxide (GO) and biomolecules is very crucialfor the development of biomedical applications based on GO. This study reports the first observation of thespontaneous formation of self-assembled liquid crystals and three-dimensional hydrogels of graphene oxidewith double-stranded DNA by simple mixing in an aqueous buffer media without unwinding double-strandedDNA to single-stranded DNA. The GO/dsDNA hydrogels have shown controlled porosity by changing the concentration of the components. The strong binding between dsDNA and graphene is proved by Ramanspectroscopy

  6. Interaction of phenazinium dyes with double-stranded poly(A): spectroscopy and isothermal titration calorimetry studies.

    Science.gov (United States)

    Khan, Asma Yasmeen; Saha, Baishakhi; Kumar, Gopinatha Suresh

    2014-10-15

    A comprehensive study on the binding of phenazinium dyes viz. janus green B, indoine blue, safranine O and phenosafranine with double stranded poly(A) using various spectroscopic and calorimetric techniques is presented. A higher binding of janus green B and indoine blue over safranine O and phenosafranine to poly(A) was observed from all experiments. Intercalative mode of binding of the dyes was inferred from fluorescence polarization anisotropy, iodide quenching and viscosity experiments. Circular dichroism study revealed significant perturbation of the secondary structure of poly(A) on binding of these dyes. Results from isothermal titration calorimetry experiments suggested that the binding was predominantly entropy driven with a minor contribution of enthalpy to the standard molar Gibbs energy. The results presented here may open new opportunities in the application of these dyes as RNA targeted therapeutic agents.

  7. Oral delivery of double-stranded RNAs induces mortality in nymphs and adults of the Asian citrus psyllid, Diaphorina citri

    Science.gov (United States)

    Galdeano, Diogo Manzano; Breton, Michèle Claire; Lopes, João Roberto Spotti; Falk, Bryce W.

    2017-01-01

    The Asian citrus psyllid (ACP), Diaphorina citri Kuwayama, is one of the most important citrus pests. ACP is the vector of the phloem-limited bacteria Candidatus Liberibacter americanus and Candidatus Liberibacter asiaticus, the causal agents of the devastating citrus disease huanglongbing (HLB). The management of HLB is based on the use of healthy young plants, eradication of infected plants and chemical control of the vector. RNA interference (RNAi) has proven to be a promising tool to control pests and explore gene functions. Recently, studies have reported that target mRNA knockdown in many insects can be induced through feeding with double-stranded RNA (dsRNA). In the current study, we targeted the cathepsin D, chitin synthase and inhibitor of apoptosis genes of adult and nymph ACP by feeding artificial diets mixed with dsRNAs and Murraya paniculata leaves placed in dsRNAs solutions, respectively. Adult ACP mortality was positively correlated with the amount of dsRNA used. Both nymphs and adult ACP fed dsRNAs exhibited significantly increased mortality over time compared with that of the controls. Moreover, qRT-PCR analysis confirmed the dsRNA-mediated RNAi effects on target mRNAs. These results showed that RNAi can be a powerful tool for gene function studies in ACP and perhaps for HLB control. PMID:28282380

  8. Torsional regulation of hRPA-induced unwinding of double-stranded DNA

    NARCIS (Netherlands)

    De Vlaminck, I.; Vidic, I.; Van Loenhout, M.T.J.; Kanaar, R.; Lebbink, J.H.G.; Dekker, C.

    2010-01-01

    All cellular single-stranded (ss) DNA is rapidly bound and stabilized by single stranded DNA-binding proteins (SSBs). Replication protein A, the main eukaryotic SSB, is able to unwind double-stranded (ds) DNA by binding and stabilizing transiently forming bubbles of ssDNA. Here, we study the dynamic

  9. DNA double strand breaks repair pathways in mouse male germ cells

    NARCIS (Netherlands)

    Ahmed, E.A.

    2009-01-01

    DNA double strand breaks (DSBs) are induced by ionizing radiation, and during meiotic recombination. DSBs are repaired via two main pathways, homologous recombination (HR) and non homologous end-joining (NHEJ). There are three main types of male germ cells, spermatogonia, spermatocytes and spermatid

  10. Folding DNA origami from a double-stranded source of scaffold

    Science.gov (United States)

    Högberg, Björn; Liedl, Tim; Shih, William M.

    2009-01-01

    Combined heat and chemical denaturation of double-stranded DNA scaffold strands in the presence of staple strands, followed by a sudden temperature drop and then stepwise dialysis to remove the chemical denaturant, leads to self-assembly of two distinct DNA-origami structures. PMID:19566089

  11. DNA double-strand break rejoining in human follicular lymphoma and glioblastoma tumor cells

    NARCIS (Netherlands)

    Macann, AMJ; Britten, RA; Poppema, S; Pearcey, R; Rosenberg, E; Allalunis-Turner, MJ; Murray, D

    2000-01-01

    Follicle center cell lymphoma is among the most radioresponsive of human cancers. To assess whether this radioresponsiveness might be a result of a compromised ability of the tumor cells to accomplish the biologically-effective repair of DNA double-strand breaks (DSBs), we have measured i) the exten

  12. Chromatin mobility is increased at sites of DNA double-strand breaks

    NARCIS (Netherlands)

    P.M. Krawczyk (Przemek); T. Borovski (Tijana); J. Stap (Jan); T. Cijsouw (Tony); R. ten Cate (Rebecca); J.P. Medema (Jan Paul); R. Kanaar (Roland); N.A.P. Franken (Nicolaas); J.A. Aten (Jacob)

    2012-01-01

    textabstractDNA double-strand breaks (DSBs) can efficiently kill cancer cells, but they can also produce unwanted chromosome rearrangements when DNA ends from different DSBs are erroneously joined. Movement of DSB-containing chromatin domains might facilitate these DSB interactions and promote the f

  13. Meiotic and mitotic functions of mammalian RAD 18 in DNA double-strand break repair

    NARCIS (Netherlands)

    A. Inagaki (Akiko)

    2010-01-01

    textabstractThis thesis focuses on the role of RAD 18 in DNA double-strand break (DSB ) repair. Much is known about the role of RAD 18, and its critical substrate PCNA in replication damage bypass (RDB ) repair. However, the roles of RAD 18 in DSB repair are still elusive, although several interacti

  14. Mouse RAD54 affects DNA double-strand break repair and sister chromatid exchange

    NARCIS (Netherlands)

    H.B. Beverloo (Berna); R.D. Johnson (Roger); M. Jasin (Maria); R. Kanaar (Roland); J.H.J. Hoeijmakers (Jan); M.L.G. Dronkert (Mies)

    2000-01-01

    textabstractCells can achieve error-free repair of DNA double-strand breaks (DSBs) by homologous recombination through gene conversion with or without crossover. In contrast, an alternative homology-dependent DSB repair pathway, single-strand annealing (SSA), results in deletions. In this study, we

  15. Double-strand break repair and G4 DNA stability in Caenorhabditis elegans

    NARCIS (Netherlands)

    Pontier, D.B.

    2010-01-01

    DNA double-strand breaks (DSBs) can be repaired by three canonical repair pathways. Homologous recombination (HR) uses the sister chromatid or homologous chromosome as a template to repair the DSB in an error-free manner. In non-homologous end-joining (NHEJ), the broken ends are ligated with little

  16. Nanoconstructions on the base of double-stranded DNA molecules and their optical properties

    Science.gov (United States)

    Skuridin, S. G.; Yevdokimov, Yu. M.; Chulkov, D. P.; Gusev, V. M.; Kompanets, O. N.; Vereschagin, F. V.

    2016-12-01

    Experimental results have been presented on studying optical properties of nanoconstructions formed of orientationally ordered neighbouring double-stranded DNA molecules in the structure of their liquid-crystalline phases and dispersion particles of these phases including ones cured with intercalators.

  17. The role of homologous recombination in mitotic and meiotic double-strand break repair

    NARCIS (Netherlands)

    Vries, Femke Adriana Theodora de

    2007-01-01

    All organisms are composed of cells and the cell’s nucleus contains DNA. The induction of DNA damage is a threat to organisms. Signalling of DNA damage and subsequent repair is of substantial importance. Double-strand breaks (DSBs) in DNA can be induced by ionising radiation and DNA damaging agents

  18. Branch migration prevents DNA loss during double-strand break repair.

    Directory of Open Access Journals (Sweden)

    Julia S P Mawer

    2014-08-01

    Full Text Available The repair of DNA double-strand breaks must be accurate to avoid genomic rearrangements that can lead to cell death and disease. This can be accomplished by promoting homologous recombination between correctly aligned sister chromosomes. Here, using a unique system for generating a site-specific DNA double-strand break in one copy of two replicating Escherichia coli sister chromosomes, we analyse the intermediates of sister-sister double-strand break repair. Using two-dimensional agarose gel electrophoresis, we show that when double-strand breaks are formed in the absence of RuvAB, 4-way DNA (Holliday junctions are accumulated in a RecG-dependent manner, arguing against the long-standing view that the redundancy of RuvAB and RecG is in the resolution of Holliday junctions. Using pulsed-field gel electrophoresis, we explain the redundancy by showing that branch migration catalysed by RuvAB and RecG is required for stabilising the intermediates of repair as, when branch migration cannot take place, repair is aborted and DNA is lost at the break locus. We demonstrate that in the repair of correctly aligned sister chromosomes, an unstable early intermediate is stabilised by branch migration. This reliance on branch migration may have evolved to help promote recombination between correctly aligned sister chromosomes to prevent genomic rearrangements.

  19. Differential regulation of the cellular response to DNA double-strand breaks in G1

    DEFF Research Database (Denmark)

    Barlow, Jacqueline H; Lisby, Michael; Rothstein, Rodney

    2008-01-01

    Double-strand breaks (DSBs) are potentially lethal DNA lesions that can be repaired by either homologous recombination (HR) or nonhomologous end-joining (NHEJ). We show that DSBs induced by ionizing radiation (IR) are efficiently processed for HR and bound by Rfa1 during G1, while endonuclease-in...

  20. Genetics of x-ray induced double strand break repair in saccharomyces cerevisiae

    Energy Technology Data Exchange (ETDEWEB)

    Budd, M.E.

    1982-07-01

    The possible fates of x-ray-induced double-strand breaks in Saccharomyces cerevisiae were examined. One possible pathway which breaks can follow, the repair pathway, was studied by assaying strains with mutations in the RAD51, RAD54, and RAD57 loci for double-strand break repair. In order of increasing radiation sensitivity one finds: rad57-1(23/sup 0/)> rad51-1(30/sup 0/)> rad54-3(36/sup 0/). At 36/sup 0/, rad54-3 cells cannot repair double-strand breaks, while 23/sup 0/, they can. Strains with the rad57-1 mutation can rejoin broken chromosomes at both temperatures. However, the low survival at 36/sup 0/ shows that the assay is not distinguishing large DNA fragments which allow cell survival from those which cause cell death. A rad51-1 strain could also rejoin broken chromosomes, and was thus capable of incomplete repair. The data can be explained with the hypothesis that rad54-3 cells are blocked in an early step of repair, while rad51-1 and rad57-1 strains are blocked in a later step of repair. The fate of double-strand breaks when they are left unrepaired was investigated with the rad54-3 mutation. If breaks are prevented from entering the RAD54 repair pathway they become uncommitted lesions. These lesions are repaired slower than the original breaks. One possible fate for an uncommitted lesion is conversion into a fixed lesion, which is likely to be an unrepairable or misrepaired double-strand break. The presence of protein synthesis after irradiation increases the probability that a break will enter the repair pathway. Evidence shows that increased probability of repair results from enhanced synthesis of repair proteins shortly after radiation. (ERB)

  1. Genetics of x-ray induced double strand break repair in Saccharomyces cerevisiae

    Energy Technology Data Exchange (ETDEWEB)

    Budd, M.E.

    1982-01-01

    This thesis examined the possible fates of x-ray induced double strand breaks in Saccharomyces cerevisiae. One possible pathway which breaks can follow is the repair pathway and this pathway was studied by assaying strains with mutations in RAD51, RAD54, and RAD57 loci for double strand break repair using neutral sucrose sedimentation. Rad54-3 strains were sensitive to x-ray at 36/sup 0/ and resistant at 23/sup 0/, while rad57-1 strains are sensitive to radiation at 23/sup 0/ and resistant at 36/sup 0/. In order of increasing radiation sensitivity one finds: rad57-1(23/sup 0/)> rad51-1(30/sup 0/)>rad54-3(36/sup 0/). At the restrictive temperature 36/sup 0/, rad54-3 cells are unaable to repair double strand breaks, while at the permissive temperature, 23/sup 0/, these strains are able to repair double strand breaks. On the other hand, strains with the rad57-1 mutation appear to be able to rejoin broken chromosomes at both the permissive and restrictive temperature. However, the low assay is not distinguishing large DNA fragments which allow cell survival from large DNA fragments which cause cell death. A rad51-1 strain also appeared able to rejoin broken chromosomes, and is thus capable of incomplete repair. The data can be explained with the hypotheses that rad54-3 cells are blocked in a later step of repair. The fate of double strand breaks when they are left unrepaired was also investigated with the temperature conditional rad54-3 mutation. If breaks are prevented from entering the RAD54 repair pathway they are modified and become uncommitted lesions. The rate these uncommitted lesions are repaired is slower than the rate the original breaks are repaired.

  2. Detection of double-stranded RNA viruses in fecal samples of dogs with gastroenteritis in Rio de Janeiro, Brazil Detecção de vírus com genoma de RNA fita dupla em fezes de cães com gastrenterite no Rio de Janeiro, Brasil

    Directory of Open Access Journals (Sweden)

    A.P. Costa

    2004-08-01

    Full Text Available Colheram-se 163 amostras fecais no período de 1995 a 2001 para investigar a ocorrência da infecção por parvovírus e rotavírus em cães com gastrenterite utilizando-se a técnica de eletroforese em gel de poliacrilamida. Em três amostras observou-se a presença do genoma bisegmentado similar ao perfil eletroforético dos picobirnavírus (PBV e em uma, três segmentos de RNA dupla fita, característico de picotrirnavírus. Das amostras positivas para PBV, duas foram obtidas de filhotes e uma foi positiva para parvovírus canino. Este é o primeiro relato da detecção de vírus com genoma bisegmentado em cães com diarréia no Estado do Rio de Janeiro.

  3. Amplification methods bias metagenomic libraries of uncultured single-stranded and double-stranded DNA viruses.

    Science.gov (United States)

    Kim, Kyoung-Ho; Bae, Jin-Woo

    2011-11-01

    Investigation of viruses in the environment often requires the amplification of viral DNA before sequencing of viral metagenomes. In this study, two of the most widely used amplification methods, the linker amplified shotgun library (LASL) and multiple displacement amplification (MDA) methods, were applied to a sample from the seawater surface. Viral DNA was extracted from viruses concentrated by tangential flow filtration and amplified by these two methods. 454 pyrosequencing was used to read the metagenomic sequences from different libraries. The resulting taxonomic classifications of the viruses, their functional assignments, and assembly patterns differed substantially depending on the amplification method. Only double-stranded DNA viruses were retrieved from the LASL, whereas most sequences in the MDA library were from single-stranded DNA viruses, and double-stranded DNA viral sequences were minorities. Thus, the two amplification methods reveal different aspects of viral diversity.

  4. Modular construction of DNA nanotubes of tunable geometry and single- or double-stranded character.

    Science.gov (United States)

    Aldaye, Faisal A; Lo, Pik Kwan; Karam, Pierre; McLaughlin, Christopher K; Cosa, Gonzalo; Sleiman, Hanadi F

    2009-06-01

    DNA nanotubes can template the growth of nanowires, orient transmembrane proteins for nuclear magnetic resonance determination, and can potentially act as stiff interconnects, tracks for molecular motors and nanoscale drug carriers. Current methods for the construction of DNA nanotubes result in symmetrical and cylindrical assemblies that are entirely double-stranded. Here, we report a modular approach to DNA nanotube synthesis that provides access to geometrically well-defined triangular and square-shaped DNA nanotubes. We also construct the first nanotube assemblies that can exist in double- and single-stranded forms with significantly different stiffness. This approach allows for parameters such as geometry, stiffness, and single- or double-stranded character to be fine-tuned, and could enable the creation of designer nanotubes for a range of applications, including the growth of nanowires of controlled shape, the loading and release of cargo, and the real-time modulation of stiffness and persistence length within DNA interconnects.

  5. Colocalization of multiple DNA double-strand breaks at a single Rad52 repair centre

    DEFF Research Database (Denmark)

    Lisby, M.; Mortensen, Uffe Hasbro; Rothstein, R.

    2003-01-01

    DNA double-strand break repair (DSBR) is an essential process for preserving genomic integrity in all organisms. To investigate this process at the cellular level, we engineered a system of fluorescently marked DNA double-strand breaks (DSBs) in the yeast Saccharomyces cerevisiae to visualize...... in vivo DSBR in single cells. Using this system, we demonstrate for the first time that Rad52 DNA repair foci and DSBs colocalize. Time-lapse microscopy reveals that the relocalization of Rad52 protein into a focal assembly is a rapid and reversible process. In addition, analysis of DNA damage checkpoint......-deficient cells provides direct evidence for coordination between DNA repair and subsequent release from checkpoint arrest. Finally, analyses of cells experiencing multiple DSBs demonstrate that Rad52 foci are centres of DNA repair capable of simultaneously recruiting more than one DSB....

  6. The radioresistance kinase TLK1B protects the cells by promoting repair of double strand breaks

    Directory of Open Access Journals (Sweden)

    De Benedetti Arrigo

    2005-09-01

    Full Text Available Abstract Background The mammalian protein kinase TLK1 is a homologue of Tousled, a gene involved in flower development in Arabidopsis thaliana. The function of TLK1 is not well known, although knockout of the gene in Drosophila or expression of a dominant negative mutant in mouse cells causes loss of nuclear divisions and missegregation of chromosomes probably, due to alterations in chromatin remodeling capacity. Overexpression of TLK1B, a spliced variant of the TLK1 mRNA, in a model mouse cell line increases it's resistance to ionizing radiation (IR or the radiomimetic drug doxorubicin, also likely due to changes in chromatin remodeling. TLK1B is translationally regulated by the availability of the translation factor eIF4E, and its synthesis is activated by IR. The reason for this mechanism of regulation is likely to provide a rapid means of promoting repair of DSBs. TLK1B specifically phosphorylates histone H3 and Asf1, likely resulting in changes in chromatin structure, particularly at double strand breaks (DSB sites. Results In this work, we provide several lines of evidence that TLK1B protects the cells from IR by facilitating the repair of DSBs. First, the pattern of phosphorylation and dephosphorylation of H2AX and H3 indicated that cells overexpressing TLK1B return to pre-IR steady state much more rapidly than controls. Second, the repair of episomes damaged with DSBs was much more rapid in cells overexpressing TLK1B. This was also true for repair of genomic damage. Lastly, we demonstrate with an in vitro repair system that the addition of recombinant TLK1B promotes repair of a linearized plasmid incubated with nuclear extract. In addition, TLK1B in this in vitro system promotes the assembly of chromatin as shown by the formation of more highly supercoiled topomers of the plasmid. Conclusion In this work, we provide evidence that TLK1B promotes the repair of DSBs, likely as a consequence of a change in chromatin remodeling capacity that

  7. Structure of the replicative form of bacteriophage φX174 : VI. Studies on alkali-denatured double-stranded φX DNA

    NARCIS (Netherlands)

    Pouwels, P.H.; Knijnenburg, C.M.; Rotterdam, J. van; Cohen, J.A.; Jansz, H.S.

    1968-01-01

    Double-stranded φX DNA which accumulates after infection with bacteriophage φX174 in the presence of chloramphenicol consists mainly of twisted circular double-stranded DNA with no single-strand breaks (component I) and of circular double-stranded DNA, in which single-strand breaks are present (comp

  8. Evaluation of The Interaction between Netropsin and Double Stranded DNA by Capillary Zone Electrophoresis

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    Capillary zone electrophoresis (CZE) was applied to study the interaction between netropsin and a 14mer double stranded DNA (dsDNA). The binding constant of this interaction calculated from Scatchard plot was (1.07±0.10)×105 (mol/L)-1. The binding stoichiometry was 1:1. The use of polyacrylamide coated capillary showed better effect in the analysis of DNA than noncoated capillary.

  9. Effects of heavy ions on inactivation and DNA double strand breaks in Deinococcus radiodurans R1.

    Science.gov (United States)

    Zimmermann, H; Schafer, M; Schmitz, C; Bucker, H

    1994-10-01

    Inactivation and double strand break (dsb) induction after heavy ion irradiation were studied in stationary phase cells of the highly radiation resistant bacterium Deinococcus radiodurans R1. There is evidence that the radiation sensitivity of this bacterium is nearly independent on energy in the range of up to 15 MeV/u for lighter ions (Ar). The responses to dsb induction for charged particles show direct relationship between increasing radiation dose and residual intact DNA.

  10. Double-strand break damage and associated DNA repair genes predispose smokers to gene methylation

    OpenAIRE

    Leng, Shuguang; Stidley, Christine A.; Willink, Randy; Bernauer, Amanda; Do, Kieu; Picchi, Maria A.; Sheng, Xin; Frasco, Melissa, A.; Berg, David Van Den; Gilliland, Frank D.; Zima, Christopher; Crowell, Richard E.; Belinsky, Steven A.

    2008-01-01

    Gene promoter hypermethylation in sputum is a promising biomarker for predicting lung cancer. Identifying factors that predispose smokers to methylation of multiple gene promoters in the lung could impact strategies for early detection and chemoprevention. This study evaluated the hypothesis that double-strand break repair capacity and sequence variation in genes in this pathway are associated with a high methylation index in a cohort of current and former cancer-free smokers. A 50% reduction...

  11. Mechanistic Modelling and Bayesian Inference Elucidates the Variable Dynamics of Double-Strand Break Repair

    Science.gov (United States)

    2016-01-01

    DNA double-strand breaks are lesions that form during metabolism, DNA replication and exposure to mutagens. When a double-strand break occurs one of a number of repair mechanisms is recruited, all of which have differing propensities for mutational events. Despite DNA repair being of crucial importance, the relative contribution of these mechanisms and their regulatory interactions remain to be fully elucidated. Understanding these mutational processes will have a profound impact on our knowledge of genomic instability, with implications across health, disease and evolution. Here we present a new method to model the combined activation of non-homologous end joining, single strand annealing and alternative end joining, following exposure to ionising radiation. We use Bayesian statistics to integrate eight biological data sets of double-strand break repair curves under varying genetic knockouts and confirm that our model is predictive by re-simulating and comparing to additional data. Analysis of the model suggests that there are at least three disjoint modes of repair, which we assign as fast, slow and intermediate. Our results show that when multiple data sets are combined, the rate for intermediate repair is variable amongst genetic knockouts. Further analysis suggests that the ratio between slow and intermediate repair depends on the presence or absence of DNA-PKcs and Ku70, which implies that non-homologous end joining and alternative end joining are not independent. Finally, we consider the proportion of double-strand breaks within each mechanism as a time series and predict activity as a function of repair rate. We outline how our insights can be directly tested using imaging and sequencing techniques and conclude that there is evidence of variable dynamics in alternative repair pathways. Our approach is an important step towards providing a unifying theoretical framework for the dynamics of DNA repair processes. PMID:27741226

  12. Double-strand break repair on sex chromosomes: challenges during male meiotic prophase

    OpenAIRE

    Lu, Lin-Yu; Yu, Xiaochun

    2015-01-01

    During meiotic prophase, DNA double-strand break (DSB) repair-mediated homologous recombination (HR) occurs for exchange of genetic information between homologous chromosomes. Unlike autosomes or female sex chromosomes, human male sex chromosomes X and Y share little homology. Although DSBs are generated throughout male sex chromosomes, homologous recombination does not occur for most regions and DSB repair process is significantly prolonged. As a result, male sex chromosomes are coated with ...

  13. The ability of sperm selection techniques to remove single-or double-strand DNA damage

    Institute of Scientific and Technical Information of China (English)

    Maria Enciso; Miriam Iglesias; Isabel Galin; Jonas Sarasa; Antonio Gosalvez; Jaime Gosalvez

    2011-01-01

    @@ A wide variety of techniques for the preparation of sperm are currently available,of which the most commonly employed are densitygradient centrifugation (DGC) and swim-up (SUP).To date,these methods appear to be effective in selecting functional sperm for assisted reproduction techniques (ART),but they may have negative effects on sperm DNA.In this study,the ability of these semen processing techniques to eliminate spermatozoa containing single- and double-strand DNA damage was assessed by the two-tailed comet assay and the sperm chromatin dispersion test in 1[57]semen samples from patients seeking assisted reproduction treatment.Our results indicated that SUP and DGC are equally efficient in eliminating spermatozoa containing double-strand DNA damage and sperm with highly damaged (degraded) DNA,as characterized by the presence of both single- and double-strand DNA breaks.However,DGC is more efficient than SUP in selecting spermatozoa that are free from single-strand DNA damage.Future studies should characterise the importance of the various types of DNA damage and examine the sperm processing protocols used in each laboratory to determine their ability to eliminate DNA damage and hence,prevent the potential transmission of genetic mutations via ART.

  14. Opening of DNA double strands by helicases. Active versus passive opening

    CERN Document Server

    Betterton, M D

    2002-01-01

    Helicase opening of double-stranded nucleic acids may be "active" (the helicase directly destabilizes the dsNA to promote opening) or "passive" (the helicase binds ssNA available due to a thermal fluctuation which opens part of the dsNA). We describe helicase opening of dsNA, based on helicases which bind single NA strands and move towards the double-stranded region, using a discrete ``hopping'' model. The interaction between the helicase and the junction where the double strand opens is characterized by an interaction potential. The form of the potential determines whether the opening is active or passive. We calculate the rate of passive opening for the helicase PcrA, and show that the rate increases when the opening is active. Finally, we examine how to choose the interaction potential to optimize the rate of strand separation. One important result is our finding that active opening can increase the unwinding rate by 7 fold compared to passive opening.

  15. Double-stranded endonuclease activity in Bacillus halodurans clustered regularly interspaced short palindromic repeats (CRISPR)-associated Cas2 protein.

    Science.gov (United States)

    Nam, Ki Hyun; Ding, Fran; Haitjema, Charles; Huang, Qingqiu; DeLisa, Matthew P; Ke, Ailong

    2012-10-19

    The CRISPR (clustered regularly interspaced short palindromic repeats) system is a prokaryotic RNA-based adaptive immune system against extrachromosomal genetic elements. Cas2 is a universally conserved core CRISPR-associated protein required for the acquisition of new spacers for CRISPR adaptation. It was previously characterized as an endoribonuclease with preference for single-stranded (ss)RNA. Here, we show using crystallography, mutagenesis, and isothermal titration calorimetry that the Bacillus halodurans Cas2 (Bha_Cas2) from the subtype I-C/Dvulg CRISPR instead possesses metal-dependent endonuclease activity against double-stranded (ds)DNA. This activity is consistent with its putative function in producing new spacers for insertion into the 5'-end of the CRISPR locus. Mutagenesis and isothermal titration calorimetry studies revealed that a single divalent metal ion (Mg(2+) or Mn(2+)), coordinated by a symmetric Asp pair in the Bha_Cas2 dimer, is involved in the catalysis. We envision that a pH-dependent conformational change switches Cas2 into a metal-binding competent conformation for catalysis. We further propose that the distinct substrate preferences among Cas2 proteins may be determined by the sequence and structure in the β1-α1 loop.

  16. Lyn tyrosine kinase promotes silencing of ATM-dependent checkpoint signaling during recovery from DNA double-strand breaks

    Energy Technology Data Exchange (ETDEWEB)

    Fukumoto, Yasunori, E-mail: fukumoto@faculty.chiba-u.jp; Kuki, Kazumasa; Morii, Mariko; Miura, Takahito; Honda, Takuya; Ishibashi, Kenichi; Hasegawa, Hitomi; Kubota, Sho; Ide, Yudai; Yamaguchi, Noritaka; Nakayama, Yuji; Yamaguchi, Naoto, E-mail: nyama@faculty.chiba-u.jp

    2014-09-26

    Highlights: • Inhibition of Src family kinases decreased γ-H2AX signal. • Inhibition of Src family increased ATM-dependent phosphorylation of Chk2 and Kap1. • shRNA-mediated knockdown of Lyn increased phosphorylation of Kap1 by ATM. • Ectopic expression of Src family kinase suppressed ATM-mediated Kap1 phosphorylation. • Src is involved in upstream signaling for inactivation of ATM signaling. - Abstract: DNA damage activates the DNA damage checkpoint and the DNA repair machinery. After initial activation of DNA damage responses, cells recover to their original states through completion of DNA repair and termination of checkpoint signaling. Currently, little is known about the process by which cells recover from the DNA damage checkpoint, a process called checkpoint recovery. Here, we show that Src family kinases promote inactivation of ataxia telangiectasia mutated (ATM)-dependent checkpoint signaling during recovery from DNA double-strand breaks. Inhibition of Src activity increased ATM-dependent phosphorylation of Chk2 and Kap1. Src inhibition increased ATM signaling both in G2 phase and during asynchronous growth. shRNA knockdown of Lyn increased ATM signaling. Src-dependent nuclear tyrosine phosphorylation suppressed ATM-mediated Kap1 phosphorylation. These results suggest that Src family kinases are involved in upstream signaling that leads to inactivation of the ATM-dependent DNA damage checkpoint.

  17. Processing of 3'-Phosphoglycolate-Terminated DNA Double-StrandBreaks by Artemis Nuclease

    Energy Technology Data Exchange (ETDEWEB)

    Povrik, Lawrence F.; Zhou, Tong; Zhou, Ruizhe; Cowan, Morton J.; Yannone, Steven M.

    2005-10-01

    The Artemis nuclease is required for V(D)J recombination and for repair of an as yet undefined subset of radiation-induced DNA double-strand breaks. To assess the possibility that Artemis functions on oxidatively modified double-strand break termini, its activity toward model DNA substrates, bearing either 3{prime}-hydroxyl or 3{prime}-phosphoglycolate moieties, was examined. A 3{prime}-phosphoglycolate had little effect on Artemis-mediated trimming of long 3{prime} overhangs (>9 nucleotides), which were efficiently trimmed to 4-5 nucleotides. However, 3{prime}-phosphoglycolates on overhangs of 4-5 bases promoted selective Artemis-mediated trimming of a single 3{prime}-terminal nucleotide, while at least 2 nucleotides were trimmed from identical hydroxyl-terminated substrates. Artemis also efficiently removed a single nucleotide from a phosphoglycolate-terminated 3-base 3{prime} overhang, while leaving an analogous hydroxyl-terminated overhang largely intact. Such removal was dependent upon Ku, DNA-dependent protein kinase, and ATP. Together, these data suggest that Artemis-mediated cleavage of 3{prime} overhangs requires a minimum of 2 nucleotides, or a nucleotide plus a phosphoglycolate, 3{prime} to the cleavage site. Shorter 3{prime}-phosphoglycolate-terminated overhangs and blunt ends were also processed by Artemis, but much less efficiently. Consistent with the in vitro substrate specificity of Artemis, human cells lacking Artemis exhibited hypersensitivity to X-rays, bleomycin and neocarzinostatin, which all induce 3{prime}-phosphoglycolate-terminated double-strand breaks. Collectively, these results suggest that 3{prime}-phosphoglycolate termini and/or specific classes of DNA ends that arise from such blocked termini are relevant Artemis substrates in vivo.

  18. RecG Directs DNA Synthesis during Double-Strand Break Repair.

    Directory of Open Access Journals (Sweden)

    Benura Azeroglu

    2016-02-01

    Full Text Available Homologous recombination provides a mechanism of DNA double-strand break repair (DSBR that requires an intact, homologous template for DNA synthesis. When DNA synthesis associated with DSBR is convergent, the broken DNA strands are replaced and repair is accurate. However, if divergent DNA synthesis is established, over-replication of flanking DNA may occur with deleterious consequences. The RecG protein of Escherichia coli is a helicase and translocase that can re-model 3-way and 4-way DNA structures such as replication forks and Holliday junctions. However, the primary role of RecG in live cells has remained elusive. Here we show that, in the absence of RecG, attempted DSBR is accompanied by divergent DNA replication at the site of an induced chromosomal DNA double-strand break. Furthermore, DNA double-stand ends are generated in a recG mutant at sites known to block replication forks. These double-strand ends, also trigger DSBR and the divergent DNA replication characteristic of this mutant, which can explain over-replication of the terminus region of the chromosome. The loss of DNA associated with unwinding joint molecules previously observed in the absence of RuvAB and RecG, is suppressed by a helicase deficient PriA mutation (priA300, arguing that the action of RecG ensures that PriA is bound correctly on D-loops to direct DNA replication rather than to unwind joint molecules. This has led us to put forward a revised model of homologous recombination in which the re-modelling of branched intermediates by RecG plays a fundamental role in directing DNA synthesis and thus maintaining genomic stability.

  19. Translocation frequency of double-stranded DNA through a solid-state nanopore

    CERN Document Server

    Bell, Nicholas A W; Keyser, Ulrich F

    2015-01-01

    Solid-state nanopores are single molecule sensors that measure changes in ionic current as charged polymers such as DNA pass through. Here, we present comprehensive experiments on the length, voltage and salt dependence of the frequency of double-stranded DNA translocations through conical quartz nanopores with mean opening diameter 15 nm. We observe an entropic barrier limited, length dependent translocation frequency at 4M LiCl salt concentration and a drift-dominated, length independent translocation frequency at 1M KCl salt concentration. These observations are described by a unifying convection-diffusion equation which includes the contribution of an entropic barrier for polymer entry.

  20. Double strand breaks in DNA resulting from double-electron-emission events

    CERN Document Server

    Surdutovich, Eugene

    2012-01-01

    A mechanism of double strand breaking (DSB) in DNA due to the action of two electrons is considered. These are the electrons produced in the vicinity of DNA molecules due to ionization of water molecules with a consecutive emission of two electrons, making such a mechanism possible. This effect qualitatively solves a puzzle of large yields of DSBs following irradiation of DNA molecules. The transport of secondary electrons, including the additional electrons, is studied in relation to the assessment of radiation damage due to incident ions. This work is a stage in the inclusion of Auger mechanism and like effects into the multiscale approach to ion-beam cancer therapy.

  1. Regulation of DNA double-strand break repair by ubiquitin and ubiquitin-like modifiers

    DEFF Research Database (Denmark)

    Schwertman, Petra; Bekker-Jensen, Simon; Mailand, Niels

    2016-01-01

    DNA double-strand breaks (DSBs) are highly cytotoxic DNA lesions. The swift recognition and faithful repair of such damage is crucial for the maintenance of genomic stability, as well as for cell and organismal fitness. Signalling by ubiquitin, SUMO and other ubiquitin-like modifiers (UBLs......) orchestrates and regulates cellular responses to DSBs at multiple levels, often involving extensive crosstalk between these modifications. Recent findings have revealed compelling insights into the complex mechanisms by which ubiquitin and UBLs regulate protein interactions with DSB sites to promote accurate...

  2. Double strand break (DSB) repair in heterochromatin and heterochromatin proteins in DSB repair.

    Science.gov (United States)

    Lemaître, Charlène; Soutoglou, Evi

    2014-07-01

    Chromosomal translocations are a hallmark of cancer cells and they represent a major cause of tumorigenesis. To avoid chromosomal translocations, faithful repair of DNA double strand breaks (DSBs) has to be ensured in the context of high ordered chromatin structure. However, chromatin compaction is proposed to represent a barrier for DSB repair. Here we review the different mechanisms cells use to alleviate the heterochromatic barrier for DNA repair. At the same time, we discuss the activating role of heterochromatin-associated proteins in this process, therefore proposing that chromatin structure, more than being a simple barrier, is a key modulator of DNA repair.

  3. DNA double strand break repair, chromosome synapsis and transcriptional silencing in meiosis.

    Science.gov (United States)

    Inagaki, Akiko; Schoenmakers, Sam; Baarends, Willy M

    2010-05-16

    Chromosome pairing and synapsis during meiotic prophase requires the formation and repair of DNA double-strand breaks (DSBs) by the topoisomerase-like enzyme SPO11. Chromosomes, or chromosomal regions, that lack a pairing partner, such as the largely heterologous X and Y chromosomes, show delayed meiotic DSB repair and are transcriptionally silenced. Herein, we review meiosis-specific aspects of DSB repair in relation to homology recognition and meiotic silencing of heterologous regions. We propose a dynamic interplay between progression of synapsis and persistent meiotic DSBs. Signaling from these persistent breaks could inhibit heterologous synapsis and stimulate meiotic silencing of the X and Y chromosomes.

  4. Sae2, Exo1 and Sgs1 collaborate in DNA double-strand break processing

    OpenAIRE

    Mimitou, Eleni P.; Symington, Lorraine S.

    2008-01-01

    DNA ends exposed after introduction of double-strand breaks (DSBs) undergo 5′-3′ nucleolytic degradation to generate single-stranded DNA (ssDNA), the substrate for binding by the Rad51 protein to initiate homologous recombination. This process is poorly understood in eukaryotes, but several factors have been implicated, including the Mre11 complex (Mre11-Rad50-Xrs2/NBS1), Sae2/CtIP/Ctp1 and Exo1. Here we demonstrate that yeast Exo1 nuclease and Sgs1 helicase function in alternate pathways for...

  5. DNA Double-Strand Breaks,Potential Targets for HBV Integration

    Institute of Scientific and Technical Information of China (English)

    胡晓文; 林菊生; 谢琼慧; 任精华; 常莹; 吴文杰; 夏羽佳

    2010-01-01

    Hepatitis B virus(HBV)-induced hepatocellular carcinoma(HCC) is one of the most fre-quently occurring cancers.Hepadnaviral DNA integrations are considered to be essential agents which can promote the process of the hepatocarcinogenesis.More and more researches were designed to find the relationship of the two.In this study,we investigated whether HBV DNA integration occurred at sites of DNA double-strand breaks(DSBs),one of the most detrimental DNA damage.An 18-bp I-SceI homing endonuclease recognition site...

  6. Asymmetric Quantum Transport in a Double-Stranded Kronig-Penney Model

    Science.gov (United States)

    Cheon, Taksu; Poghosyan, Sergey S.

    2015-06-01

    We introduce a double-stranded Kronig-Penney model and analyze its transport properties. Asymmetric fluxes between two strands with suddenly alternating localization patterns are found as the energy is varied. The zero-size limit of the internal lines connecting two strands is examined using quantum graph vertices with four edges. We also consider a two-dimensional Kronig-Penney lattice with two types of alternating layer with δ and δ' connections, and show the existence of energy bands in which the quantum flux can flow only in selected directions.

  7. SCAI promotes DNA double-strand break repair in distinct chromosomal contexts

    DEFF Research Database (Denmark)

    Hansen, Rebecca Kring; Mund, Andreas; Poulsen, Sara Lund

    2016-01-01

    DNA double-strand breaks (DSBs) are highly cytotoxic DNA lesions, whose accurate repair by non-homologous end-joining (NHEJ) or homologous recombination (HR) is crucial for genome integrity and is strongly influenced by the local chromatin environment. Here, we identify SCAI (suppressor of cancer...... in repressive chromatin environments. Moreover, we establish an important role of SCAI in meiotic recombination, as SCAI deficiency in mice leads to germ cell loss and subfertility associated with impaired retention of the DMC1 recombinase on meiotic chromosomes. Collectively, our findings uncover SCAI...

  8. Ago2 facilitates Rad51 recruitment and DNA double-strand break repair by homologous recombination

    DEFF Research Database (Denmark)

    Gao, Min; Wei, Wei; Li, Ming Hua

    2014-01-01

    DNA double-strand breaks (DSBs) are highly cytotoxic lesions and pose a major threat to genome stability if not properly repaired. We and others have previously shown that a class of DSB-induced small RNAs (diRNAs) is produced from sequences around DSB sites. DiRNAs are associated with Argonaute...... (Ago) proteins and play an important role in DSB repair, though the mechanism through which they act remains unclear. Here, we report that the role of diRNAs in DSB repair is restricted to repair by homologous recombination (HR) and that it specifically relies on the effector protein Ago2 in mammalian...

  9. New insights on single-stranded versus double-stranded DNA library preparation for ancient DNA

    DEFF Research Database (Denmark)

    Wales, Nathan; Carøe, Christian; Sandoval-Velasco, Marcela

    2015-01-01

    An innovative single-stranded DNA (ssDNA) library preparation method has sparked great interest among ancient DNA (aDNA) researchers, especially after reports of endogenous DNA content increases >20-fold in some samples. To investigate the behavior of this method, we generated ss......DNA and conventional double-stranded DNA (dsDNA) libraries from 23 ancient and historic plant and animal specimens. We found ssDNA library preparation substantially increased endogenous content when dsDNA libraries contained...

  10. The democratization of gene editing: Insights from site-specific cleavage and double-strand break repair.

    Science.gov (United States)

    Jasin, Maria; Haber, James E

    2016-08-01

    DNA double-strand breaks (DSBs) are dangerous lesions that if not properly repaired can lead to genomic change or cell death. Organisms have developed several pathways and have many factors devoted to repairing DSBs, which broadly occurs by homologous recombination, which relies on an identical or homologous sequence to template repair, or nonhomologous end-joining. Much of our understanding of these repair mechanisms has come from the study of induced DNA cleavage by site-specific endonucleases. In addition to their biological role, these cellular pathways can be co-opted for gene editing to study gene function or for gene therapy or other applications. While the first gene editing experiments were done more than 20 years ago, the recent discovery of RNA-guided endonucleases has simplified approaches developed over the years to make gene editing an approach that is available to the entire biomedical research community. Here, we review DSB repair mechanisms and site-specific cleavage systems that have provided insight into these mechanisms and led to the current gene editing revolution.

  11. Computer programming for nucleic acid studies. II. Total chemical shifts calculation of all protons of double-stranded helices.

    Science.gov (United States)

    Giessner-Prettre, C; Ribas Prado, F; Pullman, B; Kan, L; Kast, J R; Ts'o, P O

    1981-01-01

    A FORTRAN computer program called SHIFTS is described. Through SHIFTS, one can calculate the NMR chemical shifts of the proton resonances of single and double-stranded nucleic acids of known sequences and of predetermined conformations. The program can handle RNA and DNA for an arbitrary sequence of a set of 4 out of the 6 base types A,U,G,C,I and T. Data files for the geometrical parameters are available for A-, A'-, B-, D- and S-conformations. The positions of all the atoms are calculated using a modified version of the SEQ program [1]. Then, based on this defined geometry three chemical shift effects exerted by the atoms of the neighboring nucleotides on the protons of each monomeric unit are calculated separately: the ring current shielding effect: the local atomic magnetic susceptibility effect (including both diamagnetic and paramagnetic terms); and the polarization or electric field effect. Results of the program are compared with experimental results for a gamma (ApApGpCpUpU) 2 helical duplex and with calculated results on this same helix based on model building of A'-form and B-form and on graphical procedure for evaluating the ring current effects.

  12. High-resolution profiling of gammaH2AX around DNA double strand breaks in the mammalian genome.

    Science.gov (United States)

    Iacovoni, Jason S; Caron, Pierre; Lassadi, Imen; Nicolas, Estelle; Massip, Laurent; Trouche, Didier; Legube, Gaëlle

    2010-04-21

    Chromatin acts as a key regulator of DNA-related processes such as DNA damage repair. Although ChIP-chip is a powerful technique to provide high-resolution maps of protein-genome interactions, its use to study DNA double strand break (DSB) repair has been hindered by the limitations of the available damage induction methods. We have developed a human cell line that permits induction of multiple DSBs randomly distributed and unambiguously positioned within the genome. Using this system, we have generated the first genome-wide mapping of gammaH2AX around DSBs. We found that all DSBs trigger large gammaH2AX domains, which spread out from the DSB in a bidirectional, discontinuous and not necessarily symmetrical manner. The distribution of gammaH2AX within domains is influenced by gene transcription, as parallel mappings of RNA Polymerase II and strand-specific expression showed that gammaH2AX does not propagate on active genes. In addition, we showed that transcription is accurately maintained within gammaH2AX domains, indicating that mechanisms may exist to protect gene transcription from gammaH2AX spreading and from the chromatin rearrangements induced by DSBs.

  13. TRF2 is required for repair of nontelomeric DNA double-strand breaks by homologous recombination

    Science.gov (United States)

    Mao, Zhiyong; Seluanov, Andrei; Jiang, Ying; Gorbunova, Vera

    2007-01-01

    TRF2 (telomeric repeat binding factor 2) is an essential component of the telomeric cap, where it forms and stabilizes the T-loop junctions. TRF2 forms the T-loops by stimulating strand invasion of the 3′ overhang into duplex DNA. TRF2 also has been shown to localize to nontelomeric DNA double-strand breaks, but its functional role in DNA repair has not been examined. Here, we present evidence that TRF2 is involved in homologous recombination (HR) repair of nontelomeric double-strand breaks. Depletion of TRF2 strongly inhibited HR and delayed the formation of Rad51 foci after γ-irradiation, whereas overexpression of TRF2 stimulated HR. Depletion of TRF2 had no effect on nonhomologous end-joining, and overexpression of TRF2 inhibited nonhomologous end-joining. We propose, based on our results and on the ability of TRF2 to mediate strand invasion, that TRF2 plays an essential role in HR by facilitating the formation of early recombination intermediates. PMID:17670947

  14. Electrochemical molecular beacon biosensor for sequence-specific recognition of double-stranded DNA.

    Science.gov (United States)

    Miao, Xiangmin; Guo, Xiaoting; Xiao, Zhiyou; Ling, Liansheng

    2014-09-15

    Direct recognition of double-stranded DNA (dsDNA) was crucial to disease diagnosis and gene therapy, because DNA in its natural state is double stranded. Here, a novel sensor for the sequence-specific recognition of dsDNA was developed based on the structure change of ferrocene (Fc) redox probe modified molecular beacon (MB). For constructing such a sensor, gold nanoparticles (AuNPs) were initially electrochemical-deposited onto glass carbon electrode (GCE) surface to immobilize thiolated MB in their folded states with Au-S bond. Hybridization of MB with target dsDNA induced the formation of parallel triplex DNA and opened the stem-loop structure of it, which resulted in the redox probe (Fc) away from the electrode and triggered the decrease of current signals. Under optimal conditions, dsDNA detection could be realized in the range from 350 pM to 25 nM, with a detection limit of 275 pM. Moreover, the proposed method has good sequence-specificity for target dsDNA compared with single base pair mismatch and two base pairs mismatches.

  15. Meiotic double-strand breaks uncover and protect against mitotic errors in the C. elegans germline.

    Science.gov (United States)

    Stevens, Deanna; Oegema, Karen; Desai, Arshad

    2013-12-01

    In sexually reproducing multicellular organisms, genetic information is propagated via the germline, the specialized tissue that generates haploid gametes. The C. elegans germline generates gametes in an assembly line-like process-mitotic divisions under the control of the stem cell niche produce nuclei that, upon leaving the niche, enter into meiosis and progress through meiotic prophase [1]. Here, we characterize the effects of perturbing cell division in the mitotic region of the C. elegans germline. We show that mitotic errors result in a spindle checkpoint-dependent cell-cycle delay, but defective nuclei are eventually formed and enter meiosis. These defective nuclei are eliminated by programmed cell death during meiotic prophase. The cell death-based removal of defective nuclei does not require the spindle checkpoint but instead depends on the DNA damage checkpoint. Removal of nuclei resulting from errors in mitosis also requires Spo11, the enzyme that creates double-strand breaks to initiate meiotic recombination. Consistent with this, double-strand breaks are increased in number and persist longer in germlines with mitotic defects. These findings reveal that the process of initiating meiotic recombination inherently selects against nuclei with abnormal chromosomal content generated by mitotic errors, thereby ensuring the genomic integrity of gametes.

  16. How quantum entanglement in DNA synchronizes double-strand breakage by type II restriction endonucleases.

    Science.gov (United States)

    Kurian, P; Dunston, G; Lindesay, J

    2016-02-21

    Macroscopic quantum effects in living systems have been studied widely in pursuit of fundamental explanations for biological energy transport and sensing. While it is known that type II endonucleases, the largest class of restriction enzymes, induce DNA double-strand breaks by attacking phosphodiester bonds, the mechanism by which simultaneous cutting is coordinated between the catalytic centers remains unclear. We propose a quantum mechanical model for collective electronic behavior in the DNA helix, where dipole-dipole oscillations are quantized through boundary conditions imposed by the enzyme. Zero-point modes of coherent oscillations would provide the energy required for double-strand breakage. Such quanta may be preserved in the presence of thermal noise by the enzyme's displacement of water surrounding the DNA recognition sequence. The enzyme thus serves as a decoherence shield. Palindromic mirror symmetry of the enzyme-DNA complex should conserve parity, because symmetric bond-breaking ceases when the symmetry of the complex is violated or when physiological parameters are perturbed from optima. Persistent correlations in DNA across longer spatial separations-a possible signature of quantum entanglement-may be explained by such a mechanism.

  17. Design and implementation of a microfluidic half adder chip based on double-stranded DNA.

    Science.gov (United States)

    Wang, Jing; Huang, Yourui

    2014-06-01

    In recent years, DNA computing has gained significant research interest. The design of a biochip with DNA computing as a carrier has become a key area in the development of a DNA molecular computer. The half adder, as the basic unit of various arithmetic units, has a complex structure that directly affects the overall complexity of a computer's structure. In this study, a half adder on a microfluidic chip is developed by means of bio-reaction. This technology is combined with a biochip and adopts glass and polydimethylsiloxane to fabricate a microscale hybrid chip. Using a DNA strand as an operand, realization of the half adder on a microfluidic chip is achieved by controlling the annealing and denaturation of double-stranded DNA. The computing results are rapidly and accurately obtained by detecting the presence of double-stranded DNA in a solution by agarose gel electrophoresis. The microfluidic half-adder chip accurately realizes half-adder computations and overcomes the shortcomings of traditional integrated circuit half adders, optical half adders, and chemical molecule half adders, such as complex structure, limited component size, and low accuracy.

  18. Assembly and function of DNA double-strand break repair foci in mammalian cells

    DEFF Research Database (Denmark)

    Bekker-Jensen, Simon; Mailand, Niels

    2010-01-01

    DNA double-strand breaks (DSBs) are among the most cytotoxic types of DNA damage, which if left unrepaired can lead to mutations or gross chromosomal aberrations, and promote the onset of diseases associated with genomic instability such as cancer. One of the most discernible hallmarks of the cel......DNA double-strand breaks (DSBs) are among the most cytotoxic types of DNA damage, which if left unrepaired can lead to mutations or gross chromosomal aberrations, and promote the onset of diseases associated with genomic instability such as cancer. One of the most discernible hallmarks...... of the cellular response to DSBs is the accumulation and local concentration of a plethora of DNA damage signaling and repair proteins in the vicinity of the lesion, initiated by ATM-mediated phosphorylation of H2AX (¿-H2AX) and culminating in the generation of distinct nuclear compartments, so-called Ionizing...... Radiation-Induced Foci (IRIF). The assembly of proteins at the DSB-flanking chromatin occurs in a highly ordered and strictly hierarchical fashion. To a large extent, this is achieved by regulation of protein-protein interactions triggered by a variety of post-translational modifications including...

  19. New tools to study DNA double-strand break repair pathway choice.

    Directory of Open Access Journals (Sweden)

    Daniel Gomez-Cabello

    Full Text Available A broken DNA molecule is difficult to repair, highly mutagenic, and extremely cytotoxic. Such breaks can be repaired by homology-independent or homology-directed mechanisms. Little is known about the network that controls the repair pathway choice except that a licensing step for homology-mediated repair exists, called DNA-end resection. The choice between these two repair pathways is a key event for genomic stability maintenance, and an imbalance of the ratio is directly linked with human diseases, including cancer. Here we present novel reporters to study the balance between both repair options in human cells. In these systems, a double-strand break can be alternatively repaired by homology-independent or -dependent mechanisms, leading to the accumulation of distinct fluorescent proteins. These reporters thus allow the balance between both repair pathways to be analyzed in different experimental setups. We validated the reporters by analyzing the effect of protein downregulation of the DNA end resection and non-homologous end-joining pathways. Finally, we analyzed the role of the DNA damage response on double-strand break (DSB repair mechanism selection. Our reporters could be used in the future to understand the roles of specific factors, whole pathways, or drugs in DSB repair pathway choice, or for genome-wide screening. Moreover, our findings can be applied to increase gene-targeting efficiency, making it a beneficial tool for a broad audience in the biological sciences.

  20. Resection is responsible for loss of transcription around a double-strand break in Saccharomyces cerevisiae.

    Science.gov (United States)

    Manfrini, Nicola; Clerici, Michela; Wery, Maxime; Colombo, Chiara Vittoria; Descrimes, Marc; Morillon, Antonin; d'Adda di Fagagna, Fabrizio; Longhese, Maria Pia

    2015-07-31

    Emerging evidence indicate that the mammalian checkpoint kinase ATM induces transcriptional silencing in cis to DNA double-strand breaks (DSBs) through a poorly understood mechanism. Here we show that in Saccharomyces cerevisiae a single DSB causes transcriptional inhibition of proximal genes independently of Tel1/ATM and Mec1/ATR. Since the DSB ends undergo nucleolytic degradation (resection) of their 5'-ending strands, we investigated the contribution of resection in this DSB-induced transcriptional inhibition. We discovered that resection-defective mutants fail to stop transcription around a DSB, and the extent of this failure correlates with the severity of the resection defect. Furthermore, Rad9 and generation of γH2A reduce this DSB-induced transcriptional inhibition by counteracting DSB resection. Therefore, the conversion of the DSB ends from double-stranded to single-stranded DNA, which is necessary to initiate DSB repair by homologous recombination, is responsible for loss of transcription around a DSB in S. cerevisiae.

  1. Sequence-Dependent Fluorescence of Cy3- and Cy5-Labeled Double-Stranded DNA.

    Science.gov (United States)

    Kretschy, Nicole; Sack, Matej; Somoza, Mark M

    2016-03-16

    The fluorescent intensity of Cy3 and Cy5 dyes is strongly dependent on the nucleobase sequence of the labeled oligonucleotides. Sequence-dependent fluorescence may significantly influence the data obtained from many common experimental methods based on fluorescence detection of nucleic acids, such as sequencing, PCR, FRET, and FISH. To quantify sequence dependent fluorescence, we have measured the fluorescence intensity of Cy3 and Cy5 bound to the 5' end of all 1024 possible double-stranded DNA 5mers. The fluorescence intensity was also determined for these dyes bound to the 5' end of fixed-sequence double-stranded DNA with a variable sequence 3' overhang adjacent to the dye. The labeled DNA oligonucleotides were made using light-directed, in situ microarray synthesis. The results indicate that the fluorescence intensity of both dyes is sensitive to all five bases or base pairs, that the sequence dependence is stronger for double- (vs single-) stranded DNA, and that the dyes are sensitive to both the adjacent dsDNA sequence and the 3'-ssDNA overhang. Purine-rich sequences result in higher fluorescence. The results can be used to estimate measurement error in experiments with fluorescent-labeled DNA, as well as to optimize the fluorescent signal by considering the nucleobase environment of the labeling cyanine dye.

  2. Reprint of "Sequence and phylogenetic analyses of novel totivirus-like double-stranded RNAs from field-collected powdery mildew fungi".

    Science.gov (United States)

    Kondo, Hideki; Hisano, Sakae; Chiba, Sotaro; Maruyama, Kazuyuki; Andika, Ida Bagus; Toyoda, Kazuhiro; Fujimori, Fumihiro; Suzuki, Nobuhiro

    2016-07-02

    The identification of mycoviruses contributes greatly to understanding of the diversity and evolutionary aspects of viruses. Powdery mildew fungi are important and widely studied obligate phytopathogenic agents, but there has been no report on mycoviruses infecting these fungi. In this study, we used a deep sequencing approach to analyze the double-stranded RNA (dsRNA) segments isolated from field-collected samples of powdery mildew fungus-infected red clover plants in Japan. Database searches identified the presence of at least ten totivirus (genus Totivirus)-like sequences, termed red clover powdery mildew-associated totiviruses (RPaTVs). The majority of these sequences shared moderate amino acid sequence identity with each other (powdery mildew fungus populations infecting red clover plants in the field.

  3. Double-stranded RNA viral infection of Trichomonas vaginalis isolates and its effect on the metronidazole resistance of parasites%阴道毛滴虫双链RNA病毒感染及其对甲硝唑耐药性的影响

    Institute of Scientific and Technical Information of China (English)

    王丽娟; 王东; 袁玉青; 杜纪英; 薛长责

    2011-01-01

    Objective To investigate Trichomonas vaginalis isolates from Henan Province for the presence of T. vagi-nalis virus(TVV) and analyze the effect of TVV of the metronidazole resistance of T. vaginalis. Methods DNA and RNA were simultaneously extracted after T. vaginalis isolates were grown on TYM (trypticase-yeast extract-maltose) medium and maintained as axenic cultures. Samples were then analyzed on 1 % agarose gel. The MLC of metronidazole of T. vaginalis isolates was tested using serial dilution. Results TVV was detected in 6 of 30 T. vaginalis isolates; the rate of TVV infection in T. vaginalis isolates was 20. 0%. The MLC of metronidazole against TVV-negative T. vaginalis strains in vitro was (24. 27 ±20. 899)μg/ml. This was significantly higher than that against TVV-positive strains (5. 68±3. 588)μg/ml), and the difference between the two was statistically significant(t = 2. 143,P<0. 05). Conclusion T. vaginalis virus was detected in T. vaginalis isolates from Henan. T. vaginalis isolates not harboring TVV were more likely to be resistant to metronidazole.%目的 调查河南地区阴道毛滴虫临床分离株阴道毛滴虫病毒感染情况,探索病毒感染对阴道毛滴虫甲硝唑耐药性的影响.方法 TYM(trypticase-yeast extract-maltose)无菌培养基培养阴道毛滴虫临床分离株,达到纯培养后提取虫体总核酸(DNA和RNA),进行1%琼脂糖凝胶电泳分析;连续稀释法测量每株虫体的甲硝唑最小致死浓度.结果 对30株阴道毛滴虫总核酸进行电泳,其中6株有5.5 kb双链RNA病毒带,病毒感染率为20.0%.阴道毛滴虫病毒阴性组甲硝唑最小致死浓度为( 24.27±20.899)μg/ml,病毒阳性组为(5.68±3.588)μg/ml,差异有统计学意义(t=2.143,P<0.05).结论 河南地区阴道毛滴虫临床分离株中检测到阴道毛滴虫病毒,无阴道毛滴虫病毒寄生的虫体易发生甲硝唑抵抗.

  4. Influence of insect cell apoptosis by expression of Sf-caspase-1 double-stranded RNA in recombinant baculovirus%重组杆状病毒表达Sf-caspase-1双链RNA对昆虫细胞凋亡的影响

    Institute of Scientific and Technical Information of China (English)

    欧艳梅; 许晓东

    2016-01-01

    [目的]构建包含Sf-caspase-1反向重复序列的重组杆状病毒vAcMNPV-dsCasp,并在草地贪夜蛾(Sf9)细胞中表达Sf-caspase-1双链RNA,用以抑制Sf9细胞的凋亡,为未来优化杆状病毒表达系统提供试验依据.[方法]将Sf-caspase-1反向重复序列构建到pBac5上,与AcMNPV Bacmid共转染Sf9细胞产生重组杆状病毒,用RT-PCR和PI活细胞染色方法,分别检测Sf-caspase-1 mRNA含量及Sf9细胞的凋亡情况.[结果]PCR和双酶切检测结果表明,成功构建了重组质粒pBacS-dsCasp,并得到重组杆状病毒vAcMNPV-dsCasp.PI染色结果表明,相比野生型病毒v,AcMNPV,重组杆状病毒vAcMNPV-dsCasp能够抑制Sf9细胞的凋亡.RT-PCR结果表明,vAcMNPV-dsCasp感染的Sf9细胞中Sf-caspase-1 mRNA含量明显下降.[结论]在重组杆状病毒上表达Sf-caspase-1双链RNA确实能够沉默Sf-caspase-1,从而抑制细胞凋亡.

  5. Molecular dynamics simulations of double-stranded DNA in an explicit solvent model with the zero-dipole summation method.

    Directory of Open Access Journals (Sweden)

    Takamasa Arakawa

    Full Text Available Molecular dynamics (MD simulations of a double-stranded DNA with explicit water and small ions were performed with the zero-dipole summation (ZD method, which was recently developed as one of the non-Ewald methods. Double-stranded DNA is highly charged and polar, with phosphate groups in its backbone and their counterions, and thus precise treatment for the long-range electrostatic interactions is always required to maintain the stable and native double-stranded form. A simple truncation method deforms it profoundly. On the contrary, the ZD method, which considers the neutralities of charges and dipoles in a truncated subset, well reproduced the electrostatic energies of the DNA system calculated by the Ewald method. The MD simulations using the ZD method provided a stable DNA system, with similar structures and dynamic properties to those produced by the conventional Particle mesh Ewald method.

  6. Exposure to the viral by-product dsRNA or Coxsackievirus B5 triggers pancreatic beta cell apoptosis via a Bim / Mcl-1 imbalance.

    Directory of Open Access Journals (Sweden)

    Maikel L Colli

    2011-09-01

    Full Text Available The rise in type 1 diabetes (T1D incidence in recent decades is probably related to modifications in environmental factors. Viruses are among the putative environmental triggers of T1D. The mechanisms regulating beta cell responses to viruses, however, remain to be defined. We have presently clarified the signaling pathways leading to beta cell apoptosis following exposure to the viral mimetic double-stranded RNA (dsRNA and a diabetogenic enterovirus (Coxsackievirus B5. Internal dsRNA induces cell death via the intrinsic mitochondrial pathway. In this process, activation of the dsRNA-dependent protein kinase (PKR promotes eIF2α phosphorylation and protein synthesis inhibition, leading to downregulation of the antiapoptotic Bcl-2 protein myeloid cell leukemia sequence 1 (Mcl-1. Mcl-1 decrease results in the release of the BH3-only protein Bim, which activates the mitochondrial pathway of apoptosis. Indeed, Bim knockdown prevented both dsRNA- and Coxsackievirus B5-induced beta cell death, and counteracted the proapoptotic effects of Mcl-1 silencing. These observations indicate that the balance between Mcl-1 and Bim is a key factor regulating beta cell survival during diabetogenic viral infections.

  7. Dissimilar Kinetic Behavior of Electrically Manipulated Single- and Double-Stranded DNA Tethered to a Gold Surface

    Science.gov (United States)

    Rant, Ulrich; Arinaga, Kenji; Tornow, Marc; Kim, Yong Woon; Netz, Roland R.; Fujita, Shozo; Yokoyama, Naoki; Abstreiter, Gerhard

    2006-01-01

    We report on the electrical manipulation of single- and double-stranded oligodeoxynucleotides that are end tethered to gold surfaces in electrolyte solution. The response to alternating repulsive and attractive electric surface fields is studied by time-resolved fluorescence measurements, revealing markedly distinct dynamics for the flexible single-stranded and stiff double-stranded DNA, respectively. Hydrodynamic simulations rationalize this finding and disclose two different kinetic mechanisms: stiff polymers undergo rotation around the anchoring pivot point; flexible polymers, on the other hand, are pulled onto the attracting surface segment by segment. PMID:16473909

  8. Double-strand break repair on sex chromosomes: challenges during male meiotic prophase.

    Science.gov (United States)

    Lu, Lin-Yu; Yu, Xiaochun

    2015-01-01

    During meiotic prophase, DNA double-strand break (DSB) repair-mediated homologous recombination (HR) occurs for exchange of genetic information between homologous chromosomes. Unlike autosomes or female sex chromosomes, human male sex chromosomes X and Y share little homology. Although DSBs are generated throughout male sex chromosomes, homologous recombination does not occur for most regions and DSB repair process is significantly prolonged. As a result, male sex chromosomes are coated with many DNA damage response proteins and form a unique chromatin structure known as the XY body. Interestingly, associated with the prolonged DSB repair, transcription is repressed in the XY body but not in autosomes, a phenomenon known as meiotic sex chromosome inactivation (MSCI), which is critical for male meiosis. Here using mice as model organisms, we briefly summarize recent progress on DSB repair in meiotic prophase and focus on the mechanism and function of DNA damage response in the XY body.

  9. Influence of solitons on the conductance properties of double-stranded deoxyribonucleic acid

    Indian Academy of Sciences (India)

    S A Ketabi; T Ghane; N Shahtahmasebi

    2010-01-01

    A numerical study is presented to investigate the role of solitons in the electronic states of double-stranded DNA (dsDNA) molecule in the metal/DNA/metal system. Based on tight-binding Hamiltonian model and within the framework of a generalized Green’s function technique, we consider a ladder model for poly(dG)-poly(dC) DNA molecule containing M cells with four sites (two base pair sites and two backbone sites) in each cell. In the presence of a sublattice of solitons, our results show that the homogeneous soliton distributions induce the electronic states in the band gap of DNA molecule. In addition, the room temperature current–voltage characteristic of the system shows a linear and ohmic-like behaviour.

  10. A Single Nucleotide Resolution Model for Large-Scale Simulations of Double Stranded DNA

    CERN Document Server

    Fosado, Y A G; Allan, J; Brackley, C; Henrich, O; Marenduzzo, D

    2016-01-01

    The computational modelling of DNA is becoming crucial in light of new advances in DNA nanotechnology, single-molecule experiments and in vivo DNA tampering. Here we present a mesoscopic model for double stranded DNA (dsDNA) at the single nucleotide level which retains the characteristic helical structure, while being able to simulate large molecules -- up to a million base pairs -- for time-scales which are relevant to physiological processes. This is made possible by an efficient and highly-parallelised implementation of the model which we discuss here. We compare the behaviour of our model with single molecule experiments where dsDNA is manipulated by external forces or torques. We also present some results on the kinetics of denaturation of linear DNA.

  11. Understanding the origin of liquid crystal ordering of ultrashort double-stranded DNA

    Science.gov (United States)

    Saurabh, Suman; Lansac, Yves; Jang, Yun Hee; Glaser, Matthew A.; Clark, Noel A.; Maiti, Prabal K.

    2017-03-01

    Recent experiments have shown that short double-stranded DNA (dsDNA) fragments having six- to 20-base pairs exhibit various liquid crystalline phases. This violates the condition of minimum molecular shape anisotropy that analytical theories demand for liquid crystalline ordering. It has been hypothesized that the liquid crystalline ordering is the result of end-to-end stacking of dsDNA to form long supramolecular columns which satisfy the shape anisotropy criterion necessary for ordering. To probe the thermodynamic feasibility of this process, we perform molecular dynamics simulations on ultrashort (four base pair long) dsDNA fragments, quantify the strong end-to-end attraction between them, and demonstrate that the nematic ordering of the self-assembled stacked columns is retained for a large range of temperature and salt concentration.

  12. γ-H2AX as a biomarker for DNA double-strand breaks in ecotoxicology.

    Science.gov (United States)

    Gerić, Marko; Gajski, Goran; Garaj-Vrhovac, Vera

    2014-07-01

    The visualisation of DNA damage response proteins enables the indirect measurement of DNA damage. Soon after the occurrence of a DNA double-strand break (DSB), the formation of γ-H2AX histone variants is to be expected. This review is focused on the potential use of the γ-H2AX foci assay in assessing the genotoxicity of environmental contaminants including cytostatic pharmaceuticals, since standard methods may not be sensitive enough to detect the damaging effect of low environmental concentrations of such drugs. These compounds are constantly released into the environment, potentially representing a threat to water quality, aquatic organisms, and, ultimately, human health. Our review of the literature revealed that this method could be used in the biomonitoring and risk assessment of aquatic systems affected by wastewater from the production, usage, and disposal of cytostatic pharmaceuticals.

  13. Biotinylation of Deoxyguanosine at the Abasic Site in Double-Stranded Oligodeoxynucleotides

    Directory of Open Access Journals (Sweden)

    Chun Wu

    2016-01-01

    Full Text Available Biotinylation of deoxyguanosine at an abasic site in double-stranded oligodeoxynucleotides was studied. The biotinylation of deoxyguanosine is achieved by copper-catalyzed click reaction after the conjugation of the oligodeoxynucleotide with 2-oxohex-5-ynal. The biotinylation enables visualization of the biotinylated oligodeoxynucleotides by chemiluminescence on a nylon membrane. In order to investigate the biotinylated site, the biotinylated oligodeoxynucleotides were amplified by the DNA polymerase chain reaction. Replacement of guanine opposing the abasic site with adenine generated by the activity of the terminal deoxynucleotidyl transferase of DNA polymerase was detected by DNA sequencing analysis and restriction endonuclease digestion. This study suggests that 2-oxohex-5-ynal may be useful for the detection of the unpaired deoxyguanosine endogenously generated at abasic sites in genomic DNA.

  14. Compound Poisson Processes and Clustered Damage of Radiation Induced DNA Double Strand Breaks

    Science.gov (United States)

    Gudowska-Nowak, E.; Ritter, S.; Taucher-Scholz, G.; Kraft, G.

    2000-05-01

    Recent experimental data have demonstrated that DNA damage induced by densely ionizing radiation in mammalian cells is distributed along the DNA molecule in the form of clusters. The principal constituent of DNA damage are double-strand breaks (DSB) which are formed when the breaks occur in both DNA strands and are directly opposite or separated by only a few base pairs. DSBs are believed to be most important lesions produced in chromosomes by radiation; interaction between DSBs can lead to cell killing, mutation or carcinogenesis. The paper discusses a model of clustered DSB formation viewed in terms of compound Poisson process along with the predictive essay of the formalism in application to experimental data.

  15. Repair Pathway Choices and Consequences at the Double-Strand Break.

    Science.gov (United States)

    Ceccaldi, Raphael; Rondinelli, Beatrice; D'Andrea, Alan D

    2016-01-01

    DNA double-strand breaks (DSBs) are cytotoxic lesions that threaten genomic integrity. Failure to repair a DSB has deleterious consequences, including genomic instability and cell death. Indeed, misrepair of DSBs can lead to inappropriate end-joining events, which commonly underlie oncogenic transformation due to chromosomal translocations. Typically, cells employ two main mechanisms to repair DSBs: homologous recombination (HR) and classical nonhomologous end joining (C-NHEJ). In addition, alternative error-prone DSB repair pathways, namely alternative end joining (alt-EJ) and single-strand annealing (SSA), have been recently shown to operate in many different conditions and to contribute to genome rearrangements and oncogenic transformation. Here, we review the mechanisms regulating DSB repair pathway choice, together with the potential interconnections between HR and the annealing-dependent error-prone DSB repair pathways.

  16. [Bacterial infections as seen from the eukaryotic genome: DNA double strand breaks, inflammation and cancer].

    Science.gov (United States)

    Lemercier, Claudie

    2014-01-01

    An increasing number of studies report that infection by pathogenic bacteria alters the host genome, producing highly hazardous DNA double strand breaks for the eukaryotic cell. Even when DNA repair occurs, it often leaves "scars" on chromosomes that might generate genomic instability at the next cell division. Chronic intestinal inflammation promotes the expansion of genotoxic bacteria in the intestinal microbiote which in turn triggers tumor formation and colon carcinomas. Bacteria act at the level of the host DNA repair machinery. They also highjack the host cell cycle to allow themselves time for replication in an appropriate reservoir. However, except in the case of bacteria carrying the CDT nuclease, the molecular mechanisms responsible for DNA lesions are not well understood, even if reactive oxygen species released during infection make good candidates.

  17. Anthracyclines induce double-strand DNA breaks at active gene promoters.

    Science.gov (United States)

    Yang, Fan; Kemp, Christopher J; Henikoff, Steven

    2015-03-01

    Doxorubicin is a widely used chemotherapeutic drug that intercalates between DNA base-pairs and poisons Topoisomerase II, although the mechanistic basis for cell killing remains speculative. Doxorubicin and related anthracycline compounds have been shown to increase nucleosome turnover and/or eviction around promoters, which suggests that the resulting enhanced exposure of DNA might underlie cell killing. Previously, we showed that low doses of anthracyclines increase nucleosome turnover around active gene promoters, which suggests that loss of nucleosomes might contribute to cancer cell killing. Here we apply a genome-wide method to precisely map DNA double-strand breaks (DSBs) in cancer cells. We find that spontaneous DSBs occur preferentially around promoters of active genes, and that both anthracyclines and etoposide, a Topoisomerase II poison, increase DSBs around promoters, although CpG islands are conspicuously protected from DSBs. We propose that torsion-based enhancement of nucleosome turnover by anthracyclines exposes promoter DNA, ultimately causing DSBs around promoters.

  18. SCAI promotes DNA double-strand break repair in distinct chromosomal contexts

    DEFF Research Database (Denmark)

    Hansen, Rebecca Kring; Mund, Andreas; Poulsen, Sara Lund;

    2016-01-01

    DNA double-strand breaks (DSBs) are highly cytotoxic DNA lesions, whose accurate repair by non-homologous end-joining (NHEJ) or homologous recombination (HR) is crucial for genome integrity and is strongly influenced by the local chromatin environment. Here, we identify SCAI (suppressor of cancer...... cell invasion) as a 53BP1-interacting chromatin-associated protein that promotes the functionality of several DSB repair pathways in mammalian cells. SCAI undergoes prominent enrichment at DSB sites through dual mechanisms involving 53BP1-dependent recruitment to DSB-surrounding chromatin and 53BP1......-independent accumulation at resected DSBs. Cells lacking SCAI display reduced DSB repair capacity, hypersensitivity to DSB-inflicting agents and genome instability. We demonstrate that SCAI is a mediator of 53BP1-dependent repair of heterochromatin-associated DSBs, facilitating ATM kinase signalling at DSBs...

  19. Writers, Readers, and Erasers of Histone Ubiquitylation in DNA Double-Strand Break Repair

    DEFF Research Database (Denmark)

    Smeenk, Godelieve; Mailand, Niels

    2016-01-01

    DNA double-strand breaks (DSBs) are highly cytotoxic DNA lesions, whose faulty repair may alter the content and organization of cellular genomes. To counteract this threat, numerous signaling and repair proteins are recruited hierarchically to the chromatin areas surrounding DSBs to facilitate...... accurate lesion repair and restoration of genome integrity. In vertebrate cells, ubiquitin-dependent modifications of histones adjacent to DSBs by RNF8, RNF168, and other ubiquitin ligases have a key role in promoting the assembly of repair protein complexes, serving as direct recruitment platforms...... for a range of genome caretaker proteins and their associated factors. These DNA damage-induced chromatin ubiquitylation marks provide an essential component of a histone code for DSB repair that is controlled by multifaceted regulatory circuits, underscoring its importance for genome stability maintenance...

  20. Have a break: determinants of meiotic DNA double strand break (DSB) formation and processing in plants.

    Science.gov (United States)

    Edlinger, Bernd; Schlögelhofer, Peter

    2011-03-01

    Meiosis is an essential process for sexually reproducing organisms, leading to the formation of specialized generative cells. This review intends to highlight current knowledge of early events during meiosis derived from various model organisms, including plants. It will particularly focus on cis- and trans-requirements of meiotic DNA double strand break (DSB) formation, a hallmark event during meiosis and a prerequisite for recombination of genetic traits. Proteins involved in DSB formation in different organisms, emphasizing the known factors from plants, will be introduced and their functions outlined. Recent technical advances in DSB detection and meiotic recombination analysis will be reviewed, as these new tools now allow analysis of early meiotic recombination in plants with incredible accuracy. To anticipate future directions in plant meiosis research, unpublished results will be included wherever possible.

  1. The ubiquitin- and SUMO-dependent signaling response to DNA double-strand breaks

    DEFF Research Database (Denmark)

    Bekker-Jensen, Simon; Mailand, Niels

    2011-01-01

    DNA double-strand breaks (DSBs) represent the most destructive type of chromosomal lesion and trigger rapid chromatin restructuring accompanied by accumulation of proteins in the vicinity of the DSB. Non-proteolytic ubiquitylation of chromatin surrounding DSBs, mediated by the RNF8/RNF168 ubiquitin...... ligase cascade, has emerged as a key mechanism for restoration of genome integrity by licensing the DSB-modified chromatin to concentrate genome caretaker proteins such as 53BP1 and BRCA1 near the lesions. In parallel, SUMOylation of upstream DSB regulators is also required for execution...... of this ubiquitin-dependent chromatin response, but its molecular basis is currently unclear. Here, we discuss recent insights into how ubiquitin- and SUMO-dependent signaling processes cooperate to orchestrate protein interactions with sites of DNA damage to facilitate DSB repair....

  2. Radiation induced DNA double-strand breaks in radiology; Strahleninduzierte DNA-Doppelstrangbrueche in der Radiologie

    Energy Technology Data Exchange (ETDEWEB)

    Kuefner, M.A. [Dornbirn Hospital (Austria). Dept. of Radiology; Brand, M.; Engert, C.; Uder, M. [Erlangen University Hospital (Germany). Dept. of Radiology; Schwab, S.A. [Radiologis, Oberasbach (Germany)

    2015-10-15

    Shortly after the discovery of X-rays, their damaging effect on biological tissues was observed. The determination of radiation exposure in diagnostic and interventional radiology is usually based on physical measurements or mathematical algorithms with standardized dose simulations. γ-H2AX immunofluorescence microscopy is a reliable and sensitive method for the quantification of radiation induced DNA double-strand breaks (DSB) in blood lymphocytes. The detectable amount of these DNA damages correlates well with the dose received. However, the biological radiation damage depends not only on dose but also on other individual factors like radiation sensitivity and DNA repair capacity. Iodinated contrast agents can enhance the x-ray induced DNA damage level. After their induction DSB are quickly repaired. A protective effect of antioxidants has been postulated in experimental studies. This review explains the principle of the γ-H2AX technique and provides an overview on studies evaluating DSB in radiologic examinations.

  3. Stiffer double-stranded DNA in two-dimensional confinement due to bending anisotropy

    Science.gov (United States)

    Salari, H.; Eslami-Mossallam, B.; Ranjbar, H. F.; Ejtehadi, M. R.

    2016-12-01

    Using analytical approach and Monte Carlo (MC) simulations, we study the elastic behavior of the intrinsically twisted elastic ribbons with bending anisotropy, such as double-stranded DNA (dsDNA), in two-dimensional (2D) confinement. We show that, due to the bending anisotropy, the persistence length of dsDNA in 2D conformations is always greater than three-dimensional (3D) conformations. This result is in consistence with the measured values for DNA persistence length in 2D and 3D in equal biological conditions. We also show that in two dimensions, an anisotropic, intrinsically twisted polymer exhibits an implicit twist-bend coupling, which leads to the transient curvature increasing with a half helical turn periodicity along the bent polymer.

  4. Constitutional Chromothripsis Rearrangements Involve Clustered Double-Stranded DNA Breaks and Nonhomologous Repair Mechanisms

    Directory of Open Access Journals (Sweden)

    Wigard P. Kloosterman

    2012-06-01

    Full Text Available Chromothripsis represents a novel phenomenon in the structural variation landscape of cancer genomes. Here, we analyze the genomes of ten patients with congenital disease who were preselected to carry complex chromosomal rearrangements with more than two breakpoints. The rearrangements displayed unanticipated complexity resembling chromothripsis. We find that eight of them contain hallmarks of multiple clustered double-stranded DNA breaks (DSBs on one or more chromosomes. In addition, nucleotide resolution analysis of 98 breakpoint junctions indicates that break repair involves nonhomologous or microhomology-mediated end joining. We observed that these eight rearrangements are balanced or contain sporadic deletions ranging in size between a few hundred base pairs and several megabases. The two remaining complex rearrangements did not display signs of DSBs and contain duplications, indicative of rearrangement processes involving template switching. Our work provides detailed insight into the characteristics of chromothripsis and supports a role for clustered DSBs driving some constitutional chromothripsis rearrangements.

  5. Carbon ion induced DNA double-strand breaks in melanophore B{sub 16}

    Energy Technology Data Exchange (ETDEWEB)

    Wei Zengquan; Zhou Guangming; Wang Jufang; He Jing; Li Qiang; Li Wenjian; Xie Hongmei; Cai Xichen; Tao Huang; Dang Bingrong; Han Guangwu [Chinese Academy of Sciences, Lanzhou (China). Inst. of Modern Physics; Gao Qingxiang [Lanzhou Univ. (China)

    1997-09-01

    DNA double-strand breaks (DSBs) in melanophore B{sub 16} induced by plateau and extended Bragg peak of 75 MeV/u {sup 12}C{sup 6+} ions were studied by using a technique of inverse pulsed-field gel electrophoresis (PIGE). DNA fragment lengths were distributed in two ranges: the larger in 1.4 Mbp-3.2 Mbp and the smaller in less than 1.2 Mbp. It indicates that distribution of DNA fragments induced by heavy ion irradiation is not stochastic and there probably are sensitive sites to heavy ions in DNA molecules of B{sub 16}. Percentage of DNA released from plug (PR) increased and trended towards a quasi-plateau {proportional_to}85% as dose increased. Content of the larger fragments decreased and flattened with increasing dose while content of the smaller ones increased and trended towards saturation. (orig.)

  6. Transcriptionally active chromatin recruits homologous recombination at DNA double-strand breaks.

    Science.gov (United States)

    Aymard, François; Bugler, Beatrix; Schmidt, Christine K; Guillou, Emmanuelle; Caron, Pierre; Briois, Sébastien; Iacovoni, Jason S; Daburon, Virginie; Miller, Kyle M; Jackson, Stephen P; Legube, Gaëlle

    2014-04-01

    Although both homologous recombination (HR) and nonhomologous end joining can repair DNA double-strand breaks (DSBs), the mechanisms by which one of these pathways is chosen over the other remain unclear. Here we show that transcriptionally active chromatin is preferentially repaired by HR. Using chromatin immunoprecipitation-sequencing (ChIP-seq) to analyze repair of multiple DSBs induced throughout the human genome, we identify an HR-prone subset of DSBs that recruit the HR protein RAD51, undergo resection and rely on RAD51 for efficient repair. These DSBs are located in actively transcribed genes and are targeted to HR repair via the transcription elongation-associated mark trimethylated histone H3 K36. Concordantly, depletion of SETD2, the main H3 K36 trimethyltransferase, severely impedes HR at such DSBs. Our study thereby demonstrates a primary role in DSB repair of the chromatin context in which a break occurs.

  7. DNA double-strand breaks and ATM activation by transcription-blocking DNA lesions.

    Science.gov (United States)

    Sordet, Olivier; Nakamura, Asako J; Redon, Christophe E; Pommier, Yves

    2010-01-15

    A taxia telangiectasia mutated (ATM), the deficiency of which causes a severe neurodegenerative disease, is a crucial mediator for the DNA double-strand break (DSB) response. We recently showed that transcription-blocking topoisomerase I cleavage complexes (TOP1cc) produce DSBs related to R-loop formation and activate ATM in post-mitotic neurons and lymphocytes. Here we discuss how TOP1cc can produce transcription arrest with R-loop formation and generate DSBs that activate ATM, as well as data suggesting that those transcription-dependent DSBs tend to form at the IgH locus and at specific genomic sites. We also address the potential roles of ATM in response to transcription-blocking TOP1cc.

  8. DNA double-strand break repair: a tale of pathway choices

    Institute of Scientific and Technical Information of China (English)

    Jing Li; Xingzhi Xu

    2016-01-01

    Deoxyribonucleic acid double-strand breaks (DSBs) are cytotoxic lesions that must be repaired either through homologous recombination (HR) or non-homologous end-joining (NHEJ) pathways.DSB repair is critical for genome integrity,cellular homeostasis and also constitutes the biological foundation for radiotherapy and the majority of chemotherapy.The choice between HR and NHEJ is a complex yet not completely understood process that will entail more future efforts.Herein we review our current understandings about how the choice is made over an antagonizing balance between p53-binding protein 1 and breast cancer 1 in the context of cell cycle stages,downstream effects,and distinct chromosomal histone marks.These exciting areas of research will surely bring more mechanistic insights about DSB repair and be utilized in the clinical settings.

  9. Targeted molecular trait stacking in cotton through targeted double-strand break induction.

    Science.gov (United States)

    D'Halluin, Kathleen; Vanderstraeten, Chantal; Van Hulle, Jolien; Rosolowska, Joanna; Van Den Brande, Ilse; Pennewaert, Anouk; D'Hont, Kristel; Bossut, Martine; Jantz, Derek; Ruiter, Rene; Broadhvest, Jean

    2013-10-01

    Recent developments of tools for targeted genome modification have led to new concepts in how multiple traits can be combined. Targeted genome modification is based on the use of nucleases with tailor-made specificities to introduce a DNA double-strand break (DSB) at specific target loci. A re-engineered meganuclease was designed for specific cleavage of an endogenous target sequence adjacent to a transgenic insect control locus in cotton. The combination of targeted DNA cleavage and homologous recombination-mediated repair made precise targeted insertion of additional trait genes (hppd, epsps) feasible in cotton. Targeted insertion events were recovered at a frequency of about 2% of the independently transformed embryogenic callus lines. We further demonstrated that all trait genes were inherited as a single genetic unit, which will simplify future multiple-trait introgression.

  10. The involvement of human RECQL4 in DNA double-strand break repair

    DEFF Research Database (Denmark)

    Singh, Dharmendra Kumar; Karmakar, Parimal; Aamann, Maria Diget

    2010-01-01

    Rothmund-Thomson syndrome (RTS) is an autosomal recessive hereditary disorder associated with mutation in RECQL4 gene, a member of the human RecQ helicases. The disease is characterized by genomic instability, skeletal abnormalities and predisposition to malignant tumors, especially osteosarcomas....... The precise role of RECQL4 in cellular pathways is largely unknown; however, recent evidence suggests its involvement in multiple DNA metabolic pathways. This study investigates the roles of RECQL4 in DNA double-strand break (DSB) repair. The results show that RECQL4-deficient fibroblasts are moderately...... sensitive to gamma-irradiation and accumulate more gammaH2AX and 53BP1 foci than control fibroblasts. This is suggestive of defects in efficient repair of DSB's in the RECQL4-deficient fibroblasts. Real time imaging of live cells using laser confocal microscopy shows that RECQL4 is recruited early to laser...

  11. Electronic transport in double-strand poly(dG)-poly(dC) DNA segments

    Energy Technology Data Exchange (ETDEWEB)

    Sarmento, R.G. [Departamento de Fisica, Universidade Federal do Rio Grande do Norte, 59072-970 Natal, RN (Brazil); Albuquerque, E.L. [Departamento de Fisica, Universidade Federal do Rio Grande do Norte, 59072-970 Natal, RN (Brazil)], E-mail: eudenilson@dfte.ufrn.br; Sesion, P.D. [Departamento de Fisica, Universidade Federal do Rio Grande do Norte, 59072-970 Natal, RN (Brazil); Fulco, U.L. [Departamento de Biofisica e Farmacologia, Universidade Federal do Rio Grande do Norte, 59072-970 Natal, RN (Brazil); Oliveira, B.P.W. de [Department of Physics and Astronomy, University of Southern California, Los Angeles, CA 90089 (United States)

    2009-04-06

    We study the electronic properties of a double-strand quasiperiodic DNA molecule modeled by a one-dimensional effective Hamiltonian, which includes contributions from the nucleobasis system as well as the sugar-phosphate backbone. Our theoretical approach makes use of Dyson's equation together with a transfer-matrix treatment, considering an electronic tight-binding Hamiltonian model to investigate the electronic density of states (DOS) and the electronic transmissivity of sequences of DNA finite segments. To mimic the DNA segments, we consider the finite quasiperiodic sequences of Fibonacci's type, in a poly(dG)-poly(dC) configuration, whose building blocks are the bases guanine G and cytosine C. We compared the electronic transport found for the quasiperiodic structure to those using a sequence of natural DNA, as part of the human chromosome Ch22.

  12. Electronic transport in double-strand poly(dG)-poly(dC) DNA segments

    Science.gov (United States)

    Sarmento, R. G.; Albuquerque, E. L.; Sesion, P. D.; Fulco, U. L.; de Oliveira, B. P. W.

    2009-04-01

    We study the electronic properties of a double-strand quasiperiodic DNA molecule modeled by a one-dimensional effective Hamiltonian, which includes contributions from the nucleobasis system as well as the sugar-phosphate backbone. Our theoretical approach makes use of Dyson's equation together with a transfer-matrix treatment, considering an electronic tight-binding Hamiltonian model to investigate the electronic density of states (DOS) and the electronic transmissivity of sequences of DNA finite segments. To mimic the DNA segments, we consider the finite quasiperiodic sequences of Fibonacci's type, in a poly(dG)-poly(dC) configuration, whose building blocks are the bases guanine G and cytosine C. We compared the electronic transport found for the quasiperiodic structure to those using a sequence of natural DNA, as part of the human chromosome Ch22.

  13. Studies on the interaction of the food colorant tartrazine with double stranded deoxyribonucleic acid.

    Science.gov (United States)

    Basu, Anirban; Suresh Kumar, Gopinatha

    2016-05-01

    Interaction of the food additive tartrazine with double-stranded DNA was studied by spectroscopic and calorimetric techniques. Absorbance studies revealed that tartrazine exhibited hypochromism in the presence of DNA without any bathochromic effects. Minor groove displacement assay of DAPI and Hoechst 33258 suggested that tartrazine binds in the minor groove of DNA. The complexation was predominantly entropy driven with a smaller but favorable enthalpic contribution to the standard molar Gibbs energy. The equilibrium constant was evaluated to be (3.68 ± .08) × 10(4) M(-1) at 298.15 K. The negative standard molar heat capacity value along with an enthalpy-entropy compensation phenomenon proposed the involvement of dominant hydrophobic forces in the binding process. Tartrazine enhanced the thermal stability of DNA by 7.53 K under saturation conditions.

  14. Non-canonical uracil processing in DNA gives rise to double-strand breaks and deletions

    DEFF Research Database (Denmark)

    Bregenhorn, Stephanie; Kallenberger, Lia; Artola-Borán, Mariela

    2016-01-01

    During class switch recombination (CSR), antigen-stimulated B-cells rearrange their immunoglobulin constant heavy chain (CH) loci to generate antibodies with different effector functions. CSR is initiated by activation-induced deaminase (AID), which converts cytosines in switch (S) regions......, repetitive sequences flanking the CHloci, to uracils. Although U/G mispairs arising in this way are generally efficiently repaired to C/Gs by uracil DNA glycosylase (UNG)-initiated base excision repair (BER), uracil processing in S-regions of activated B-cells occasionally gives rise to double strand breaks...... (DSBs), which trigger CSR. Surprisingly, genetic experiments revealed that CSR is dependent not only on AID and UNG, but also on mismatch repair (MMR). To elucidate the role of MMR in CSR, we studied the processing of uracil-containing DNA substrates in extracts of MMR-proficient and -deficient human...

  15. RNF4 is required for DNA double-strand break repair in vivo

    DEFF Research Database (Denmark)

    Vyas, R; Kumar, R; Clermont, F

    2013-01-01

    for both homologous recombination (HR) and non-homologous end joining repair. To establish a link between Rnf4 and the DNA damage response (DDR) in vivo, we generated an Rnf4 allelic series in mice. We show that Rnf4-deficiency causes persistent ionizing radiation-induced DNA damage and signaling......Unrepaired DNA double-strand breaks (DSBs) cause genetic instability that leads to malignant transformation or cell death. Cells respond to DSBs with the ordered recruitment of signaling and repair proteins to the sites of DNA lesions. Coordinated protein SUMOylation and ubiquitylation have crucial......, and that Rnf4-deficient cells and mice exhibit increased sensitivity to genotoxic stress. Mechanistically, we show that Rnf4 targets SUMOylated MDC1 and SUMOylated BRCA1, and is required for the loading of Rad51, an enzyme required for HR repair, onto sites of DNA damage. Similarly to inactivating mutations...

  16. The role of DNA double-strand breaks in spontaneous homologous recombination in S. cerevisiae

    DEFF Research Database (Denmark)

    Lettier, Gaëlle; Feng, Q.; Mayolo, A.A. de

    2006-01-01

    Homologous recombination (HR) is a source of genomic instability and the loss of heterozygosity in mitotic cells. Since these events pose a severe health risk, it is important to understand the molecular events that cause spontaneous HR. In eukaryotes, high levels of HR are a normal feature...... of meiosis and result from the induction of a large number of DNA double-strand breaks (DSBs). By analogy, it is generally believed that the rare spontaneous mitotic HR events are due to repair of DNA DSBs that accidentally occur during mitotic growth. Here we provide the first direct evidence that most...... spontaneous mitotic HR in Saccharomyces cerevisiae is initiated by DNA lesions other than DSBs. Specifically, we describe a class of rad52 mutants that are fully proficient in inter- and intra-chromosomal mitotic HR, yet at the same time fail to repair DNA DSBs. The conclusions are drawn from genetic analyses...

  17. New insights on single-stranded versus double-stranded DNA library preparation for ancient DNA.

    Science.gov (United States)

    Wales, Nathan; Carøe, Christian; Sandoval-Velasco, Marcela; Gamba, Cristina; Barnett, Ross; Samaniego, José Alfredo; Madrigal, Jazmín Ramos; Orlando, Ludovic; Gilbert, M Thomas P

    2015-12-01

    An innovative single-stranded DNA (ssDNA) library preparation method has sparked great interest among ancient DNA (aDNA) researchers, especially after reports of endogenous DNA content increases >20-fold in some samples. To investigate the behavior of this method, we generated ssDNA and conventional double-stranded DNA (dsDNA) libraries from 23 ancient and historic plant and animal specimens. We found ssDNA library preparation substantially increased endogenous content when dsDNA libraries contained DNA, but this enrichment is less pronounced when dsDNA preparations successfully recover short endogenous DNA fragments (mean size < 70 bp). Our findings can help researchers determine when to utilize the time- and resource-intensive ssDNA library preparation method.

  18. Recognition, signaling, and repair of DNA double-strand breaks produced by ionizing radiation in mammalian cells: the molecular choreography.

    Science.gov (United States)

    Thompson, Larry H

    2012-01-01

    The faithful maintenance of chromosome continuity in human cells during DNA replication and repair is critical for preventing the conversion of normal diploid cells to an oncogenic state. The evolution of higher eukaryotic cells endowed them with a large genetic investment in the molecular machinery that ensures chromosome stability. In mammalian and other vertebrate cells, the elimination of double-strand breaks with minimal nucleotide sequence change involves the spatiotemporal orchestration of a seemingly endless number of proteins ranging in their action from the nucleotide level to nucleosome organization and chromosome architecture. DNA DSBs trigger a myriad of post-translational modifications that alter catalytic activities and the specificity of protein interactions: phosphorylation, acetylation, methylation, ubiquitylation, and SUMOylation, followed by the reversal of these changes as repair is completed. "Superfluous" protein recruitment to damage sites, functional redundancy, and alternative pathways ensure that DSB repair is extremely efficient, both quantitatively and qualitatively. This review strives to integrate the information about the molecular mechanisms of DSB repair that has emerged over the last two decades with a focus on DSBs produced by the prototype agent ionizing radiation (IR). The exponential growth of molecular studies, heavily driven by RNA knockdown technology, now reveals an outline of how many key protein players in genome stability and cancer biology perform their interwoven tasks, e.g. ATM, ATR, DNA-PK, Chk1, Chk2, PARP1/2/3, 53BP1, BRCA1, BRCA2, BLM, RAD51, and the MRE11-RAD50-NBS1 complex. Thus, the nature of the intricate coordination of repair processes with cell cycle progression is becoming apparent. This review also links molecular abnormalities to cellular pathology as much a possible and provides a framework of temporal relationships.

  19. Depletion of the bloom syndrome helicase stimulates homology-dependent repair at double-strand breaks in human chromosomes.

    Science.gov (United States)

    Wang, Yibin; Smith, Krissy; Waldman, Barbara Criscuolo; Waldman, Alan S

    2011-04-03

    Mutation of BLM helicase causes Blooms syndrome, a disorder associated with genome instability, high levels of sister chromatid exchanges, and cancer predisposition. To study the influence of BLM on double-strand break (DSB) repair in human chromosomes, we stably transfected a normal human cell line with a DNA substrate that contained a thymidine kinase (tk)-neo fusion gene disrupted by the recognition site for endonuclease I-SceI. The substrate also contained a closely linked functional tk gene to serve as a recombination partner for the tk-neo fusion gene. We derived two cell lines each containing a single integrated copy of the DNA substrate. In these cell lines, a DSB was introduced within the tk-neo fusion gene by expression of I-SceI. DSB repair events that occurred via homologous recombination (HR) or nonhomologous end-joining (NHEJ) were recovered by selection for G418-resistant clones. DSB repair was examined under conditions of either normal BLM expression or reduced BLM expression brought about by RNA interference. We report that BLM knockdown in both cell lines specifically increased the frequency of HR events that produced deletions by crossovers or single-strand annealing while leaving the frequency of gene conversions unchanged or reduced. We observed no change in the accuracy of individual HR events and no substantial alteration of the nature of individual NHEJ events when BLM expression was reduced. Our work provides the first direct evidence that BLM influences DSB repair pathway choice in human chromosomes and suggests that BLM deficiency can engender genomic instability by provoking an increased frequency of HR events of a potentially deleterious nature.

  20. A robust network of double-strand break repair pathways governs genome integrity during C. elegans development.

    NARCIS (Netherlands)

    Pontier, D.B.; Tijsterman, M.

    2009-01-01

    To preserve genomic integrity, various mechanisms have evolved to repair DNA double-strand breaks (DSBs). Depending on cell type or cell cycle phase, DSBs can be repaired error-free, by homologous recombination, or with concomitant loss of sequence information, via nonhomologous end-joining (NHEJ) o

  1. Mre11 ATLD17/18 mutation retains Tel1/ATM activity but blocks DNA double-strand break repair

    NARCIS (Netherlands)

    O. Limbo (Oliver); D. Moiani (Davide); A. Kertokalio (Aryandi); C. Wyman (Claire); J.A. Tainer (John); P. Russell (Paul)

    2012-01-01

    textabstractThe Mre11 complex (Mre11-Rad50-Nbs1 or MRN) binds double-strand breaks where it interacts with CtIP/Ctp1/Sae2 and ATM/Tel1 to preserve genome stability through its functions in homology-directed repair, checkpoint signaling and telomere maintenance. Here, we combine biochemical, structur

  2. The human RAD54 recombinational DNA repair protein is a double-stranded DNA-dependent ATPase

    NARCIS (Netherlands)

    J. Essers (Jeroen); J. de Wit (Jan); R. Kanaar (Roland); J.H.J. Hoeijmakers (Jan); S.M.A. Swagemakers (Sigrid)

    1998-01-01

    textabstractDNA double-strand break repair through the RAD52 homologous recombination pathway in the yeast Saccharomyces cerevisiae requires, among others, the RAD51, RAD52, and RAD54 genes. The biological importance of homologous recombination is underscored by the conservation of

  3. The structure-specific endonuclease Mus81-Eme1 promotes conversion of interstrand DNA crosslinks into double-strands breaks

    NARCIS (Netherlands)

    K. Hanada (Katsuhiro); M. Budzowska (Magdalena); M. Modesti (Mauro); A. Maas (Alex); C. Wyman (Claire); J. Essers (Jeroen); R. Kanaar (Roland)

    2006-01-01

    textabstractRepair of interstrand crosslinks (ICLs) requires multiple-strand incisions to separate the two covalently attached strands of DNA. It is unclear how these incisions are generated. DNA double-strand breaks (DSBs) have been identified as intermediates in ICL repair, but enzymes responsible

  4. Poly(ADP-ribose polymerase (PARP-1 is not involved in DNA double-strand break recovery

    Directory of Open Access Journals (Sweden)

    Fernet Marie

    2003-07-01

    Full Text Available Abstract Background The cytotoxicity and the rejoining of DNA double-strand breaks induced by γ-rays, H2O2 and neocarzinostatin, were investigated in normal and PARP-1 knockout mouse 3T3 fibroblasts to determine the role of poly(ADP-ribose polymerase (PARP-1 in DNA double-strand break repair. Results PARP-1-/- were considerably more sensitive than PARP-1+/+ 3T3s to induced cell kill by γ-rays and H2O2. However, the two cell lines did not show any significant difference in the susceptibility to neocarzinostatin below 1.5 nM drug. Restoration of PARP-1 expression in PARP-1-/- 3T3s by retroviral transfection of the full PARP-1 cDNA did not induce any change in neocarzinostatin response. Moreover the incidence and the rejoining kinetics of neocarzinostatin-induced DNA double-strand breaks were identical in PARP-1+/+ and PARP-1-/- 3T3s. Poly(ADP-ribose synthesis following γ-rays and H2O2 was observed in PARP-1-proficient cells only. In contrast neocarzinostatin, even at supra-lethal concentration, was unable to initiate PARP-1 activation yet it induced H2AX histone phosphorylation in both PARP1+/+ and PARP-1-/- 3T3s as efficiently as γ-rays and H2O2. Conclusions The results show that PARP-1 is not a major determinant of DNA double-strand break recovery with either strand break rejoining or cell survival as an endpoint. Even though both PARP-1 and ATM activation are major determinants of the cell response to γ-rays and H2O2, data suggest that PARP-1-dependent poly(ADP-ribose synthesis and ATM-dependent H2AX phosphorylation, are not inter-related in the repair pathway of neocarzinostatin-induced DNA double-strand breaks.

  5. Zinc Finger Nuclease induced DNA double stranded breaks and rearrangements in MLL

    Energy Technology Data Exchange (ETDEWEB)

    Do, To Uyen [Graduate Group in Immunology, University of California Davis, Davis, CA 95616 (United States); Department of Radiation Oncology, University of California Davis, Sacramento CA 95817 (United States); Ho, Bay; Shih, Shyh-Jen [Department of Radiation Oncology, University of California Davis, Sacramento CA 95817 (United States); Vaughan, Andrew, E-mail: Andrew.vaughan@ucdmc.ucdavis.edu [Graduate Group in Immunology, University of California Davis, Davis, CA 95616 (United States); Department of Radiation Oncology, University of California Davis, Sacramento CA 95817 (United States)

    2012-12-15

    Highlights: ► A Zinc Finger Nuclease (ZFN) targeting a leukemogenic hot spot for rearrangement in MLL is created. ► The novel ZFN efficiently cleaves MLL exon 13. ► Despite MLL cleavage and evidence of mis-repair, no leukemogenic translocations were produced. ► MLL cleavage alone is insufficient to generate leukemogenic translocations. - Abstract: Radiation treatment or chemotherapy has been linked with a higher risk of secondary cancers such as therapy related Acute Myeloid Leukemia (tAML). Several of these cancers have been shown to be correlated to the introduction of double stranded breaks (DSB) and rearrangements within the Mixed Lineage Leukemia (MLL) gene. We used Zinc Finger Nucleases (ZFNs) to introduce precise cuts within MLL to examine how a single DNA DSB might lead to chromosomal rearrangements. A ZFN targeting exon 13 within the Breakpoint Cluster Region of MLL was transiently expressed in a human lymphoblast cell line originating from a CML patient. Although FISH analysis showed ZFN DSB at this region increased the rate of MLL fragmentation, we were unable to detect leukemogenic rearrangements or translocations via inverse PCR. Interestingly, gene fragmentation as well as small interstitial deletions, insertions and base substitutions increased with the inhibition of DNA-PK, suggesting repair of this particular DSB is linked to non-homologous end joining (NHEJ). Although mis-repair of DSBs may be necessary for the initiation of leukemogenic translocations, a MLL targeted DNA break alone is insufficient.

  6. [Double-strand DNA breaks induction and repair in human blood lymphocytes irradiated with adapting dose].

    Science.gov (United States)

    Osipov, A N; Lizunova, E Iu; Vorob'eva, N Iu; Pelevina, I I

    2009-01-01

    Using a DNA-comet assay was shown that irradiation of human blood lymphocytes at G1 cell cycle with a low conditioning dose (5 cGy) induces an adaptive response (AR) manifested in reduction of the double-strand DNA (DSB) amount induced by challenging dose at 10 Gy. 24 h after conditioning irradiation (48 h after PHA addition) in cells irradiated at both conditioning and challenging doses a relative DBS amount was approximately 24% less in comparison to versus a control irradiated at challenging dose only. 48 h after adapting irradiation this index increased to approximately 35%, while 72 h after was decreased to approximately 29%. AR observed by us during 72 h after its induction did not accompanied by statistically significant changes in DBS repair enhancing. It is possible to assume that basic role in AR forming in lymphocytes under experimental conditions used by us playing the processes preventing radiation-induced DBS formation (antioxidant defense system activation, chromatin conformation changes ets).

  7. The role of DNA double-strand breaks in spontaneous homologous recombination in S. cerevisiae.

    Directory of Open Access Journals (Sweden)

    Gaëlle Lettier

    2006-11-01

    Full Text Available Homologous recombination (HR is a source of genomic instability and the loss of heterozygosity in mitotic cells. Since these events pose a severe health risk, it is important to understand the molecular events that cause spontaneous HR. In eukaryotes, high levels of HR are a normal feature of meiosis and result from the induction of a large number of DNA double-strand breaks (DSBs. By analogy, it is generally believed that the rare spontaneous mitotic HR events are due to repair of DNA DSBs that accidentally occur during mitotic growth. Here we provide the first direct evidence that most spontaneous mitotic HR in Saccharomyces cerevisiae is initiated by DNA lesions other than DSBs. Specifically, we describe a class of rad52 mutants that are fully proficient in inter- and intra-chromosomal mitotic HR, yet at the same time fail to repair DNA DSBs. The conclusions are drawn from genetic analyses, evaluation of the consequences of DSB repair failure at the DNA level, and examination of the cellular re-localization of Rad51 and mutant Rad52 proteins after introduction of specific DSBs. In further support of our conclusions, we show that, as in wild-type strains, UV-irradiation induces HR in these rad52 mutants, supporting the view that DNA nicks and single-stranded gaps, rather than DSBs, are major sources of spontaneous HR in mitotic yeast cells.

  8. Analysis of DNA Double-strand Break (DSB) Repair in Mammalian Cells

    Science.gov (United States)

    Seluanov, Andrei; Mao, Zhiyong; Gorbunova, Vera

    2010-01-01

    DNA double-strand breaks are the most dangerous DNA lesions that may lead to massive loss of genetic information and cell death. Cells repair DSBs using two major pathways: nonhomologous end joining (NHEJ) and homologous recombination (HR). Perturbations of NHEJ and HR are often associated with premature aging and tumorigenesis, hence it is important to have a quantitative way of measuring each DSB repair pathway. Our laboratory has developed fluorescent reporter constructs that allow sensitive and quantitative measurement of NHEJ and HR. The constructs are based on an engineered GFP gene containing recognition sites for a rare-cutting I-SceI endonuclease for induction of DSBs. The starting constructs are GFP negative as the GFP gene is inactivated by an additional exon, or by mutations. Successful repair of the I-SceI-induced breaks by NHEJ or HR restores the functional GFP gene. The number of GFP positive cells counted by flow cytometry provides quantitative measure of NHEJ or HR efficiency. PMID:20864925

  9. Relative biological effectiveness for photons: implication of complex DNA double-strand breaks as critical lesions

    Science.gov (United States)

    Liang, Ying; Fu, Qibin; Wang, Xudong; Liu, Feng; Yang, Gen; Luo, Chunxiong; Ouyang, Qi; Wang, Yugang

    2017-03-01

    Current knowledge in radiobiology ascribes the adverse biological effects of ionizing radiation primarily to the induction of DNA double-strand breaks (DSBs), which is supposed to be potentially lethal and may be converted to lethal damage due to misrepair. Soft and ultrasoft x-rays have been found to bear elevated biological effectiveness for cell killing compared with conventional x-rays or 60Co γ-rays. This phenomenon is qualitatively interpreted as the increased level of DSB induction for low energy photons, however, a thorough quantitative reasoning is lacking. Here, we systematically compared the relative biological effectiveness (RBE) with relative DSB induction for photons from several hundreds of eV up to MeV. Although there is an approximate two-fold increase in the yields of DSB for low energy photons found in our calculation and a large number of experimental measurements, it is far from enough to account for the three- to four-fold increase in RBE. Further theoretical investigations show that DSB complexity (additional single-strand breaks and base damage within 10 base pairs) increases notably for low energy photons, which largely reconciles the discrepancy between RBE and DSB induction. Our theoretical results are in line with accumulating experimental evidence that complex DSBs are refractory to repair machinery and may contribute predominantly to the formation of lethal damage.

  10. Activating Akt1 mutations alter DNA double strand break repair and radiosensitivity

    Science.gov (United States)

    Oeck, S.; Al-Refae, K.; Riffkin, H.; Wiel, G.; Handrick, R.; Klein, D.; Iliakis, G.; Jendrossek, V.

    2017-01-01

    The survival kinase Akt has clinical relevance to radioresistance. However, its contributions to the DNA damage response, DNA double strand break (DSB) repair and apoptosis remain poorly defined and often contradictory. We used a genetic approach to explore the consequences of genetic alterations of Akt1 for the cellular radiation response. While two activation-associated mutants with prominent nuclear access, the phospho-mimicking Akt1-TDSD and the clinically relevant PH-domain mutation Akt1-E17K, accelerated DSB repair and improved survival of irradiated Tramp-C1 murine prostate cancer cells and Akt1-knockout murine embryonic fibroblasts in vitro, the classical constitutively active membrane-targeted myrAkt1 mutant had the opposite effects. Interestingly, DNA-PKcs directly phosphorylated Akt1 at S473 in an in vitro kinase assay but not vice-versa. Pharmacological inhibition of DNA-PKcs or Akt restored radiosensitivity in tumour cells expressing Akt1-E17K or Akt1-TDSD. In conclusion, Akt1-mediated radioresistance depends on its activation state and nuclear localization and is accessible to pharmacologic inhibition. PMID:28209968

  11. The conformation of double-stranded DNA inside bacteriophages depends on capsid size and shape.

    Science.gov (United States)

    Petrov, Anton S; Boz, Mustafa Burak; Harvey, Stephen C

    2007-11-01

    The packaging of double-stranded DNA into bacteriophages leads to the arrangement of the genetic material into highly-packed and ordered structures. Although modern experimental techniques reveal the most probable location of DNA inside viral capsids, the individual conformations of DNA are yet to be determined. In the current study we present the results of molecular dynamics simulations of the DNA packaging into several bacteriophages performed within the framework of a coarse-grained model. The final DNA conformations depend on the size and shape of the capsid, as well as the size of the protein portal, if any. In particular, isometric capsids with small or absent portals tend to form concentric spools, whereas the presence of a large portal favors coaxial spooling; slightly and highly elongated capsids result in folded and twisted toroidal conformations, respectively. The results of the simulations also suggest that the predominant factor in defining the global DNA arrangement inside bacteriophages is the minimization of the bending stress upon packaging.

  12. Dynamic control of chirality and self-assembly of double-stranded helicates with light.

    Science.gov (United States)

    Zhao, Depeng; van Leeuwen, Thomas; Cheng, Jinling; Feringa, Ben L

    2017-03-01

    Helicity switching in biological and artificial systems is a fundamental process that allows for the dynamic control of structures and their functions. In contrast to chemical approaches to responsive behaviour in helicates, the use of light as an external stimulus offers unique opportunities to invert the chirality of helical structures in a non-invasive manner with high spatiotemporal precision. Here, we report that unidirectional rotary motors with connecting oligobipyridyl ligands, which can dynamically change their chirality upon irradiation, assemble into metal helicates that are responsive to light. The motor function controls the self-assembly process as well as the helical chirality, allowing switching between oligomers and double-stranded helicates with distinct handedness. The unidirectionality of the light-induced motion governs the sequence of programmable steps, enabling the highly regulated self-assembly of fully responsive helical structures. This discovery paves the way for the future development of new chirality-dependent photoresponsive systems including smart materials, enantioselective catalysts and light-driven molecular machines.

  13. The Heterochromatic Barrier to DNA Double Strand Break Repair: How to Get the Entry Visa

    Directory of Open Access Journals (Sweden)

    Aaron A. Goodarzi

    2012-09-01

    Full Text Available Over recent decades, a deep understanding of pathways that repair DNA double strand breaks (DSB has been gained from biochemical, structural, biophysical and cellular studies. DNA non-homologous end-joining (NHEJ and homologous recombination (HR represent the two major DSB repair pathways, and both processes are now well understood. Recent work has demonstrated that the chromatin environment at a DSB significantly impacts upon DSB repair and that, moreover, dramatic modifications arise in the chromatin surrounding a DSB. Chromatin is broadly divided into open, transcriptionally active, euchromatin (EC and highly compacted, transcriptionally inert, heterochromatin (HC, although these represent extremes of a spectrum. The HC superstructure restricts both DSB repair and damage response signaling. Moreover, DSBs within HC (HC-DSBs are rapidly relocalized to the EC-HC interface. The damage response protein kinase, ataxia telangiectasia mutated (ATM, is required for HC-DSB repair but is dispensable for the relocalization of HC-DSBs. It has been proposed that ATM signaling enhances HC relaxation in the DSB vicinity and that this is a prerequisite for HC-DSB repair. Hence, ATM is essential for repair of HC-DSBs. Here, we discuss how HC impacts upon the response to DSBs and how ATM overcomes the barrier that HC poses to repair.

  14. Simulating Molecular Interactions of Carbon Nanoparticles with a Double-Stranded DNA Fragment

    Directory of Open Access Journals (Sweden)

    Zhuang Wang

    2015-01-01

    Full Text Available Molecular interactions between carbon nanoparticles (CNPs and a double-stranded deoxyribonucleic acid (dsDNA fragment were investigated using molecular dynamics (MD simulations. Six types of CNPs including fullerenes (C60 and C70, (8,0 single-walled carbon nanotube (SWNT, (8,0 double-walled carbon nanotube (DWNT, graphene quantum dot (GQD, and graphene oxide quantum dot (GOQD were studied. Analysis of the best geometry indicates that the dsDNA fragment can bind to CNPs through pi-stacking and T-shape. Moreover, C60, DWNT, and GOQD bind to the dsDNA molecules at the minor groove of the nucleotide, and C70, SWNT, and GQD bind to the dsDNA molecules at the hydrophobic ends. Estimated interaction energy implies that van der Waals force may mainly contribute to the mechanisms for the dsDNA-C60, dsDNA-C70, and dsDNA-SWNT interactions and electrostatic force may contribute considerably to the dsDNA-DWNT, dsDNA-GQD, and dsDNA-GOQD interactions. On the basis of the results from large-scale MD simulations, it was found that the presence of the dsDNA enhances the dispersion of C60, C70, and SWNT in water and has a slight impact on DWNT, GQD, and GOQD.

  15. MOF Phosphorylation by ATM Regulates 53BP1-Mediated Double-Strand Break Repair Pathway Choice

    Directory of Open Access Journals (Sweden)

    Arun Gupta

    2014-07-01

    Full Text Available Cell-cycle phase is a critical determinant of the choice between DNA damage repair by nonhomologous end-joining (NHEJ or homologous recombination (HR. Here, we report that double-strand breaks (DSBs induce ATM-dependent MOF (a histone H4 acetyl-transferase phosphorylation (p-T392-MOF and that phosphorylated MOF colocalizes with γ-H2AX, ATM, and 53BP1 foci. Mutation of the phosphorylation site (MOF-T392A impedes DNA repair in S and G2 phase but not G1 phase cells. Expression of MOF-T392A also blocks the reduction in DSB-associated 53BP1 seen in wild-type S/G2 phase cells, resulting in enhanced 53BP1 and reduced BRCA1 association. Decreased BRCA1 levels at DSB sites correlates with defective repairosome formation, reduced HR repair, and decreased cell survival following irradiation. These data support a model whereby ATM-mediated MOF-T392 phosphorylation modulates 53BP1 function to facilitate the subsequent recruitment of HR repair proteins, uncovering a regulatory role for MOF in DSB repair pathway choice during S/G2 phase.

  16. Analysis of DNA double-strand break (DSB) repair in mammalian cells.

    Science.gov (United States)

    Seluanov, Andrei; Mao, Zhiyong; Gorbunova, Vera

    2010-09-08

    DNA double-strand breaks are the most dangerous DNA lesions that may lead to massive loss of genetic information and cell death. Cells repair DSBs using two major pathways: nonhomologous end joining (NHEJ) and homologous recombination (HR). Perturbations of NHEJ and HR are often associated with premature aging and tumorigenesis, hence it is important to have a quantitative way of measuring each DSB repair pathway. Our laboratory has developed fluorescent reporter constructs that allow sensitive and quantitative measurement of NHEJ and HR. The constructs are based on an engineered GFP gene containing recognition sites for a rare-cutting I-SceI endonuclease for induction of DSBs. The starting constructs are GFP negative as the GFP gene is inactivated by an additional exon, or by mutations. Successful repair of the I-SceI-induced breaks by NHEJ or HR restores the functional GFP gene. The number of GFP positive cells counted by flow cytometry provides quantitative measure of NHEJ or HR efficiency.

  17. DNA double-strand breaks induced along the trajectory of particles

    Science.gov (United States)

    Cho, I. C.; Niu, H.; Chen, C. H.; Yu, Y. C.; Hsu, C. H.

    2011-12-01

    It is well-known that the DNA damage caused by charged particles considerably differs from damage due to electromagnetic radiation. In the case of irradiation by charged particles the DNA lesions are more complex and clustered. Such clustered damage is presumed difficult to be repaired, and is potentially lethal. In this study, we utilize a 90°-scattering system and related imaging techniques to investigate the accumulation of γ-H2AX along the trajectory of charged particles. By immunostaining the γ-H2AX protein, optical images of corresponding double strand breaks were observed using a high resolution confocal microscope. We demonstrate the difference in the accumulation of γ-H2AX from irradiation by 1 MeV protons and that of 150 keV X-rays. The acquired images were arranged and reconstructed into a 3D image using ImageJ software. We discovered that the γ-H2AX foci, following irradiation by protons, have a tendency to extend in the beam direction, while those from X-ray irradiation tend to be smaller and more randomly distributed. These results can be explained by the physical model of energy deposition.

  18. Mechanical properties of double-stranded DNA biolayers immobilized on microcantilever under axial compression.

    Science.gov (United States)

    Zhang, Neng-Hui; Chen, Jian-Zhong

    2009-07-22

    In label-free biodetections based on microcantilever technology, double-stranded DNA (dsDNA) structures form through the linkage between probe single-stranded DNA (ssDNA) molecules immobilized on solid substrates and target ssDNA molecules in solutions. Mechanical/electrical properties of these biolayers are important factors for nanomechanical deflections of microcantilevers. In this paper, the biolayer immobilized on microcantilever is treated as a bar with a macroscopic elastic modulus on the basis of continuum mechanics viewpoints. In consideration of hydration force, screened electrostatic repulsion and conformational fluctuation in biolayers, load-deformation curves of dsDNA biolayers under axial compression are depicted with the help of the energy conservation law and a mesoscopic free energy presented by Strey et al. (1997, 1999) [Strey, H.H., Parsegian, V.A., Podgornik, R., 1997. Equation of state for DNA liquid crystals: fluctuation enhanced electrostatic double layer repulsion. Physical Review Letters 78, 895-898; Strey, H.H., Parsegian, V.A., Podgornik, R., 1999. Equation of state for polymer liquid crystals: theory and experiment. Physical Review E 59, 999-1008] from a liquid crystal theory. And the analytical relation between macroscopic Young's modulus of biolayers and nanoscopic geometrical properties of dsDNA, packing density, buffer salt solution concentration, etc. is also formulated.

  19. Generation of Gross Chromosomal Rearrangements by a Single Engineered DNA Double Strand Break

    Science.gov (United States)

    Qiu, Zhijun; Zhang, Zhenhua; Roschke, Anna; Varga, Tamas; Aplan, Peter D.

    2017-01-01

    Gross chromosomal rearrangements (GCRs), including translocations, inversions amplifications, and deletions, can be causal events leading to malignant transformation. GCRs are thought to be triggered by DNA double strand breaks (DSBs), which in turn can be spontaneous or induced by external agents (eg. cytotoxic chemotherapy, ionizing radiation). It has been shown that induction of DNA DSBs at two defined loci can produce stable balanced chromosomal translocations, however, a single engineered DNA DSB could not. Herein, we report that although a single engineered DNA DSB in H2AX “knockdown” cells did not generate GCRs, repair of a single engineered DNA DSB in fibroblasts that had ablated H2ax did produce clonal, stable GCRs, including balanced translocations and megabase-pair inversions. Upon correction of the H2ax deficiency, cells no longer generated GCRs following a single engineered DNA DSB. These findings demonstrate that clonal, stable GCRs can be produced by a single engineered DNA DSB in H2ax knockout cells, and that the production of these GCRs is ameliorated by H2ax expression. PMID:28225067

  20. Phosphorylation of Ku dictates DNA double-strand break (DSB) repair pathway choice in S phase.

    Science.gov (United States)

    Lee, Kyung-Jong; Saha, Janapriya; Sun, Jingxin; Fattah, Kazi R; Wang, Shu-Chi; Jakob, Burkhard; Chi, Linfeng; Wang, Shih-Ya; Taucher-Scholz, Gisela; Davis, Anthony J; Chen, David J

    2016-02-29

    Multiple DNA double-strand break (DSB) repair pathways are active in S phase of the cell cycle; however, DSBs are primarily repaired by homologous recombination (HR) in this cell cycle phase. As the non-homologous end-joining (NHEJ) factor, Ku70/80 (Ku), is quickly recruited to DSBs in S phase, we hypothesized that an orchestrated mechanism modulates pathway choice between HR and NHEJ via displacement of the Ku heterodimer from DSBs to allow HR. Here, we provide evidence that phosphorylation at a cluster of sites in the junction of the pillar and bridge regions of Ku70 mediates the dissociation of Ku from DSBs. Mimicking phosphorylation at these sites reduces Ku's affinity for DSB ends, suggesting that phosphorylation of Ku70 induces a conformational change responsible for the dissociation of the Ku heterodimer from DNA ends. Ablating phosphorylation of Ku70 leads to the sustained retention of Ku at DSBs, resulting in a significant decrease in DNA end resection and HR, specifically in S phase. This decrease in HR is specific as these phosphorylation sites are not required for NHEJ. Our results demonstrate that the phosphorylation-mediated dissociation of Ku70/80 from DSBs frees DNA ends, allowing the initiation of HR in S phase and providing a mechanism of DSB repair pathway choice in mammalian cells.

  1. Electrochemical Study on the Interaction of Irinotecan with Calf Thymus Double Stranded DNA

    Institute of Scientific and Technical Information of China (English)

    Hajian, Reza; Huat, Tan Guan

    2012-01-01

    Voltammetric behavior of Irinotecan (CPT-11) was studied in a phosphate buffer (0.002 mol.L^-1, pH 7.5) solution at the hanging mercury drop electrode (HMDE) using cyclic voltammetry (CV). CPT-11 showed two irreversible cathodic peaks at - 1.01 V and - 1.09 V which involved two electrons and two protons in each reduction step. In addition, the interaction of Irinotecan with double-stranded calf thymus DNA (ds-DNA) was studied by CV at the HMDE employing an irreversible electrochemical equation. As a result of the reaction with ds-DNA, the reduc- tion peaks related to CPT-11 were shifted in a negative direction and the peak currents were decreased. The diffu- sion coefficients of CPT- 11 in the absence (Dr) and presence (Db) of ds-DNA were calculated as 2.8 ×10 5 cm2.s^- 1 and 1.6 × 10^-5 cm2·s^-1 respectively. The binding constant (K=1.0×10^4 L·mol^-1), and binding site size (s=0.60) of CPT-11 interacting with ds-DNA were obtained simultaneously by non-linear fit analysis. The results demon strate that the main interaction mode of CPT-11 with ds-DNA is electrostatic.

  2. Deletion-bias in DNA double-strand break repair differentially contributes to plant genome shrinkage.

    Science.gov (United States)

    Vu, Giang T H; Cao, Hieu X; Reiss, Bernd; Schubert, Ingo

    2017-02-28

    In order to prevent genome instability, cells need to be protected by a number of repair mechanisms, including DNA double-strand break (DSB) repair. The extent to which DSB repair, biased towards deletions or insertions, contributes to evolutionary diversification of genome size is still under debate. We analyzed mutation spectra in Arabidopsis thaliana and in barley (Hordeum vulgare) by PacBio sequencing of three DSB-targeted loci each, uncovering repair via gene conversion, single strand annealing (SSA) or nonhomologous end-joining (NHEJ). Furthermore, phylogenomic comparisons between A. thaliana and two related species were used to detect naturally occurring deletions during Arabidopsis evolution. Arabidopsis thaliana revealed significantly more and larger deletions after DSB repair than barley, and barley displayed more and larger insertions. Arabidopsis displayed a clear net loss of DNA after DSB repair, mainly via SSA and NHEJ. Barley revealed a very weak net loss of DNA, apparently due to less active break-end resection and easier copying of template sequences into breaks. Comparative phylogenomics revealed several footprints of SSA in the A. thaliana genome. Quantitative assessment of DNA gain and loss through DSB repair processes suggests deletion-biased DSB repair causing ongoing genome shrinking in A. thaliana, whereas genome size in barley remains nearly constant.

  3. Multiple-pathway analysis of double-strand break repair mutations in Drosophila.

    Directory of Open Access Journals (Sweden)

    Dena M Johnson-Schlitz

    2007-04-01

    Full Text Available The analysis of double-strand break (DSB repair is complicated by the existence of several pathways utilizing a large number of genes. Moreover, many of these genes have been shown to have multiple roles in DSB repair. To address this complexity we used a repair reporter construct designed to measure multiple repair outcomes simultaneously. This approach provides estimates of the relative usage of several DSB repair pathways in the premeiotic male germline of Drosophila. We applied this system to mutations at each of 11 repair loci plus various double mutants and altered dosage genotypes. Most of the mutants were found to suppress one of the pathways with a compensating increase in one or more of the others. Perhaps surprisingly, none of the single mutants suppressed more than one pathway, but they varied widely in how the suppression was compensated. We found several cases in which two or more loci were similar in which pathway was suppressed while differing in how this suppression was compensated. Taken as a whole, the data suggest that the choice of which repair pathway is used for a given DSB occurs by a two-stage "decision circuit" in which the DSB is first placed into one of two pools from which a specific pathway is then selected.

  4. Radiation dose determines the method for quantification of DNA double strand breaks

    Energy Technology Data Exchange (ETDEWEB)

    Bulat, Tanja; Keta, Olitija; Korićanac, Lela; Žakula, Jelena; Petrović, Ivan; Ristić-Fira, Aleksandra [University of Belgrade, Vinča Institute of Nuclear Sciences, Belgrade (Serbia); Todorović, Danijela, E-mail: dtodorovic@medf.kg.ac.rs [University of Kragujevac, Faculty of Medical Sciences, Kragujevac (Serbia)

    2016-03-15

    Ionizing radiation induces DNA double strand breaks (DSBs) that trigger phosphorylation of the histone protein H2AX (γH2AX). Immunofluorescent staining visualizes formation of γH2AX foci, allowing their quantification. This method, as opposed to Western blot assay and Flow cytometry, provides more accurate analysis, by showing exact position and intensity of fluorescent signal in each single cell. In practice there are problems in quantification of γH2AX. This paper is based on two issues: the determination of which technique should be applied concerning the radiation dose, and how to analyze fluorescent microscopy images obtained by different microscopes. HTB140 melanoma cells were exposed to γ-rays, in the dose range from 1 to 16 Gy. Radiation effects on the DNA level were analyzed at different time intervals after irradiation by Western blot analysis and immunofluorescence microscopy. Immunochemically stained cells were visualized with two types of microscopes: AxioVision (Zeiss, Germany) microscope, comprising an ApoTome software, and AxioImagerA1 microscope (Zeiss, Germany). Obtained results show that the level of γH2AX is time and dose dependent. Immunofluorescence microscopy provided better detection of DSBs for lower irradiation doses, while Western blot analysis was more reliable for higher irradiation doses. AxioVision microscope containing ApoTome software was more suitable for the detection of γH2AX foci. (author)

  5. The dynamic DNA methylomes of double-stranded DNA viruses associated with human cancer

    Science.gov (United States)

    Fernandez, Agustin F.; Rosales, Cecilia; Lopez-Nieva, Pilar; Graña, Osvaldo; Ballestar, Esteban; Ropero, Santiago; Espada, Jesus; Melo, Sonia A.; Lujambio, Amaia; Fraga, Mario F.; Pino, Irene; Javierre, Biola; Carmona, Francisco J.; Acquadro, Francesco; Steenbergen, Renske D.M.; Snijders, Peter J.F.; Meijer, Chris J.; Pineau, Pascal; Dejean, Anne; Lloveras, Belen; Capella, Gabriel; Quer, Josep; Buti, Maria; Esteban, Juan-Ignacio; Allende, Helena; Rodriguez-Frias, Francisco; Castellsague, Xavier; Minarovits, Janos; Ponce, Jordi; Capello, Daniela; Gaidano, Gianluca; Cigudosa, Juan Cruz; Gomez-Lopez, Gonzalo; Pisano, David G.; Valencia, Alfonso; Piris, Miguel Angel; Bosch, Francesc X.; Cahir-McFarland, Ellen; Kieff, Elliott; Esteller, Manel

    2009-01-01

    The natural history of cancers associated with virus exposure is intriguing, since only a minority of human tissues infected with these viruses inevitably progress to cancer. However, the molecular reasons why the infection is controlled or instead progresses to subsequent stages of tumorigenesis are largely unknown. In this article, we provide the first complete DNA methylomes of double-stranded DNA viruses associated with human cancer that might provide important clues to help us understand the described process. Using bisulfite genomic sequencing of multiple clones, we have obtained the DNA methylation status of every CpG dinucleotide in the genome of the Human Papilloma Viruses 16 and 18 and Human Hepatitis B Virus, and in all the transcription start sites of the Epstein-Barr Virus. These viruses are associated with infectious diseases (such as hepatitis B and infectious mononucleosis) and the development of human tumors (cervical, hepatic, and nasopharyngeal cancers, and lymphoma), and are responsible for 1 million deaths worldwide every year. The DNA methylomes presented provide evidence of the dynamic nature of the epigenome in contrast to the genome. We observed that the DNA methylome of these viruses evolves from an unmethylated to a highly methylated genome in association with the progression of the disease, from asymptomatic healthy carriers, through chronically infected tissues and pre-malignant lesions, to the full-blown invasive tumor. The observed DNA methylation changes have a major functional impact on the biological behavior of the viruses. PMID:19208682

  6. Using carbon nanotubes to induce micronuclei and double strand breaks of the DNA in human cells

    Energy Technology Data Exchange (ETDEWEB)

    Cveticanin, Jelena; Joksic, Gordana; Leskovac, Andreja; Petrovic, Sandra; Sobot, Ana Valenta; Neskovic, Olivera, E-mail: oliveran@vinca.rs [Vinca Institute of Nuclear Sciences, PO Box 522, Belgrade (Serbia)

    2010-01-08

    Carbon nanotubes are unique one-dimensional macromolecules with promising applications in biology and medicine. Since their toxicity is still under debate, here we present a study investigating the genotoxic properties of purified single wall carbon nanotubes (SWCNTs), multiwall carbon nanotubes (MWCNTs), and amide functionalized purified SWCNTs on cultured human lymphocytes employing cytokinesis block micronucleus assay and enumeration of {gamma}H2AX foci as a measure of double strand breaks (DSBs) of the DNA in normal human fibroblasts. SWCNTs induce micronuclei (MN) formation in lymphocytes and decrease the proliferation potential (CBPI) of cells. In a fibroblast cell line the same dose of SWCNTs induces {gamma}H2AX foci 2.7-fold higher than in a control. Amide functionalized purified SWCNTs behave differently: they do not disturb the cell proliferation potential of harvested lymphocytes, but induce micronuclei to a higher extent than SWCNTs. When applied on fibroblasts, amide functionalized SWCNTs also induce {gamma}H2AX foci, 3.18-fold higher than the control. The cellular effects of MWCNTs display the broad spectrum of clastogenic properties seen as the highest incidence of induced lymphocyte micronuclei and anaphase bridges among nuclei in binucleated cells. Surprisingly, the incidence of induced {gamma}H2AX foci was not as high as was expected by the micronucleus test, which indicates that MWCNTs act as clastogen and aneugen agents simultaneously. Biological endpoints investigated in this study indicate a close relationship between the electrochemical properties of carbon nanotubes and observed genotoxicity.

  7. The yeast Saccharomyces cerevisiae DNA polymerase IV: possible involvement in double strand break DNA repair.

    Science.gov (United States)

    Leem, S H; Ropp, P A; Sugino, A

    1994-08-11

    We identified and purified a new DNA polymerase (DNA polymerase IV), which is similar to mammalian DNA polymerase beta, from Saccharomyces cerevisiae and suggested that it is encoded by YCR14C (POLX) on chromosome III. Here, we provided a direct evidence that the purified DNA polymerase IV is indeed encoded by POLX. Strains harboring a pol4 deletion mutation exhibit neither mitotic growth defect nor a meiosis defect, suggesting that DNA polymerase IV participates in nonessential functions in DNA metabolism. The deletion strains did not exhibit UV-sensitivity. However, they did show weak sensitivity to MMS-treatment and exhibited a hyper-recombination phenotype when intragenic recombination was measured during meiosis. Furthermore, MAT alpha pol4 delta segregants had a higher frequency of illegitimate mating with a MAT alpha tester strain than that of wild-type cells. These results suggest that DNA polymerase IV participates in a double-strand break repair pathway. A 3.2kb of the POL4 transcript was weakly expressed in mitotically growing cells. During meiosis, a 2.2 kb POL4 transcript was greatly induced, while the 3.2 kb transcript stayed at constant levels. This induction was delayed in a swi4 delta strain during meiosis, while no effect was observed in a swi6 delta strain.

  8. Dynamic control of chirality and self-assembly of double-stranded helicates with light

    Science.gov (United States)

    Zhao, Depeng; van Leeuwen, Thomas; Cheng, Jinling; Feringa, Ben L.

    2016-11-01

    Helicity switching in biological and artificial systems is a fundamental process that allows for the dynamic control of structures and their functions. In contrast to chemical approaches to responsive behaviour in helicates, the use of light as an external stimulus offers unique opportunities to invert the chirality of helical structures in a non-invasive manner with high spatiotemporal precision. Here, we report that unidirectional rotary motors with connecting oligobipyridyl ligands, which can dynamically change their chirality upon irradiation, assemble into metal helicates that are responsive to light. The motor function controls the self-assembly process as well as the helical chirality, allowing switching between oligomers and double-stranded helicates with distinct handedness. The unidirectionality of the light-induced motion governs the sequence of programmable steps, enabling the highly regulated self-assembly of fully responsive helical structures. This discovery paves the way for the future development of new chirality-dependent photoresponsive systems including smart materials, enantioselective catalysts and light-driven molecular machines.

  9. Radiation dose determines the method for quantification of DNA double strand breaks

    Directory of Open Access Journals (Sweden)

    TANJA BULAT

    2016-03-01

    Full Text Available ABSTRACT Ionizing radiation induces DNA double strand breaks (DSBs that trigger phosphorylation of the histone protein H2AX (γH2AX. Immunofluorescent staining visualizes formation of γH2AX foci, allowing their quantification. This method, as opposed to Western blot assay and Flow cytometry, provides more accurate analysis, by showing exact position and intensity of fluorescent signal in each single cell. In practice there are problems in quantification of γH2AX. This paper is based on two issues: the determination of which technique should be applied concerning the radiation dose, and how to analyze fluorescent microscopy images obtained by different microscopes. HTB140 melanoma cells were exposed to γ-rays, in the dose range from 1 to 16 Gy. Radiation effects on the DNA level were analyzed at different time intervals after irradiation by Western blot analysis and immunofluorescence microscopy. Immunochemically stained cells were visualized with two types of microscopes: AxioVision (Zeiss, Germany microscope, comprising an ApoTome software, and AxioImagerA1 microscope (Zeiss, Germany. Obtained results show that the level of γH2AX is time and dose dependent. Immunofluorescence microscopy provided better detection of DSBs for lower irradiation doses, while Western blot analysis was more reliable for higher irradiation doses. AxioVision microscope containing ApoTome software was more suitable for the detection of γH2AX foci.

  10. Characteristics of {gamma}-H2AX foci at DNA double-strand breaks sites

    Energy Technology Data Exchange (ETDEWEB)

    Pilch, D.R.; Sedelnikova, O.A.; Redon, C. [National Cancer Institute, National Institutes of Health, Lab. of Molecular Pharmacology, Bethesda, Maryland (United States); Celeste, A.; Nussenzweig, A. [National Cancer Institute, National Institutes of Health, Experimental Immunology Branch, Bethesda, Maryland (United States); Bonner, W.M. [National Cancer Institute, National Institutes of Health, Lab. of Molecular Pharmacology, Bethesda, Maryland (United States)

    2003-06-01

    Phosphorylated H2AX ({gamma}-H2AX) is essential to the efficient recognition and (or) repair of DNA double strand breaks (DSBs), and many molecules, often thousands, of H2AX become rapidly phosphorylated at the site of each nascent DSB. An antibody to {gamma}-H2AX reveals that this highly amplified process generates nuclear foci. The phosphorylation site is a serine four residues from the C-terminus which has been evolutionarily conserved in organisms from giardia intestinalis to humans. Mice and yeast lacking the conserved serine residue demonstrate a variety of defects in DNA DSB processing. H2AX{sup {delta}}{sup /{delta}} mice are smaller, sensitive to ionizing radiation, defective in class switch recombination and spermatogenesis while cells from the mice demonstrate substantially increased numbers of genomic defects. {gamma}-H2AX foci formation is a sensitive biological dosimeter and presents new and exciting opportunities to understand important biological processes, human diseases, and individual variations in radiation sensitivity. These potentialities demonstrate the importance of understanding the parameters and functions of {gamma}-H2AX formation. (author)

  11. Analysis of DNA double-strand break repair pathways in mice

    Energy Technology Data Exchange (ETDEWEB)

    Brugmans, Linda [Department of Cell Biology and Genetics, Erasmus MC, Dr. Molewaterplein 50, PO Box 1738, Rotterdam 3015GE (Netherlands); Kanaar, Roland [Department of Cell Biology and Genetics, Erasmus MC, Dr. Molewaterplein 50, PO Box 1738, Rotterdam 3015GE (Netherlands); Department of Radiation Oncology, Erasmus MC, PO Box 1738, 3000 DR Rotterdam (Netherlands); Essers, Jeroen [Department of Cell Biology and Genetics, Erasmus MC, Dr. Molewaterplein 50, PO Box 1738, Rotterdam 3015GE (Netherlands) and Department of Radiation Oncology, Erasmus MC, PO Box 1738, 3000 DR Rotterdam (Netherlands)]. E-mail: j.essers@erasmusmc.nl

    2007-01-03

    During the last years significant new insights have been gained into the mechanism and biological relevance of DNA double-strand break (DSB) repair in relation to genome stability. DSBs are a highly toxic DNA lesion, because they can lead to chromosome fragmentation, loss and translocations, eventually resulting in cancer. DSBs can be induced by cellular processes such as V(D)J recombination or DNA replication. They can also be introduced by exogenous agents DNA damaging agents such as ionizing radiation or mitomycin C. During evolution several pathways have evolved for the repair of these DSBs. The most important DSB repair mechanisms in mammalian cells are nonhomologous end-joining and homologous recombination. By using an undamaged repair template, homologous recombination ensures accurate DSB repair, whereas the untemplated nonhomologous end-joining pathway does not. Although both pathways are active in mammals, the relative contribution of the two repair pathways to genome stability differs in the different cell types. Given the potential differences in repair fidelity, it is of interest to determine the relative contribution of homologous recombination and nonhomologous end-joining to DSB repair. In this review, we focus on the biological relevance of DSB repair in mammalian cells and the potential overlap between nonhomologous end-joining and homologous recombination in different tissues.

  12. The MRE11 GAR motif regulates DNA double-strand break processing and ATR activation

    Institute of Scientific and Technical Information of China (English)

    Zhenbao Yu; Gillian Vogel; Yan Coulombe; Danielle Dubeau; Elizabeth Spehalski; Josée Hébert; David O Ferguson; Jean Yves Masson; Stéphane Richard

    2012-01-01

    The MRE11/RAD50/NBS1 complex is the primary sensor rapidly recruited to DNA double-strand breaks (DSBs).MRE11 is known to be arginine methylated by PRMT1 within its glycine-arginine-rich (GAR) motif.In this study,we report a mouse knock-in allele of Mre11 that substitutes the arginines with lysines in the GAR motif and generates the MRE11RK protein devoid of methylated arginines.The Mre11RK/RK mice were hypersensitive to γ-irradiation (IR) and the cells from these mice displayed cell cycle checkpoint defects and chromosome instability.Moreover,the Mre11RK/RK MEFs exhibited ATR/CHK1 signaling defects and impairment in the recruitment of RPA and RAD51 to the damaged sites.The MRKRN complex formed and localized to the sites of DNA damage and normally activated the ATM pathway in response to IR.The MRKRN complex exhibited exonuclease and DNA-binding defects in vitro responsible for the impaired DNA end resection and ATR activation observed in vivo in response to IR.Our findings provide genetic evidence for the critical role of the MRE11 GAR motif in DSB repair,and demonstrate a mechanistic link between post-translational modifications at the MRE11 GAR motif and DSB processing,as well as the ATR/CHK1 checkpoint signaling.

  13. DNA Double Strand Break Repair and its Association with Inherited Predispositions to Breast Cancer

    Directory of Open Access Journals (Sweden)

    Scott Rodney J

    2004-02-01

    Full Text Available Abstract Mutations in BRCA1 account for the majority of familial aggregations of early onset breast and ovarian cancer (~70% and about 1/5 of all early onset breast cancer families; in contrast, mutations in BRCA2 account for a smaller proportion of breast/ovarian cancer families and a similar proportion of early onset breast cancer families. BRCA2 has also been shown to be associated with a much more pleiotropic disease spectrum compared to BRCA1. Since the identification of both BRCA1 and BRCA2 investigations into the functions of these genes have revealed that both are associated with the maintenance of genomic integrity via their apparent roles in cellular response to DNA damage, especially their involvement in the process of double strand DNA break repair. This review will focus on the specific roles of both genes and how functional differences may account for the diverse clinical findings observed between families that harbour BRCA1 or BRCA2 mutations.

  14. MOF phosphorylation by ATM regulates 53BP1-mediated double-strand break repair pathway choice.

    Science.gov (United States)

    Gupta, Arun; Hunt, Clayton R; Hegde, Muralidhar L; Chakraborty, Sharmistha; Chakraborty, Sharmistha; Udayakumar, Durga; Horikoshi, Nobuo; Singh, Mayank; Ramnarain, Deepti B; Hittelman, Walter N; Namjoshi, Sarita; Asaithamby, Aroumougame; Hazra, Tapas K; Ludwig, Thomas; Pandita, Raj K; Tyler, Jessica K; Pandita, Tej K

    2014-07-10

    Cell-cycle phase is a critical determinant of the choice between DNA damage repair by nonhomologous end-joining (NHEJ) or homologous recombination (HR). Here, we report that double-strand breaks (DSBs) induce ATM-dependent MOF (a histone H4 acetyl-transferase) phosphorylation (p-T392-MOF) and that phosphorylated MOF colocalizes with γ-H2AX, ATM, and 53BP1 foci. Mutation of the phosphorylation site (MOF-T392A) impedes DNA repair in S and G2 phase but not G1 phase cells. Expression of MOF-T392A also blocks the reduction in DSB-associated 53BP1 seen in wild-type S/G2 phase cells, resulting in enhanced 53BP1 and reduced BRCA1 association. Decreased BRCA1 levels at DSB sites correlates with defective repairosome formation, reduced HR repair, and decreased cell survival following irradiation. These data support a model whereby ATM-mediated MOF-T392 phosphorylation modulates 53BP1 function to facilitate the subsequent recruitment of HR repair proteins, uncovering a regulatory role for MOF in DSB repair pathway choice during S/G2 phase.

  15. Chromosomal Integrity after UV Irradiation Requires FANCD2-Mediated Repair of Double Strand Breaks

    Science.gov (United States)

    Federico, María Belén; Vallerga, María Belén; Radl, Analía; Paviolo, Natalia Soledad; Bocco, José Luis; Di Giorgio, Marina; Soria, Gastón; Gottifredi, Vanesa

    2016-01-01

    Fanconi Anemia (FA) is a rare autosomal recessive disorder characterized by hypersensitivity to inter-strand crosslinks (ICLs). FANCD2, a central factor of the FA pathway, is essential for the repair of double strand breaks (DSBs) generated during fork collapse at ICLs. While lesions different from ICLs can also trigger fork collapse, the contribution of FANCD2 to the resolution of replication-coupled DSBs generated independently from ICLs is unknown. Intriguingly, FANCD2 is readily activated after UV irradiation, a DNA-damaging agent that generates predominantly intra-strand crosslinks but not ICLs. Hence, UV irradiation is an ideal tool to explore the contribution of FANCD2 to the DNA damage response triggered by DNA lesions other than ICL repair. Here we show that, in contrast to ICL-causing agents, UV radiation compromises cell survival independently from FANCD2. In agreement, FANCD2 depletion does not increase the amount of DSBs generated during the replication of UV-damaged DNA and is dispensable for UV-induced checkpoint activation. Remarkably however, FANCD2 protects UV-dependent, replication-coupled DSBs from aberrant processing by non-homologous end joining, preventing the accumulation of micronuclei and chromatid aberrations including non-homologous chromatid exchanges. Hence, while dispensable for cell survival, FANCD2 selectively safeguards chromosomal stability after UV-triggered replication stress. PMID:26765540

  16. Properties of natural double-strand-break sites at a recombination hotspot in Saccharomyces cerevisiae.

    Science.gov (United States)

    Haring, Stuart J; Halley, George R; Jones, Alex J; Malone, Robert E

    2003-01-01

    This study addresses three questions about the properties of recombination hotspots in Saccharomyces cerevisiae: How much DNA is required for double-strand-break (DSB) site recognition? Do naturally occurring DSB sites compete with each other in meiotic recombination? What role does the sequence located at the sites of DSBs play? In S. cerevisiae, the HIS2 meiotic recombination hotspot displays a high level of gene conversion, a 3'-to-5' conversion gradient, and two DSB sites located approximately 550 bp apart. Previous studies of hotspots, including HIS2, suggest that global chromosome structure plays a significant role in recombination activity, raising the question of how much DNA is sufficient for hotspot activity. We find that 11.5 kbp of the HIS2 region is sufficient to partially restore gene conversion and both DSBs when moved to another yeast chromosome. Using a variety of different constructs, studies of hotspots have indicated that DSB sites compete with one another for DSB formation. The two naturally occurring DSBs at HIS2 afforded us the opportunity to examine whether or not competition occurs between these native DSB sites. Small deletions of DNA at each DSB site affect only that site; analyses of these deletions show no competition occurring in cis or in trans, indicating that DSB formation at each site at HIS2 is independent. These small deletions significantly affect the frequency of DSB formation at the sites, indicating that the DNA sequence located at a DSB site can play an important role in recombination initiation. PMID:14504220

  17. Ataxia telangiectasia mutated activation by transcription- and topoisomerase I-induced DNA double-strand breaks.

    Science.gov (United States)

    Sordet, Olivier; Redon, Christophe E; Guirouilh-Barbat, Josée; Smith, Susan; Solier, Stéphanie; Douarre, Céline; Conti, Chiara; Nakamura, Asako J; Das, Benu B; Nicolas, Estelle; Kohn, Kurt W; Bonner, William M; Pommier, Yves

    2009-08-01

    Ataxia telangiectasia mutated (ATM), the deficiency of which causes a severe neurodegenerative disease, is a crucial mediator for the DNA damage response (DDR). As neurons have high rates of transcription that require topoisomerase I (TOP1), we investigated whether TOP1 cleavage complexes (TOP1cc)-which are potent transcription-blocking lesions-also produce transcription-dependent DNA double-strand breaks (DSBs) with ATM activation. We show the induction of DSBs and DDR activation in post-mitotic primary neurons and lymphocytes treated with camptothecin, with the induction of nuclear DDR foci containing activated ATM, gamma-H2AX (phosphorylated histone H2AX), activated CHK2 (checkpoint kinase 2), MDC1 (mediator of DNA damage checkpoint 1) and 53BP1 (p53 binding protein 1). The DSB-ATM-DDR pathway was suppressed by inhibiting transcription and gamma-H2AX signals were reduced by RNase H1 transfection, which removes transcription-mediated R-loops. Thus, we propose that Top1cc produce transcription arrests with R-loop formation and generate DSBs that activate ATM in post-mitotic cells.

  18. Transcription and double-strand breaks induce similar mitotic recombination events in Saccharomyces cerevisiae.

    Science.gov (United States)

    González-Barrera, Sergio; García-Rubio, María; Aguilera, Andrés

    2002-10-01

    We have made a comparative analysis of double-strand-break (DSB)-induced recombination and spontaneous recombination under low- and high-transcription conditions in yeast. We constructed two different recombination substrates, one for the analysis of intermolecular gene conversions and the other for intramolecular gene conversions and inversions. Such substrates were based on the same leu2-HOr allele fused to the tet promoter and containing a 21-bp HO site. Gene conversions and inversions were differently affected by rad1, rad51, rad52, and rad59 single and double mutations, consistent with the actual view that such events occur by different recombination mechanisms. However, the effect of each mutation on each type of recombination event was the same, whether associated with transcription or induced by the HO-mediated DSB. Both the highly transcribed DNA and the HO-cut sequence acted as recipients of the gene conversion events. These results are consistent with the hypothesis that transcription promotes initiation of recombination along the DNA sequence being transcribed. The similarity between transcription-associated and DSB-induced recombination suggests that transcription promotes DNA breaks.

  19. Transcription-induced DNA double strand breaks: both oncogenic force and potential therapeutic target?

    Science.gov (United States)

    Haffner, Michael C; De Marzo, Angelo M; Meeker, Alan K; Nelson, William G; Yegnasubramanian, Srinivasan

    2011-06-15

    An emerging model of transcriptional activation suggests that induction of transcriptional programs, for instance by stimulating prostate or breast cells with androgens or estrogens, respectively, involves the formation of DNA damage, including DNA double strand breaks (DSB), recruitment of DSB repair proteins, and movement of newly activated genes to transcription hubs. The DSB can be mediated by the class II topoisomerase TOP2B, which is recruited with the androgen receptor and estrogen receptor to regulatory sites on target genes and is apparently required for efficient transcriptional activation of these genes. These DSBs are recognized by the DNA repair machinery triggering the recruitment of repair proteins such as poly(ADP-ribose) polymerase 1 (PARP1), ATM, and DNA-dependent protein kinase (DNA-PK). If illegitimately repaired, such DSBs can seed the formation of genomic rearrangements like the TMPRSS2-ERG fusion oncogene in prostate cancer. Here, we hypothesize that these transcription-induced, TOP2B-mediated DSBs can also be exploited therapeutically and propose that, in hormone-dependent tumors like breast and prostate cancers, a hormone-cycling therapy, in combination with topoisomerase II poisons or inhibitors of the DNA repair components PARP1 and DNA-PK, could overwhelm cancer cells with transcription-associated DSBs. Such strategies may find particular utility in cancers, like prostate cancer, which show low proliferation rates, in which other chemotherapeutic strategies that target rapidly proliferating cells have had limited success.

  20. Sp1 facilitates DNA double-strand break repair through a nontranscriptional mechanism.

    Science.gov (United States)

    Beishline, Kate; Kelly, Crystal M; Olofsson, Beatrix A; Koduri, Sravanthi; Emrich, Jacqueline; Greenberg, Roger A; Azizkhan-Clifford, Jane

    2012-09-01

    Sp1 is a ubiquitously expressed transcription factor that is phosphorylated by ataxia telangiectasia mutated kinase (ATM) in response to ionizing radiation and H(2)O(2). Here, we show by indirect immunofluorescence that Sp1 phosphorylated on serine 101 (pSp1) localizes to ionizing radiation-induced foci with phosphorylated histone variant γH2Ax and members of the MRN (Mre11, Rad50, and Nbs1) complex. More precise analysis of occupancy of DNA double-strand breaks (DSBs) by chromatin immunoprecipitation (ChIP) shows that Sp1, like Nbs1, resides within 200 bp of DSBs. Using laser microirradiation of cells, we demonstrate that pSp1 is present at DNA DSBs by 7.5 min after induction of damage and remains at the break site for at least 8 h. Depletion of Sp1 inhibits repair of site-specific DNA breaks, and the N-terminal 182-amino-acid peptide, which contains targets of ATM kinase but lacks the zinc finger DNA binding domain, is phosphorylated, localizes to DSBs, and rescues the repair defect resulting from Sp1 depletion. Together, these data demonstrate that Sp1 is rapidly recruited to the region immediately adjacent to sites of DNA DSBs and is required for DSB repair, through a mechanism independent of its sequence-directed transcriptional effects.

  1. Interference in DNA replication can cause mitotic chromosomal breakage unassociated with double-strand breaks.

    Directory of Open Access Journals (Sweden)

    Mari Fujita

    Full Text Available Morphological analysis of mitotic chromosomes is used to detect mutagenic chemical compounds and to estimate the dose of ionizing radiation to be administered. It has long been believed that chromosomal breaks are always associated with double-strand breaks (DSBs. We here provide compelling evidence against this canonical theory. We employed a genetic approach using two cell lines, chicken DT40 and human Nalm-6. We measured the number of chromosomal breaks induced by three replication-blocking agents (aphidicolin, 5-fluorouracil, and hydroxyurea in DSB-repair-proficient wild-type cells and cells deficient in both homologous recombination and nonhomologous end-joining (the two major DSB-repair pathways. Exposure of cells to the three replication-blocking agents for at least two cell cycles resulted in comparable numbers of chromosomal breaks for RAD54(-/-/KU70(-/- DT40 clones and wild-type cells. Likewise, the numbers of chromosomal breaks induced in RAD54(-/-/LIG4(-/- Nalm-6 clones and wild-type cells were also comparable. These data indicate that the replication-blocking agents can cause chromosomal breaks unassociated with DSBs. In contrast with DSB-repair-deficient cells, chicken DT40 cells deficient in PIF1 or ATRIP, which molecules contribute to the completion of DNA replication, displayed higher numbers of mitotic chromosomal breaks induced by aphidicolin than did wild-type cells, suggesting that single-strand gaps left unreplicated may result in mitotic chromosomal breaks.

  2. ATM release at resected double-strand breaks provides heterochromatin reconstitution to facilitate homologous recombination.

    Directory of Open Access Journals (Sweden)

    Verena Geuting

    Full Text Available Non-homologous end-joining (NHEJ and homologous recombination (HR represent the two main pathways for repairing DNA double-strand breaks (DSBs. During the G2 phase of the mammalian cell cycle, both processes can operate and chromatin structure is one important factor which determines DSB repair pathway choice. ATM facilitates the repair of heterochromatic DSBs by phosphorylating and inactivating the heterochromatin building factor KAP-1, leading to local chromatin relaxation. Here, we show that ATM accumulation and activity is strongly diminished at DSBs undergoing end-resection during HR. Such DSBs remain unrepaired in cells devoid of the HR factors BRCA2, XRCC3 or RAD51. Strikingly, depletion of KAP-1 or expression of phospho-mimic KAP-1 allows repair of resected DSBs in the absence of BRCA2, XRCC3 or RAD51 by an erroneous PARP-dependent alt-NHEJ process. We suggest that DSBs in heterochromatin elicit initial local heterochromatin relaxation which is reversed during HR due to the release of ATM from resection break ends. The restored heterochromatic structure facilitates HR and prevents usage of error-prone alternative processes.

  3. Stable gene replacement in barley by targeted double-strand break induction.

    Science.gov (United States)

    Watanabe, Koichi; Breier, Ulrike; Hensel, Götz; Kumlehn, Jochen; Schubert, Ingo; Reiss, Bernd

    2016-03-01

    Gene targeting is becoming an important tool for precision genome engineering in plants. During gene replacement, a variant of gene targeting, transformed DNA integrates into the genome by homologous recombination (HR) to replace resident sequences. We have analysed gene targeting in barley (Hordeum vulgare) using a model system based on double-strand break (DSB) induction by the meganuclease I-SceI and a transgenic, artificial target locus. In the plants we obtained, the donor construct was inserted at the target locus by homology-directed DNA integration in at least two transformants obtained in a single experiment and was stably inherited as a single Mendelian trait. Both events were produced by one-sided integration. Our data suggest that gene replacement can be achieved in barley with a frequency suitable for routine application. The use of a codon-optimized nuclease and co-transfer of the nuclease gene together with the donor construct are probably the components important for efficient gene targeting. Such an approach, employing the recently developed synthetic nucleases/nickases that allow DSB induction at almost any sequence of a genome of interest, sets the stage for precision genome engineering as a routine tool even for important crops such as barley.

  4. OsRAD51C Is Essential for Double Strand Break Repair in Rice Meiosis

    Directory of Open Access Journals (Sweden)

    Ding eTang

    2014-05-01

    Full Text Available RAD51C is one of the RAD51 paralogs that plays an important role in DNA double-strand break repair by homologous recombination. Here, we identified and characterized OsRAD51C, the rice homolog of human RAD51C. The Osrad51c mutant plant is normal in vegetative growth but exhibits complete male and female sterility. Cytological investigation revealed that homologous pairing and synapsis were severely disrupted. Massive chromosome fragmentation occurred during metaphase I in Osrad51c meiocytes, and was fully suppressed by the CRC1 mutation. Immunofluorescence analysis showed that OsRAD51C localized onto the chromosomes from leptotene to early pachytene during prophase I, and that normal loading of OsRAD51C was dependent on OsREC8, PAIR2, and PAIR3. Additionally, ZEP1 did not localize properly in Osrad51c, indicating that OsRAD51C is required for synaptonemal complex assembly. Our study also provided evidence in support of a functional divergence in RAD51C among organisms.

  5. Thermodynamics for the Formation of Double-Stranded DNA-Single-Walled Carbon Nanotube Hybrids.

    Science.gov (United States)

    Shiraki, Tomohiro; Tsuzuki, Akiko; Toshimitsu, Fumiyuki; Nakashima, Naotoshi

    2016-03-24

    For the first time, the thermodynamics are described for the formation of double-stranded DNA (ds-DNA)-single-walled carbon nanotube (SWNT) hybrids. This treatment is applied to the exchange reaction of sodium cholate (SC) molecules on SWNTs and the ds-DNAs d(A)20 -d(T)20 and nuclear factor (NF)-κB decoy. UV/Vis/near-IR spectroscopy with temperature variations was used for analyzing the exchange reaction on the SWNTs with four different chiralities: (n,m)=(8,3), (6,5), (7,5), and (8,6). Single-stranded DNAs (ss-DNAs), including d(A)20 and d(T)20, are also used for comparison. The d(A)20-d(T)20 shows a drastic change in its thermodynamic parameters around the melting temperature (Tm ) of the DNA oligomer. No such Tm dependency was measured, owing to high Tm in the NF-κB decoy DNA and no Tm in the ss-DNA.

  6. Cohesin protects genes against γH2AX Induced by DNA double-strand breaks.

    Directory of Open Access Journals (Sweden)

    Pierre Caron

    2012-01-01

    Full Text Available Chromatin undergoes major remodeling around DNA double-strand breaks (DSB to promote repair and DNA damage response (DDR activation. We recently reported a high-resolution map of γH2AX around multiple breaks on the human genome, using a new cell-based DSB inducible system. In an attempt to further characterize the chromatin landscape induced around DSBs, we now report the profile of SMC3, a subunit of the cohesin complex, previously characterized as required for repair by homologous recombination. We found that recruitment of cohesin is moderate and restricted to the immediate vicinity of DSBs in human cells. In addition, we show that cohesin controls γH2AX distribution within domains. Indeed, as we reported previously for transcription, cohesin binding antagonizes γH2AX spreading. Remarkably, depletion of cohesin leads to an increase of γH2AX at cohesin-bound genes, associated with a decrease in their expression level after DSB induction. We propose that, in agreement with their function in chromosome architecture, cohesin could also help to isolate active genes from some chromatin remodelling and modifications such as the ones that occur when a DSB is detected on the genome.

  7. Fine-tuning the ubiquitin code at DNA double-strand breaks: deubiquitinating enzymes at work

    Directory of Open Access Journals (Sweden)

    Elisabetta eCitterio

    2015-09-01

    Full Text Available Ubiquitination is a reversible protein modification broadly implicated in cellular functions. Signaling processes mediated by ubiquitin are crucial for the cellular response to DNA double-strand breaks (DSBs, one of the most dangerous types of DNA lesions. In particular, the DSB response critically relies on active ubiquitination by the RNF8 and RNF168 ubiquitin ligases at the chromatin, which is essential for proper DSB signaling and repair. How this pathway is fine-tuned and what the functional consequences are of its deregulation for genome integrity and tissue homeostasis are subject of intense investigation. One important regulatory mechanism is by reversal of substrate ubiquitination through the activity of specific deubiquitinating enzymes (DUBs, as supported by the implication of a growing number of DUBs in DNA damage response (DDR processes. Here, we discuss the current knowledge of how ubiquitin-mediated signaling at DSBs is controlled by deubiquitinating enzymes, with main focus on DUBs targeting histone H2A and on their recent implication in stem cell biology and cancer.

  8. RECQL4 Promotes DNA End Resection in Repair of DNA Double-Strand Breaks

    Directory of Open Access Journals (Sweden)

    Huiming Lu

    2016-06-01

    Full Text Available The RecQ helicase RECQL4, mutated in Rothmund-Thomson syndrome, regulates genome stability, aging, and cancer. Here, we identify a crucial role for RECQL4 in DNA end resection, which is the initial and an essential step of homologous recombination (HR-dependent DNA double-strand break repair (DSBR. Depletion of RECQL4 severely reduces HR-mediated repair and 5′ end resection in vivo. RECQL4 physically interacts with MRE11-RAD50-NBS1 (MRN, which senses DSBs and initiates DNA end resection with CtIP. The MRE11 exonuclease regulates the retention of RECQL4 at laser-induced DSBs. RECQL4 also directly interacts with CtIP via its N-terminal domain and promotes CtIP recruitment to the MRN complex at DSBs. Moreover, inactivation of RECQL4’s helicase activity impairs DNA end processing and HR-dependent DSBR without affecting its interaction with MRE11 and CtIP, suggesting an important role for RECQL4’s unwinding activity in the process. Thus, we report that RECQL4 is an important participant in HR-dependent DSBR.

  9. DNA double strand breaks and Hsp70 expression in proton irradiated living cells

    Energy Technology Data Exchange (ETDEWEB)

    Fiedler, Anja [Institute for Experimental Physics II, University of Leipzig (Germany) and Faculty of Biology, Pharmacy and Psychology, University of Leipzig (Germany)]. E-mail: afiedler@uni-leipzig.de; Reinert, Tilo [Institute for Experimental Physics II, University of Leipzig (Germany); Tanner, Judith [Clinic and Polyclinic for Radiation Oncology, University of Halle-Wittenberg (Germany); Butz, Tilman [Institute for Experimental Physics II, University of Leipzig (Germany)

    2007-07-15

    DNA double strand breaks (DSBs) in living cells can be directly provoked by ionising radiation. DSBs can be visualized by immunostaining the phosphorylated histone {gamma}H2AX. Our concern was to test the feasibility of {gamma}H2AX staining for a direct visualization of single proton hits. If single protons produce detectable foci, DNA DSBs could be used as 'biological track detectors' for protons. Ionising radiation can also damage proteins indirectly by inducing free radicals. Heat shock proteins (Hsp) help to refold or even degrade the damaged proteins. The level of the most famous heat shock protein Hsp70 is increased by ionising radiation. We investigated the expression of {gamma}H2AX and Hsp70 after cross and line patterned irradiation with counted numbers of 2.25 MeV protons on primary human skin fibroblasts. The proton induced DSBs appear more delocalised than it was expected by the ion hit accuracy. Cooling the cells before the irradiation reduces the delocalisation of DNA DSBs, which is probably caused by the reduced diffusion of DNA damaging agents. Proton irradiation seems to provoke protein damages mainly in the cytoplasm indicated by cytoplasmic Hsp70 aggregates. On the contrary, in control heat shocked cells the Hsp70 was predominantly localized in the cell nucleus. However, the irradiated area could not be recognized, all cells on the Si{sub 3}N{sub 4} window showed a homogenous Hsp70 expression pattern.

  10. Double-strand breaks at the target locus stimulate gene targeting in embryonic stem cells.

    Science.gov (United States)

    Smih, F; Rouet, P; Romanienko, P J; Jasin, M

    1995-01-01

    Double-strand breaks (DSBs) are recombinogenic lesions in chromosomal DNA in yeast, Drosophila and Caenorhabditis elegans. Recent studies in mammalian cells utilizing the I-Scel endonuclease have demonstrated that in some immortalized cell lines DSBs in chromosomal DNA are also recombinogenic. We have now tested embryonic stem (ES) cells, a non-transformed mouse cell line frequently used in gene targeting studies. We find that a DSB introduced by I-Scel stimulates gene targeting at a selectable neo locus at least 50-fold. The enhanced level of targeting is achieved by transient expression of the I-Scel endonuclease. In 97% of targeted clones a single base pair polymorphism in the transfected homologous fragment was incorporated into the target locus. Analysis of the targeted locus demonstrated that most of the homologous recombination events were 'two-sided', in contrast to previous studies in 3T3 cells in which 'one-sided' homologous events predominated. Thus ES cells may be more faithful in incorporating homologous fragments into their genome than other cells in culture. Images PMID:8559659

  11. Syntheses and structures of dinuclear double-stranded helicates of divalent manganese, iron, cobalt, and zinc.

    Science.gov (United States)

    Reid, Stuart D; Blake, Alexander J; Wilson, Claire; Love, Jason B

    2006-01-23

    The syntheses and solid-state and solution structures of a series of unusually volatile, charge neutral, [4 + 4] double-stranded helical complexes of divalent manganese, iron, cobalt, and zinc are described. Deprotonation of the N4-donor iminopyrrole ligand H2L by KH cleanly generates the salt K2(THF)2L, which displays both sigma and pi interactions between K and iminopyrrolyl fragments in the X-ray crystal structure. Transamination, salt elimination, and protonolysis reactions were found to be versatile and, in general, high-yielding routes to the dinuclear double helicates [M2(L)2] (M = Mn, Fe, Co, and Zn). These compounds are isomorphous in the solid state by X-ray crystallography and adopt dinuclear cleft motifs as a result of pi stacking between opposing iminopyrrolyl fragments. This motif was also observed in the solution structures of [Fe2(L)2] and [Zn2(L)2] below 230 and 200 K, respectively (DeltaG++ = approximately 46 and 39.0 kJ mol(-1), respectively).

  12. RECQL4 Promotes DNA End Resection in Repair of DNA Double-Strand Breaks.

    Science.gov (United States)

    Lu, Huiming; Shamanna, Raghavendra A; Keijzers, Guido; Anand, Roopesh; Rasmussen, Lene Juel; Cejka, Petr; Croteau, Deborah L; Bohr, Vilhelm A

    2016-06-28

    The RecQ helicase RECQL4, mutated in Rothmund-Thomson syndrome, regulates genome stability, aging, and cancer. Here, we identify a crucial role for RECQL4 in DNA end resection, which is the initial and an essential step of homologous recombination (HR)-dependent DNA double-strand break repair (DSBR). Depletion of RECQL4 severely reduces HR-mediated repair and 5' end resection in vivo. RECQL4 physically interacts with MRE11-RAD50-NBS1 (MRN), which senses DSBs and initiates DNA end resection with CtIP. The MRE11 exonuclease regulates the retention of RECQL4 at laser-induced DSBs. RECQL4 also directly interacts with CtIP via its N-terminal domain and promotes CtIP recruitment to the MRN complex at DSBs. Moreover, inactivation of RECQL4's helicase activity impairs DNA end processing and HR-dependent DSBR without affecting its interaction with MRE11 and CtIP, suggesting an important role for RECQL4's unwinding activity in the process. Thus, we report that RECQL4 is an important participant in HR-dependent DSBR.

  13. Mutator Phenotype and DNA Double-Strand Break Repair in BLM Helicase-Deficient Human Cells

    Science.gov (United States)

    Suzuki, Tetsuya; Yasui, Manabu

    2016-01-01

    Bloom syndrome (BS), an autosomal recessive disorder of the BLM gene, predisposes sufferers to various cancers. To investigate the mutator phenotype and genetic consequences of DNA double-strand breaks (DSBs) in BS cells, we developed BLM helicase-deficient human cells by disrupting the BLM gene. Cells with a loss of heterozygosity (LOH) due to homologous recombination (HR) or nonhomologous end joining (NHEJ) can be restored with or without site-directed DSB induction. BLM cells exhibited a high frequency of spontaneous interallelic HR with crossover, but noncrossover events with long-tract gene conversions also occurred. Despite the highly interallelic HR events, BLM cells predominantly produced hemizygous LOH by spontaneous deletion. These phenotypes manifested during repair of DSBs. Both NHEJ and HR appropriately repaired DSBs in BLM cells, resulting in hemizygous and homozygous LOHs, respectively. However, the magnitude of the LOH was exacerbated in BLM cells, as evidenced by large deletions and long-tract gene conversions with crossover. BLM helicase suppresses the elongation of branch migration and crossover of double Holliday junctions (HJs) during HR repair, and a deficiency in this enzyme causes collapse, abnormal elongation, and/or preferable resolution to crossover of double HJs, resulting in a large-scale LOH. This mechanism underlies the predisposition for cancer in BS. PMID:27601585

  14. Detection of KRAS mutations using double-stranded toehold-exchange probes.

    Science.gov (United States)

    Wu, Zhenhua; Ma, Tianle; Coll, Jean-Luc; Liu, Fangming; Zhang, Honglian; Ma, Yunfei; Wang, Zhishuo; Jin, Qinghui; Mao, Hongju; Zhao, Jianlong

    2016-06-15

    Detection of KRAS mutations in cancer tissues is immensely valuable for the identification of personalized genotype-based therapy. Here, we employed a double-stranded toehold-exchange probe, which is labeled with fluorescent molecules (FAM) and quenchers (Dabcyl), to detect KRAS mutations in cancer tissues. This probe was able to differentiate the intended mutation in a sample containing as little as 5% mutant alleles in a background of wild-type DNA. This probe also performed robustly at a wide range of conditions, for examples, from 4 °C to 37 °C, from 200 mM Na(+) to 1M Na(+), and from 200 mM K(+) to 500 mM K(+). Furthermore, we validated the practicality of this probe in a clinical setting using 8 pairs of cancer tissue samples and their NT (corresponding adjacent nontumorous tissue) samples. All the results generated from the probe detection agreed with those from direct sequencing. Combining features of extreme high specificity and robustness, this probe is a valuable tool for reliable diagnosis of cancer-related mutations.

  15. ERCC1-XPF endonuclease facilitates DNA double-strand break repair.

    Science.gov (United States)

    Ahmad, Anwaar; Robinson, Andria Rasile; Duensing, Anette; van Drunen, Ellen; Beverloo, H Berna; Weisberg, David B; Hasty, Paul; Hoeijmakers, Jan H J; Niedernhofer, Laura J

    2008-08-01

    ERCC1-XPF endonuclease is required for nucleotide excision repair (NER) of helix-distorting DNA lesions. However, mutations in ERCC1 or XPF in humans or mice cause a more severe phenotype than absence of NER, prompting a search for novel repair activities of the nuclease. In Saccharomyces cerevisiae, orthologs of ERCC1-XPF (Rad10-Rad1) participate in the repair of double-strand breaks (DSBs). Rad10-Rad1 contributes to two error-prone DSB repair pathways: microhomology-mediated end joining (a Ku86-independent mechanism) and single-strand annealing. To determine if ERCC1-XPF participates in DSB repair in mammals, mutant cells and mice were screened for sensitivity to gamma irradiation. ERCC1-XPF-deficient fibroblasts were hypersensitive to gamma irradiation, and gammaH2AX foci, a marker of DSBs, persisted in irradiated mutant cells, consistent with a defect in DSB repair. Mutant mice were also hypersensitive to irradiation, establishing an essential role for ERCC1-XPF in protecting against DSBs in vivo. Mice defective in both ERCC1-XPF and Ku86 were not viable. However, Ercc1(-/-) Ku86(-/-) fibroblasts were hypersensitive to gamma irradiation compared to single mutants and accumulated significantly greater chromosomal aberrations. Finally, in vitro repair of DSBs with 3' overhangs led to large deletions in the absence of ERCC1-XPF. These data support the conclusion that, as in yeast, ERCC1-XPF facilitates DSB repair via an end-joining mechanism that is Ku86 independent.

  16. PARP-1 and Ku compete for repair of DNA double strand breaks by distinct NHEJ pathways

    Science.gov (United States)

    Wang, Minli; Wu, Weizhong; Wu, Wenqi; Rosidi, Bustanur; Zhang, Lihua; Wang, Huichen; Iliakis, George

    2006-01-01

    Poly(ADP-ribose)polymerase 1 (PARP-1) recognizes DNA strand interruptions in vivo and triggers its own modification as well as that of other proteins by the sequential addition of ADP-ribose to form polymers. This modification causes a release of PARP-1 from DNA ends and initiates a variety of responses including DNA repair. While PARP-1 has been firmly implicated in base excision and single strand break repair, its role in the repair of DNA double strand breaks (DSBs) remains unclear. Here, we show that PARP-1, probably together with DNA ligase III, operates in an alternative pathway of non-homologous end joining (NHEJ) that functions as backup to the classical pathway of NHEJ that utilizes DNA-PKcs, Ku, DNA ligase IV, XRCC4, XLF/Cernunnos and Artemis. PARP-1 binds to DNA ends in direct competition with Ku. However, in irradiated cells the higher affinity of Ku for DSBs and an excessive number of other forms of competing DNA lesions limit its contribution to DSB repair. When essential components of the classical pathway of NHEJ are absent, PARP-1 is recruited for DSB repair, particularly in the absence of Ku and non-DSB lesions. This form of DSB repair is sensitive to PARP-1 inhibitors. The results define the function of PARP-1 in DSB repair and characterize a candidate pathway responsible for joining errors causing genomic instability and cancer. PMID:17088286

  17. Mechanisms of DNA Packaging by Large Double-Stranded DNA Viruses.

    Science.gov (United States)

    Rao, Venigalla B; Feiss, Michael

    2015-11-01

    Translocation of viral double-stranded DNA (dsDNA) into the icosahedral prohead shell is catalyzed by TerL, a motor protein that has ATPase, endonuclease, and translocase activities. TerL, following endonucleolytic cleavage of immature viral DNA concatemer recognized by TerS, assembles into a pentameric ring motor on the prohead's portal vertex and uses ATP hydrolysis energy for DNA translocation. TerL's N-terminal ATPase is connected by a hinge to the C-terminal endonuclease. Inchworm models propose that modest domain motions accompanying ATP hydrolysis are amplified, through changes in electrostatic interactions, into larger movements of the C-terminal domain bound to DNA. In phage ϕ29, four of the five TerL subunits sequentially hydrolyze ATP, each powering translocation of 2.5 bp. After one viral genome is encapsidated, the internal pressure signals termination of packaging and ejection of the motor. Current focus is on the structures of packaging complexes and the dynamics of TerL during DNA packaging, endonuclease regulation, and motor mechanics.

  18. RECQL4 Promotes DNA End Resection in Repair of DNA Double-Strand Breaks

    DEFF Research Database (Denmark)

    Lu, Huiming; Shamanna, Raghavendra A; Keijzers, Guido

    2016-01-01

    The RecQ helicase RECQL4, mutated in Rothmund-Thomson syndrome, regulates genome stability, aging, and cancer. Here, we identify a crucial role for RECQL4 in DNA end resection, which is the initial and an essential step of homologous recombination (HR)-dependent DNA double-strand break repair (DSBR......). Depletion of RECQL4 severely reduces HR-mediated repair and 5' end resection in vivo. RECQL4 physically interacts with MRE11-RAD50-NBS1 (MRN), which senses DSBs and initiates DNA end resection with CtIP. The MRE11 exonuclease regulates the retention of RECQL4 at laser-induced DSBs. RECQL4 also directly...... interacts with CtIP via its N-terminal domain and promotes CtIP recruitment to the MRN complex at DSBs. Moreover, inactivation of RECQL4's helicase activity impairs DNA end processing and HR-dependent DSBR without affecting its interaction with MRE11 and CtIP, suggesting an important role for RECQL4's...

  19. Effects of Breast Shielding during Heart Imaging on DNA Double-Strand-Break Levels: A Prospective Randomized Controlled Trial.

    Science.gov (United States)

    Cheezum, Michael K; Redon, Christophe E; Burrell, Allison S; Kaviratne, Anthony S; Bindeman, Jody; Maeda, Daisuke; Balmakhtar, Houria; Pezel, Ashly; Wisniewski, Piotr; Delacruz, Panfilo; Nguyen, Binh; Bonner, William M; Villines, Todd C

    2016-10-01

    Purpose To examine the effect of breast shielding on blood lymphocyte deoxyribonucleic acid (DNA) double-strand-break levels resulting from in vivo radiation and ex vivo radiation at breast-tissue level, and the effect of breast shielding on image quality. Materials and Methods The study was approved by institutional review and commpliant with HIPAA guidelines. Adult women who underwent 64-section coronary computed tomographic (CT) angiography and who provided informed consent were prospectively randomized to the use (n = 50) or absence (n = 51) of bismuth breast shields. Peripheral blood samples were obtained before and 30 minutes after in vivo radiation during CT angiography to compare DNA double-strand-break levels by γ-H2AX immunofluorescence in blood lymphocytes. To estimate DNA double-strand-break induction at breast-tissue level, a blood sample was taped to the sternum for ex vivo radiation with or without shielding. Data were analyzed by linear regression and independent sample t tests. Results Breast shielding had no effect on DNA double-strand-break levels from ex vivo radiation of blood samples under shields at breast-tissue level (unadjusted regression: β = .08; P = .43 versus no shielding), or in vivo radiation of circulating lymphocytes (β = -.07; P = .50). Predictors of increased DNA double-strand-break levels included total radiation dose, increasing tube potential, and tube current (P radiation exposures (median, 3.4 mSv), breast shielding yielded a 33% increase in image noise and 19% decrease in the rate of excellent quality ratings. Conclusion Among women who underwent coronary CT angiography, breast shielding had no effect on DNA double-strand-break levels in blood lymphocytes exposed to in vivo radiation, or ex vivo radiation at breast-tissue level. At present relatively low radiation exposures, breast shielding contributed to an increase in image noise and a decline in image quality. The findings support efforts to minimize radiation by

  20. [Effect of single-stranded and double-stranded breaks on the melting temperature of phage T2 DNA].

    Science.gov (United States)

    Iurgaĭtis, A P; Lazurkin, Iu S; Bannikov, Iu A

    1979-01-01

    The effect of single- and double-stranded breaks in DNA phage T2, on the melting temperature of this DNA in the 0,05 M SSC solution, was investigated. The number of cleavages per 1000 nucleotide pairs varied in the range of 0 to 10. It is shown that single- and double-stranded breaks affect the melting temperature with approximately (within 20%) the same efficiency. The relationship between the melting temperature shift (delta Tm) and the number of cleavages is non-linear. The magnitude of the effect is characterized by delta Tm of 2 +/- 0.4 degrees C for the average inter-cleavage distance of 200 base pairs. It is shown that the observed melting curves are non-equilibrium ones, which is probably due to the fact that the effect of cleavages on the melting temperature is largely results from the complete and practically irreversible separation of strands.

  1. Positive regulation of DNA double strand break repair activity during differentiation of long life span cells: the example of adipogenesis.

    Directory of Open Access Journals (Sweden)

    Aline Meulle

    Full Text Available Little information is available on the ability of terminally differentiated cells to efficiently repair DNA double strand breaks (DSBs, and one might reasonably speculate that efficient DNA repair of these threatening DNA lesions, is needed in cells of long life span with no or limited regeneration from precursor. Few tissues are available besides neurons that allow the study of DNA DSBs repair activity in very long-lived cells. Adipocytes represent a suitable model since it is generally admitted that there is a very slow turnover of adipocytes in adult. Using both Pulse Field Gel Electrophoresis (PFGE and the disappearance of the phosphorylated form of the histone variant H2AX, we demonstrated that the ability to repair DSBs is increased during adipocyte differentiation using the murine pre-adipocyte cell line, 3T3F442A. In mammalian cells, DSBs are mainly repaired by the non-homologous end-joining pathway (NHEJ that relies on the DNA dependent protein kinase (DNA-PK activity. During the first 24 h following the commitment into adipogenesis, we show an increase in the expression and activity of the catalytic sub-unit of the DNA-PK complex, DNA-PKcs. The increased in DNA DSBs repair activity observed in adipocytes was due to the increase in DNA-PK activity as shown by the use of DNA-PK inhibitor or sub-clones of 3T3F442A deficient in DNA-PKcs using long term RNA interference. Interestingly, the up-regulation of DNA-PK does not regulate the differentiation program itself. Finally, similar positive regulation of DNA-PKcs expression and activity was observed during differentiation of primary culture of pre-adipocytes isolated from human sub-cutaneous adipose tissue. Our results show that DNA DSBs repair activity is up regulated during the early commitment into adipogenesis due to an up-regulation of DNA-PK expression and activity. In opposition to the general view that DNA DSBs repair is decreased during differentiation, our results demonstrate

  2. Oral delivery of double-stranded RNAs and siRNAs induces RNAi effects in the potato/tomato psyllid, Bactericerca cockerelli.

    Directory of Open Access Journals (Sweden)

    Hada Wuriyanghan

    Full Text Available The potato/tomato psyllid, Bactericerca cockerelli (B. cockerelli, and the Asian citrus psyllid, Diaphorina citri (D. citri, are very important plant pests, but they are also vectors of phloem-limited bacteria that are associated with two devastating plant diseases. B. cockerelli is the vector of Candidatus Liberibacter psyllaurous (solanacearum, which is associated with zebra chip disease of potatoes, and D. citri is the vector of Ca. Liberibacter asiaticus, which is associated with the Huanglongbing (citrus greening disease that currently threatens the entire Florida citrus industry. Here we used EST sequence information from D. citri to identify potential targets for RNA interference in B. cockerelli. We targeted ubiquitously expressed and gut-abundant mRNAs via injection and oral acquisition of double-stranded RNAs and siRNAs and were able to induce mortality in recipient psyllids. We also showed knockdown of target mRNAs, and that oral acquisition resulted primarily in mRNA knockdown in the psyllid gut. Concurrent with gene knockdown was the accumulation of target specific ∼ 21 nucleotide siRNAs for an abundant mRNA for BC-Actin. These results showed that RNAi can be a powerful tool for gene function studies in psyllids, and give support for continued efforts for investigating RNAi approaches as possible tools for psyllid and plant disease control.

  3. Synthetic lethal targeting of DNA double strand break repair deficient cells by human apurinic/apyrimidinic endonuclease (APE1) inhibitors

    OpenAIRE

    Sultana, Rebeka; McNeill, Daniel R.; Abbotts, Rachel; Mohammed, Mohammed Z.; Zdzienicka, Małgorzata Z.; Qutob, Haitham; Seedhouse, Claire; Charles A. Laughton; Fischer, Peter M.; Patel, Poulam M.; Wilson, David M.; Madhusudan, Srinivasan

    2012-01-01

    An apurinic/apyrimidinic (AP) site is an obligatory cytotoxic intermediate in DNA Base Excision Repair (BER) that is processed by human AP endonuclease 1 (APE1). APE1 is essential for BER and an emerging drug target in cancer. We have isolated novel small molecule inhibitors of APE1. In the current study we have investigated the ability of APE1 inhibitors to induce synthetic lethality in a panel of DNA double strand break (DSB) repair deficient and proficient cells; a) Chine...

  4. Conservative Repair of a Chromosomal Double-Strand Break by Single-Strand DNA through Two Steps of Annealing▿ †

    OpenAIRE

    Storici, Francesca; Snipe, Joyce R.; Chan, Godwin K.; Dmitry A Gordenin; Michael A Resnick

    2006-01-01

    The repair of chromosomal double-strand breaks (DSBs) is essential to normal cell growth, and homologous recombination is a universal process for DSB repair. We explored DSB repair mechanisms in the yeast Saccharomyces cerevisiae using single-strand oligonucleotides with homology to both sides of a DSB. Oligonucleotide-directed repair occurred exclusively via Rad52- and Rad59-mediated single-strand annealing (SSA). Even the SSA domain of human Rad52 provided partial complementation for a null...

  5. Lack of dependence on p53 for DNA double strand break repair of episomal vectors in human lymphoblasts

    Science.gov (United States)

    Kohli, M.; Jorgensen, T. J.

    1999-01-01

    The p53 tumor suppressor gene has been shown to be involved in a variety of repair processes, and recent findings have suggested that p53 may be involved in DNA double strand break repair in irradiated cells. The role of p53 in DNA double strand break repair, however, has not been fully investigated. In this study, we have constructed a novel Epstein-Barr virus (EBV)-based shuttle vector, designated as pZEBNA, to explore the influence of p53 on DNA strand break repair in human lymphoblasts, since EBV-based vectors do not inactivate the p53 pathway. We have compared plasmid survival of irradiated, restriction enzyme linearized, and calf intestinal alkaline phosphatase (CIP)-treated pZEBNA with a Simian virus 40 (SV40)-based shuttle vector, pZ189, in TK6 (wild-type p53) and WTK1 (mutant p53) lymphoblasts and determined that p53 does not modulate DNA double strand break repair in these cell lines. Copyright 1999 Academic Press.

  6. The adsorption-desorption transition of double-stranded DNA interacting with an oppositely charged dendrimer induced by multivalent anions.

    Science.gov (United States)

    Jiang, Yangwei; Zhang, Dong; Zhang, Yaoyang; Deng, Zhenyu; Zhang, Linxi

    2014-05-28

    The adsorption-desorption transition of DNA in DNA-dendrimer solutions is observed when high-valence anions, such as hexavalent anions, are added to the DNA-dendrimer solutions. In the DNA-dendrimer solutions with low-valence anions, dendrimers bind tightly with the V-shaped double-stranded DNA. When high-valence anions, such as pentavalent or hexavalent anions, are added to the DNA-dendrimer solutions, the double-stranded DNA chains can be stretched straightly and the dendrimers are released from the double-stranded DNA chains. In fact, adding high-valence anions to the solutions can change the charge spatial distribution in the DNA-dendrimer solutions, and weaken the electrostatic interactions between the positively charged dendrimers and the oppositely charged DNA chains. Adsorption-desorption transition of DNA is induced by the overcharging of dendrimers. This investigation is capable of helping us understand how to control effectively the release of DNA in gene/drug delivery because an effective gene delivery for dendrimers includes non-covalent DNA-dendrimer binding and the effective release of DNA in gene therapy.

  7. Hypermutability of damaged single-strand DNA formed at double-strand breaks and uncapped telomeres in yeast Saccharomyces cerevisiae.

    Directory of Open Access Journals (Sweden)

    Yong Yang

    2008-11-01

    Full Text Available The major DNA repair pathways operate on damage in double-strand DNA because they use the intact strand as a template after damage removal. Therefore, lesions in transient single-strand stretches of chromosomal DNA are expected to be especially threatening to genome stability. To test this hypothesis, we designed systems in budding yeast that could generate many kilobases of persistent single-strand DNA next to double-strand breaks or uncapped telomeres. The systems allowed controlled restoration to the double-strand state after applying DNA damage. We found that lesions induced by UV-light and methyl methanesulfonate can be tolerated in long single-strand regions and are hypermutagenic. The hypermutability required PCNA monoubiquitination and was largely attributable to translesion synthesis by the error-prone DNA polymerase zeta. In support of multiple lesions in single-strand DNA being a source of hypermutability, analysis of the UV-induced mutants revealed strong strand-specific bias and unexpectedly high frequency of alleles with widely separated multiple mutations scattered over several kilobases. Hypermutability and multiple mutations associated with lesions in transient stretches of long single-strand DNA may be a source of carcinogenesis and provide selective advantage in adaptive evolution.

  8. Different fates of oocytes with DNA double-strand breaks in vitro and in vivo.

    Science.gov (United States)

    Lin, Fei; Ma, Xue-Shan; Wang, Zhen-Bo; Wang, Zhong-Wei; Luo, Yi-Bo; Huang, Lin; Jiang, Zong-Zhe; Hu, Meng-Wen; Schatten, Heide; Sun, Qing-Yuan

    2014-01-01

    In female mice, despite the presence of slight DNA double-strand breaks (DSBs), fully grown oocytes are able to undergo meiosis resumption as indicated by germinal vesicle breakdown (GVBD); however, severe DNA DSBs do reduce and delay entry into M phase through activation of the DNA damage checkpoint. But little is known about the effect of severe DNA DSBs on the spindle assembly checkpoint (SAC) during oocyte maturation. We showed that nearly no first polar body (PB1) was extruded at 12 h of in vitro maturation (IVM) in severe DNA DSBs oocytes, and the limited number of oocytes with PB1 were actually at telophase. However, about 60% of the severe DNA DSBs oocytes which underwent GVBD at 2 h of IVM released a PB1 at 18 h of IVM and these oocytes did reach the second metaphase (MII) stage. Chromosome spread at MI and MII stages showed that chromosomes fragmented after GVBD in severe DNA DSBs oocytes. The delayed PB1 extrusion was due to the disrupted attachment of microtubules to kinetochores and activation of the SAC. At the same time, misaligned chromosome fragments became obvious at the first metaphase (MI) in severe DNA DSBs oocytes. These data implied that the inactivation of SAC during the metaphase-anaphase transition of first meiosis was independent of chromosome integrity. Next, we induced DNA DSBs in vivo, and found that the number of superovulated oocytes per mouse was significantly reduced; moreover, this treatment increased the percentage of apoptotic oocytes. These results suggest that DNA DSBs oocytes undergo apoptosis in vivo.

  9. Dynamics of a double-stranded DNA segment in a shear flow

    Science.gov (United States)

    Panja, Debabrata; Barkema, Gerard T.; van Leeuwen, J. M. J.

    2016-04-01

    We study the dynamics of a double-stranded DNA (dsDNA) segment, as a semiflexible polymer, in a shear flow, the strength of which is customarily expressed in terms of the dimensionless Weissenberg number Wi. Polymer chains in shear flows are well known to undergo tumbling motion. When the chain lengths are much smaller than the persistence length, one expects a (semiflexible) chain to tumble as a rigid rod. At low Wi, a polymer segment shorter than the persistence length does indeed tumble as a rigid rod. However, for higher Wi the chain does not tumble as a rigid rod, even if the polymer segment is shorter than the persistence length. In particular, from time to time the polymer segment may assume a buckled form, a phenomenon commonly known as Euler buckling. Using a bead-spring Hamiltonian model for extensible dsDNA fragments, we first analyze Euler buckling in terms of the oriented deterministic state (ODS), which is obtained as the steady-state solution of the dynamical equations by turning off the stochastic (thermal) forces at a fixed orientation of the chain. The ODS exhibits symmetry breaking at a critical Weissenberg number Wic, analogous to a pitchfork bifurcation in dynamical systems. We then follow up the analysis with simulations and demonstrate symmetry breaking in computer experiments, characterized by a unimodal to bimodal transformation of the probability distribution of the second Rouse mode with increasing Wi. Our simulations reveal that shear can cause strong deformation for a chain that is shorter than its persistence length, similar to recent experimental observations.

  10. DNA double-strand breaks activate ATM independent of mitochondrial dysfunction in A549 cells.

    Science.gov (United States)

    Kalifa, Lidza; Gewandter, Jennifer S; Staversky, Rhonda J; Sia, Elaine A; Brookes, Paul S; O'Reilly, Michael A

    2014-10-01

    Excessive nuclear or mitochondrial DNA damage can lead to mitochondrial dysfunction, decreased energy production, and increased generation of reactive oxygen species (ROS). Although numerous cell signaling pathways are activated when cells are injured, the ataxia telangiectasia mutant (ATM) protein has emerged as a major regulator of the response to both mitochondrial dysfunction and nuclear DNA double-strand breaks (DSBs). Because mitochondrial dysfunction is often a response to excessive DNA damage, it has been difficult to determine whether nuclear and/or mitochondrial DNA DSBs activate ATM independent of mitochondrial dysfunction. In this study, mitochondrial and nuclear DNA DSBs were generated in the A549 human lung adenocarcinoma cell line by infecting with retroviruses expressing the restriction endonuclease PstI fused to a mitochondrial targeting sequence (MTS) or nuclear localization sequence (NLS) and a hemagglutinin antigen epitope tag (HA). Expression of MTS-PstI-HA or NLS-PstI-HA activated the DNA damage response defined by phosphorylation of ATM, the tumor suppressor protein p53 (TP53), KRAB-associated protein (KAP)-1, and structural maintenance of chromosomes (SMC)-1. Phosphorylated ATM and SMC1 were detected in nuclear fractions, whereas phosphorylated TP53 and KAP1 were detected in both mitochondrial and nuclear fractions. PstI also enhanced expression of the cyclin-dependent kinase inhibitor p21 and inhibited cell growth. This response to DNA damage occurred in the absence of detectable mitochondrial dysfunction and excess production of ROS. These findings reveal that DNA DSBs are sufficient to activate ATM independent of mitochondrial dysfunction and suggest that the activated form of ATM and some of its substrates are restricted to the nuclear compartment, regardless of the site of DNA damage.

  11. DNA Double Strand Break Response and Limited Repair Capacity in Mouse Elongated Spermatids

    Directory of Open Access Journals (Sweden)

    Emad A. Ahmed

    2015-12-01

    Full Text Available Spermatids are extremely sensitive to genotoxic exposures since during spermiogenesis only error-prone non homologous end joining (NHEJ repair pathways are available. Hence, genomic damage may accumulate in sperm and be transmitted to the zygote. Indirect, delayed DNA fragmentation and lesions associated with apoptotic-like processes have been observed during spermatid elongation, 27 days after irradiation. The proliferating spermatogonia and early meiotic prophase cells have been suggested to retain a memory of a radiation insult leading later to this delayed fragmentation. Here, we used meiotic spread preparations to localize phosphorylate histone H2 variant (γ-H2AX foci marking DNA double strand breaks (DSBs in elongated spermatids. This technique enabled us to determine the background level of DSB foci in elongated spermatids of RAD54/RAD54B double knockout (dko mice, severe combined immunodeficiency SCID mice, and poly adenosine diphosphate (ADP-ribose polymerase 1 (PARP1 inhibitor (DPQ-treated mice to compare them with the appropriate wild type controls. The repair kinetics data and the protein expression patterns observed indicate that the conventional NHEJ repair pathway is not available for elongated spermatids to repair the programmed and the IR-induced DSBs, reflecting the limited repair capacity of these cells. However, although elongated spermatids express the proteins of the alternative NHEJ, PARP1-inhibition had no effect on the repair kinetics after IR, suggesting that DNA damage may be passed onto sperm. Finally, our genetic mutant analysis suggests that an incomplete or defective meiotic recombinational repair of Spo11-induced DSBs may lead to a carry-over of the DSB damage or induce a delayed nuclear fragmentation during the sensitive programmed chromatin remodeling occurring in elongated spermatids.

  12. Role of Double-Strand Break End-Tethering during Gene Conversion in Saccharomyces cerevisiae.

    Directory of Open Access Journals (Sweden)

    Suvi Jain

    2016-04-01

    Full Text Available Correct repair of DNA double-strand breaks (DSBs is critical for maintaining genome stability. Whereas gene conversion (GC-mediated repair is mostly error-free, repair by break-induced replication (BIR is associated with non-reciprocal translocations and loss of heterozygosity. We have previously shown that a Recombination Execution Checkpoint (REC mediates this competition by preventing the BIR pathway from acting on DSBs that can be repaired by GC. Here, we asked if the REC can also determine whether the ends that are engaged in a GC-compatible configuration belong to the same break, since repair involving ends from different breaks will produce potentially deleterious translocations. We report that the kinetics of repair are markedly delayed when the two DSB ends that participate in GC belong to different DSBs (termed Trans compared to the case when both DSB ends come from the same break (Cis. However, repair in Trans still occurs by GC rather than BIR, and the overall efficiency of repair is comparable. Hence, the REC is not sensitive to the "origin" of the DSB ends. When the homologous ends for GC are in Trans, the delay in repair appears to reflect their tethering to sequences on the other side of the DSB that themselves recombine with other genomic locations with which they share sequence homology. These data support previous observations that the two ends of a DSB are usually tethered to each other and that this tethering facilitates both ends encountering the same donor sequence. We also found that the presence of homeologous/repetitive sequences in the vicinity of a DSB can distract the DSB end from finding its bona fide homologous donor, and that inhibition of GC by such homeologous sequences is markedly increased upon deleting Sgs1 but not Msh6.

  13. Tying the loose ends together in DNA double strand break repair with 53BP1

    Directory of Open Access Journals (Sweden)

    Carpenter Phillip B

    2006-08-01

    Full Text Available Abstract To maintain genomic stability and ensure the fidelity of chromosomal transmission, cells respond to various forms of genotoxic stress, including DNA double-stranded breaks (DSBs, through the activation of DNA damage response signaling networks. In response to DSBs as induced by ionizing radiation (IR, during DNA replication, or through immunoglobulin heavy chain (IgH rearrangements in B cells of lymphoid origin, the phosphatidyl inositol-like kinase (PIK kinases ATM (mutated in ataxia telangiectasia, ATR (ATM and Rad3-related kinase, and the DNA-dependent protein kinase (DNA-PK activate signaling pathways that lead to DSB repair. DSBs are repaired by either of two major, non-mutually exclusive pathways: homologous recombination (HR that utilizes an undamaged sister chromatid template (or homologous chromosome and non- homologous end joining (NHEJ, an error prone mechanism that processes and joins broken DNA ends through the coordinated effort of a small set of ubiquitous factors (DNA-PKcs, Ku70, Ku80, artemis, Xrcc4/DNA lig IV, and XLF/Cernunnos. The PIK kinases phosphorylate a variety of effector substrates that propagate the DNA damage signal, ultimately resulting in various biological outputs that influence cell cycle arrest, transcription, DNA repair, and apoptosis. A variety of data has revealed a critical role for p53-binding protein 1 (53BP1 in the cellular response to DSBs including various aspects of p53 function. Importantly, 53BP1 plays a major role in suppressing translocations, particularly in B and T cells. This report will review past experiments and current knowledge regarding the role of 53BP1 in the DNA damage response.

  14. Fine resolution mapping of double-strand break sites for human ribosomal DNA units

    Directory of Open Access Journals (Sweden)

    Bernard J. Pope

    2016-12-01

    Full Text Available DNA breakage arises during a variety of biological processes, including transcription, replication and genome rearrangements. In the context of disease, extensive fragmentation of DNA has been described in cancer cells and during early stages of neurodegeneration (Stephens et al., 2011 Stephens et al. (2011 [5]; Blondet et al., 2001 Blondet et al. (2001 [1]. Stults et al. (2009 Stults et al. (2009 [6] reported that human rDNA gene clusters are hotspots for recombination and that rDNA restructuring is among the most common chromosomal alterations in adult solid tumours. As such, analysis of rDNA regions is likely to have significant prognostic and predictive value, clinically. Tchurikov et al. (2015a, 2016 Tchurikov et al. (2015a, 2016 [7,9] have made major advances in this direction, reporting that sites of human genome double-strand breaks (DSBs occur frequently at sites in rDNA that are tightly linked with active transcription - the authors used a RAFT (rapid amplification of forum termini protocol that selects for blunt-ended sites. They reported the relative frequency of these rDNA DSBs within defined co-ordinate ‘windows’ of varying size and made these data (as well as the relevant ‘raw’ sequencing information available to the public (Tchurikov et al., 2015b. Assay designs targeting rDNA DSB hotspots will benefit greatly from the publication of break sites at greater resolution. Here, we re-analyse public RAFT data and make available rDNA DSB co-ordinates to the single-nucleotide level.

  15. Chromatin modification and NBS1: their relationship in DNA double-strand break repair.

    Science.gov (United States)

    Saito, Yuichiro; Zhou, Hui; Kobayashi, Junya

    2016-01-01

    The importance of chromatin modification, including histone modification and chromatin remodeling, for DNA double-strand break (DSB) repair, as well as transcription and replication, has been elucidated. Phosphorylation of H2AX to γ-H2AX is one of the first responses following DSB detection, and this histone modification is important for the DSB damage response by triggering several events, including the accumulation of DNA damage response-related proteins and subsequent homologous recombination (HR) repair. The roles of other histone modifications such as acetylation, methylation and ubiquitination have also been recently clarified, particularly in the context of HR repair. NBS1 is a multifunctional protein that is involved in various DNA damage responses. Its recently identified binding partner RNF20 is an E3 ubiquitin ligase that facilitates the monoubiquitination of histone H2B, a process that is crucial for recruitment of the chromatin remodeler SNF2h to DSB damage sites. Evidence suggests that SNF2h functions in HR repair, probably through regulation of end-resection. Moreover, several recent reports have indicated that SNF2h can function in HR repair pathways as a histone remodeler and that other known histone remodelers can also participate in DSB damage responses. On the other hand, information about the roles of such chromatin modifications and NBS1 in non-homologous end joining (NHEJ) repair of DSBs and stalled fork-related damage responses is very limited; therefore, these aspects and processes need to be further studied to advance our understanding of the mechanisms and molecular players involved.

  16. Preferential repair of DNA double-strand break at the active gene in vivo.

    Science.gov (United States)

    Chaurasia, Priyasri; Sen, Rwik; Pandita, Tej K; Bhaumik, Sukesh R

    2012-10-19

    Previous studies have demonstrated transcription-coupled nucleotide/base excision repair. We report here for the first time that DNA double-strand break (DSB) repair is also coupled to transcription. We generated a yeast strain by introducing a homing (Ho) endonuclease cut site followed by a nucleotide sequence for multiple Myc epitopes at the 3' end of the coding sequence of a highly active gene, ADH1. This yeast strain also contains the Ho cut site at the nearly silent or poorly active mating type α (MATα) locus and expresses Ho endonuclease under the galactose-inducible GAL1 promoter. Using this strain, DSBs were generated at the ADH1 and MATα loci in galactose-containing growth medium that induced HO expression. Subsequently, yeast cells were transferred to dextrose-containing growth medium to stop HO expression, and the DSB repair was monitored at the ADH1 and MATα loci by PCR, using the primer pairs flanking the Ho cut sites. Our results revealed a faster DSB repair at the highly active ADH1 than that at the nearly silent MATα locus, hence implicating a transcription-coupled DSB repair at the active gene in vivo. Subsequently, we extended this study to another gene, PHO5 (carrying the Ho cut site at its coding sequence), under transcriptionally active and inactive growth conditions. We found a fast DSB repair at the active PHO5 gene in comparison to its inactive state. Collectively, our results demonstrate a preferential DSB repair at the active gene, thus supporting transcription-coupled DSB repair in living cells.

  17. Deregulation of DNA double-strand break repair in multiple myeloma: implications for genome stability.

    Directory of Open Access Journals (Sweden)

    Ana B Herrero

    Full Text Available Multiple myeloma (MM is a hematological malignancy characterized by frequent chromosome abnormalities. However, the molecular basis for this genome instability remains unknown. Since both impaired and hyperactive double strand break (DSB repair pathways can result in DNA rearrangements, we investigated the functionality of DSB repair in MM cells. Repair kinetics of ionizing-radiation (IR-induced DSBs was similar in MM and normal control lymphoblastoid cell lines, as revealed by the comet assay. However, four out of seven MM cell lines analyzed exhibited a subset of persistent DSBs, marked by γ-H2AX and Rad51 foci that elicited a prolonged G2/M DNA damage checkpoint activation and hypersensitivity to IR, especially in the presence of checkpoint inhibitors. An analysis of the proteins involved in DSB repair in MM cells revealed upregulation of DNA-PKcs, Artemis and XRCC4, that participate in non-homologous end joining (NHEJ, and Rad51, involved in homologous recombination (HR. Accordingly, activity of both NHEJ and HR were elevated in MM cells compared to controls, as determined by in vivo functional assays. Interestingly, levels of proteins involved in a highly mutagenic, translocation-promoting, alternative NHEJ subpathway (Alt-NHEJ were also increased in all MM cell lines, with the Alt-NHEJ protein DNA ligase IIIα, also overexpressed in several plasma cell samples isolated from MM patients. Overactivation of the Alt-NHEJ pathway was revealed in MM cells by larger deletions and higher sequence microhomology at repair junctions, which were reduced by chemical inhibition of the pathway. Taken together, our results uncover a deregulated DSB repair in MM that might underlie the characteristic genome instability of the disease, and could be therapeutically exploited.

  18. Repair at single targeted DNA double-strand breaks in pluripotent and differentiated human cells.

    Directory of Open Access Journals (Sweden)

    Hua Fung

    Full Text Available Differences in ex vivo cell culture conditions can drastically affect stem cell physiology. We sought to establish an assay for measuring the effects of chemical, environmental, and genetic manipulations on the precision of repair at a single DNA double-strand break (DSB in pluripotent and somatic human cells. DSBs in mammalian cells are primarily repaired by either homologous recombination (HR or nonhomologous end-joining (NHEJ. For the most part, previous studies of DSB repair in human cells have utilized nonspecific clastogens like ionizing radiation, which are highly nonphysiologic, or assayed repair at randomly integrated reporters. Measuring repair after random integration is potentially confounded by locus-specific effects on the efficiency and precision of repair. We show that the frequency of HR at a single DSB differs up to 20-fold between otherwise isogenic human embryonic stem cells (hESCs based on the site of the DSB within the genome. To overcome locus-specific effects on DSB repair, we used zinc finger nucleases to efficiently target a DSB repair reporter to a safe-harbor locus in hESCs and a panel of somatic human cell lines. We demonstrate that repair at a targeted DSB is highly precise in hESCs, compared to either the somatic human cells or murine embryonic stem cells. Differentiation of hESCs harboring the targeted reporter into astrocytes reduces both the efficiency and precision of repair. Thus, the phenotype of repair at a single DSB can differ based on either the site of damage within the genome or the stage of cellular differentiation. Our approach to single DSB analysis has broad utility for defining the effects of genetic and environmental modifications on repair precision in pluripotent cells and their differentiated progeny.

  19. The Transcriptional Response to DNA-Double-Strand Breaks in Physcomitrella patens

    Science.gov (United States)

    Kamisugi, Yasuko; Whitaker, John W.

    2016-01-01

    The model bryophyte Physcomitrella patens is unique among plants in supporting the generation of mutant alleles by facile homologous recombination-mediated gene targeting (GT). Reasoning that targeted transgene integration occurs through the capture of transforming DNA by the homology-dependent pathway for DNA double-strand break (DNA-DSB) repair, we analysed the genome-wide transcriptomic response to bleomycin-induced DNA damage and generated mutants in candidate DNA repair genes. Massively parallel (Illumina) cDNA sequencing identified potential participants in gene targeting. Transcripts encoding DNA repair proteins active in multiple repair pathways were significantly up-regulated. These included Rad51, CtIP, DNA ligase 1, Replication protein A and ATR in homology-dependent repair, Xrcc4, DNA ligase 4, Ku70 and Ku80 in non-homologous end-joining and Rad1, Tebichi/polymerase theta, PARP in microhomology-mediated end-joining. Differentially regulated cell-cycle components included up-regulated Rad9 and Hus1 DNA-damage-related checkpoint proteins and down-regulated D-type cyclins and B-type CDKs, commensurate with the imposition of a checkpoint at G2 of the cell cycle characteristic of homology-dependent DNA-DSB repair. Candidate genes, including ATP-dependent chromatin remodelling helicases associated with repair and recombination, were knocked out and analysed for growth defects, hypersensitivity to DNA damage and reduced GT efficiency. Targeted knockout of PpCtIP, a cell-cycle activated mediator of homology-dependent DSB resection, resulted in bleomycin-hypersensitivity and greatly reduced GT efficiency. PMID:27537368

  20. ERCC1-XPF Endonuclease Facilitates DNA Double-Strand Break Repair▿ †

    Science.gov (United States)

    Ahmad, Anwaar; Robinson, Andria Rasile; Duensing, Anette; van Drunen, Ellen; Beverloo, H. Berna; Weisberg, David B.; Hasty, Paul; Hoeijmakers, Jan H. J.; Niedernhofer, Laura J.

    2008-01-01

    ERCC1-XPF endonuclease is required for nucleotide excision repair (NER) of helix-distorting DNA lesions. However, mutations in ERCC1 or XPF in humans or mice cause a more severe phenotype than absence of NER, prompting a search for novel repair activities of the nuclease. In Saccharomyces cerevisiae, orthologs of ERCC1-XPF (Rad10-Rad1) participate in the repair of double-strand breaks (DSBs). Rad10-Rad1 contributes to two error-prone DSB repair pathways: microhomology-mediated end joining (a Ku86-independent mechanism) and single-strand annealing. To determine if ERCC1-XPF participates in DSB repair in mammals, mutant cells and mice were screened for sensitivity to gamma irradiation. ERCC1-XPF-deficient fibroblasts were hypersensitive to gamma irradiation, and γH2AX foci, a marker of DSBs, persisted in irradiated mutant cells, consistent with a defect in DSB repair. Mutant mice were also hypersensitive to irradiation, establishing an essential role for ERCC1-XPF in protecting against DSBs in vivo. Mice defective in both ERCC1-XPF and Ku86 were not viable. However, Ercc1−/− Ku86−/− fibroblasts were hypersensitive to gamma irradiation compared to single mutants and accumulated significantly greater chromosomal aberrations. Finally, in vitro repair of DSBs with 3′ overhangs led to large deletions in the absence of ERCC1-XPF. These data support the conclusion that, as in yeast, ERCC1-XPF facilitates DSB repair via an end-joining mechanism that is Ku86 independent. PMID:18541667

  1. Mouse RAD54 Affects DNA Double-Strand Break Repair and Sister Chromatid Exchange

    Science.gov (United States)

    Dronkert, Mies L. G.; Beverloo, H. Berna; Johnson, Roger D.; Hoeijmakers, Jan H. J.; Jasin, Maria; Kanaar, Roland

    2000-01-01

    Cells can achieve error-free repair of DNA double-strand breaks (DSBs) by homologous recombination through gene conversion with or without crossover. In contrast, an alternative homology-dependent DSB repair pathway, single-strand annealing (SSA), results in deletions. In this study, we analyzed the effect of mRAD54, a gene involved in homologous recombination, on the repair of a site-specific I-SceI-induced DSB located in a repeated DNA sequence in the genome of mouse embryonic stem cells. We used six isogenic cell lines differing solely in the orientation of the repeats. The combination of the three recombination-test substrates used discriminated among SSA, intrachromatid gene conversion, and sister chromatid gene conversion. DSB repair was most efficient for the substrate that allowed recovery of SSA events. Gene conversion with crossover, indistinguishable from long tract gene conversion, preferentially involved the sister chromatid rather than the repeat on the same chromatid. Comparing DSB repair in mRAD54 wild-type and knockout cells revealed direct evidence for a role of mRAD54 in DSB repair. The substrate measuring SSA showed an increased efficiency of DSB repair in the absence of mRAD54. The substrate measuring sister chromatid gene conversion showed a decrease in gene conversion with and without crossover. Consistent with this observation, DNA damage-induced sister chromatid exchange was reduced in mRAD54-deficient cells. Our results suggest that mRAD54 promotes gene conversion with predominant use of the sister chromatid as the repair template at the expense of error-prone SSA. PMID:10757799

  2. Recognition of double strand breaks by a mutator protein (MU2 in Drosophila melanogaster.

    Directory of Open Access Journals (Sweden)

    Raghuvar Dronamraju

    2009-05-01

    Full Text Available Telomere capture, a rare event that stabilizes chromosome breaks, is associated with certain genetic abnormalities in humans. Studies pertaining to the generation, maintenance, and biological effects of telomere formation are limited in metazoans. A mutation, mu2(a, in Drosophila melanogaster decreases the rate of repair of double strand DNA breaks in oocytes, thus leading to chromosomes that have lost a natural telomere and gained a new telomere. Amino acid sequence, domain architecture, and protein interactions suggest that MU2 is an ortholog of human MDC1. The MU2 protein is a component of meiotic recombination foci and localizes to repair foci in S2 cells after irradiation in a manner similar to that of phosphorylated histone variant H2Av. Domain searches indicated that the protein contains an N-terminal FHA domain and a C-terminal tandem BRCT domain. Peptide pull-down studies showed that the BRCT domain interacts with phosphorylated H2Av, while the FHA domain interacts with the complex of MRE11, RAD50, and NBS. A frameshift mutation that eliminates the MU2 BRCT domain decreases the number and size of meiotic phospho-H2Av foci. MU2 is also required for the intra-S checkpoint in eye-antennal imaginal discs. MU2 participates at an early stage in the recognition of DNA damage at a step that is prerequisite for both DNA repair and cell cycle checkpoint control. We propose a model suggesting that neotelomeres may arise when radiation-induced chromosome breaks fail to be repaired, fail to arrest progression through meiosis, and are deposited in the zygote, where cell cycle control is absent and rapid rounds of replication and telomere formation ensue.

  3. Replication independent DNA double-strand break retention may prevent genomic instability

    Directory of Open Access Journals (Sweden)

    Pornthanakasem Wichai

    2010-03-01

    Full Text Available Abstract Background Global hypomethylation and genomic instability are cardinal features of cancers. Recently, we established a method for the detection of DNA methylation levels at sites close to endogenous DNA double strand breaks (EDSBs, and found that those sites have a higher level of methylation than the rest of the genome. Interestingly, the most significant differences between EDSBs and genomes were observed when cells were cultured in the absence of serum. DNA methylation levels on each genomic location are different. Therefore, there are more replication-independent EDSBs (RIND-EDSBs located in methylated genomic regions. Moreover, methylated and unmethylated RIND-EDSBs are differentially processed. Euchromatins respond rapidly to DSBs induced by irradiation with the phosphorylation of H2AX, γ-H2AX, and these initiate the DSB repair process. During G0, most DSBs are repaired by non-homologous end-joining repair (NHEJ, mediated by at least two distinct pathways; the Ku-mediated and the ataxia telangiectasia-mutated (ATM-mediated. The ATM-mediated pathway is more precise. Here we explored how cells process methylated RIND-EDSBs and if RIND-EDSBs play a role in global hypomethylation-induced genomic instability. Results We observed a significant number of methylated RIND-EDSBs that are retained within deacetylated chromatin and free from an immediate cellular response to DSBs, the γ-H2AX. When cells were treated with tricostatin A (TSA and the histones became hyperacetylated, the amount of γ-H2AX-bound DNA increased and the retained RIND-EDSBs were rapidly repaired. When NHEJ was simultaneously inhibited in TSA-treated cells, more EDSBs were detected. Without TSA, a sporadic increase in unmethylated RIND-EDSBs could be observed when Ku-mediated NHEJ was inhibited. Finally, a remarkable increase in RIND-EDSB methylation levels was observed when cells were depleted of ATM, but not of Ku86 and RAD51. Conclusions Methylated RIND-EDSBs are

  4. Parp1-XRCC1 and the repair of DNA double strand breaks in mouse round spermatids

    Energy Technology Data Exchange (ETDEWEB)

    Ahmed, Emad A. [Department of Endocrinology and Metabolism, Faculty of Science, Utrecht University, Padualaan 8, 3584 CH Utrecht (Netherlands); Boer, Peter de [Department of Obstetrics and Gynaecology, Radboud University Nijmegen Medical Centre, 6500 HB Nijmegen (Netherlands); Philippens, Marielle E.P.; Kal, Henk B. [Department of Radiotherapy, University Medical Center Utrecht, Heidelberglaan 100, 3584 CX Utrecht (Netherlands); Rooij, Dirk G. de, E-mail: d.g.derooij@uu.nl [Department of Endocrinology and Metabolism, Faculty of Science, Utrecht University, Padualaan 8, 3584 CH Utrecht (Netherlands); Center for Reproductive Medicine, Academic Medical Center, University of Amsterdam, 1105 AZ Amsterdam (Netherlands)

    2010-01-05

    The repair of DNA double strand breaks (DSBs) in male germ cells is slower and differently regulated compared to that in somatic cells. Round spermatids show DSB repair and are radioresistant to apoptosis induction. Mutation induction studies using ionizing irradiation, indicated a high frequency of chromosome aberrations (CA) in the next generation. Since they are in a G1 comparable stage of the cell cycle, haploid spermatids are expected to repair DSBs by the non-homologous end-joining pathway (NHEJ). However, immunohistochemical evidence indicates that not all components of the classical NHEJ pathway are available since the presence of DNA-PKcs cannot be shown. Here, we demonstrate that round spermatids, as well as most other types of male germ cells express both Parp1 and XRCC1. Therefore, we have determined whether the alternative Parp1/XRCC1 dependent NHEJ pathway is active in these nuclei and also have tested for classical NHEJ activity by a genetic method. To evaluate DSB repair in SCID mice, deficient for DNA-PKcs, and to study the involvement of the Parp1/XRCC1 dependent NHEJ pathway in round spermatids, the loss of {gamma}-H2AX foci after irradiation has been determined in nucleus spreads of round spermatids of SCID mice and in nucleus spreads and histological sections of Parp1-inhibited mice and their respective controls. Results show that around half of the breaks in randomly selected round spermatids are repaired between 1 and 8 h after irradiation. The repair of 16% of the induced DSBs requires DNA-PKcs and 21% Parp1. Foci numbers in the Parp1-inhibited testes tend to be higher in spermatids of all epithelial stages reaching significance in stages I-III which indicates an active Parp1/XRCC1 pathway in round spermatids and a decreased repair capacity in later round spermatid stages. In Parp1-inhibited SCID mice only 14.5% of the breaks were repaired 8 h after irradiation indicating additivity of the two NHEJ pathways in round spermatids.

  5. DNA double strand break repair enzymes function at multiple steps in retroviral infection

    Directory of Open Access Journals (Sweden)

    Agematsu Kazunaga

    2009-12-01

    Full Text Available Abstract Background DNA double strand break (DSB repair enzymes are thought to be necessary for retroviral infection, especially for the post-integration repair and circularization of viral cDNA. However, the detailed roles of DSB repair enzymes in retroviral infection remain to be elucidated. Results A GFP reporter assay showed that the infectivity of an HIV-based vector decreased in ATM- and DNA-PKcs-deficient cells when compared with their complemented cells, while that of an MLV-based vector was diminished in Mre11- and DNA-PKcs-deficient cells. By using a method based on inverse- and Alu-PCR, we analyzed sequences around 3' HIV-1 integration sites in ATM-, Mre11- and NBS1- deficient cells. Increased abnormal junctions between the HIV-1 provirus and the host DNA were found in these mutant cell lines compared to the complemented cell lines and control MRC5SV cells. The abnormal junctions contained two types of insertions: 1 GT dinucleotides, which are normally removed by integrase during integration, and 2 inserted nucleotides of unknown origin. Artemis-deficient cells also showed such abnormalities. In Mre11-deficient cells, part of a primer binding site sequence was also detected. The 5' host-virus junctions in the mutant cells also contained these types of abnormal nucleotides. Moreover, the host-virus junctions of the MLV provirus showed similar abnormalities. These findings suggest that DSB repair enzymes play roles in the 3'-processing reaction and protection of the ends of viral DNA after reverse transcription. We also identified both 5' and 3' junctional sequences of the same provirus by inverse PCR and found that only the 3' junctions were abnormal with aberrant short repeats, indicating that the integration step was partially impaired in these cells. Furthermore, the conserved base preferences around HIV-1 integration sites were partially altered in ATM-deficient cells. Conclusions These results suggest that DSB repair enzymes are

  6. Excess single-stranded DNA inhibits meiotic double-strand break repair.

    Directory of Open Access Journals (Sweden)

    Rebecca Johnson

    2007-11-01

    Full Text Available During meiosis, self-inflicted DNA double-strand breaks (DSBs are created by the protein Spo11 and repaired by homologous recombination leading to gene conversions and crossovers. Crossover formation is vital for the segregation of homologous chromosomes during the first meiotic division and requires the RecA orthologue, Dmc1. We analyzed repair during meiosis of site-specific DSBs created by another nuclease, VMA1-derived endonuclease (VDE, in cells lacking Dmc1 strand-exchange protein. Turnover and resection of the VDE-DSBs was assessed in two different reporter cassettes that can repair using flanking direct repeat sequences, thereby obviating the need for a Dmc1-dependent DNA strand invasion step. Access of the single-strand binding complex replication protein A, which is normally used in all modes of DSB repair, was checked in chromatin immunoprecipitation experiments, using antibody against Rfa1. Repair of the VDE-DSBs was severely inhibited in dmc1Delta cells, a defect that was associated with a reduction in the long tract resection required to initiate single-strand annealing between the flanking repeat sequences. Mutants that either reduce Spo11-DSB formation or abolish resection at Spo11-DSBs rescued the repair block. We also found that a replication protein A component, Rfa1, does not accumulate to expected levels at unrepaired single-stranded DNA (ssDNA in dmc1Delta cells. The requirement of Dmc1 for VDE-DSB repair using flanking repeats appears to be caused by the accumulation of large quantities of ssDNA that accumulate at Spo11-DSBs when Dmc1 is absent. We propose that these resected DSBs sequester both resection machinery and ssDNA binding proteins, which in wild-type cells would normally be recycled as Spo11-DSBs repair. The implication is that repair proteins are in limited supply, and this could reflect an underlying mechanism for regulating DSB repair in wild-type cells, providing protection from potentially harmful effects

  7. DNA double-strand break repair: a theoretical framework and its application.

    Science.gov (United States)

    Murray, Philip J; Cornelissen, Bart; Vallis, Katherine A; Chapman, S Jon

    2016-01-01

    DNA double-strand breaks (DSBs) are formed as a result of genotoxic insults, such as exogenous ionizing radiation, and are among the most serious types of DNA damage. One of the earliest molecular responses following DSB formation is the phosphorylation of the histone H2AX, giving rise to γH2AX. Many copies of γH2AX are generated at DSBs and can be detected in vitro as foci using well-established immuno-histochemical methods. It has previously been shown that anti-γH2AX antibodies, modified by the addition of the cell-penetrating peptide TAT and a fluorescent or radionuclide label, can be used to visualize and quantify DSBs in vivo. Moreover, when labelled with a high amount of the short-range, Auger electron-emitting radioisotope, (111)In, the amount of DNA damage within a cell can be increased, leading to cell death. In this report, we develop a mathematical model that describes how molecular processes at individual sites of DNA damage give rise to quantifiable foci. Equations that describe stochastic mean behaviours at individual DSB sites are derived and parametrized using population-scale, time-series measurements from two different cancer cell lines. The model is used to examine two case studies in which the introduction of an antibody (anti-γH2AX-TAT) that targets a key component in the DSB repair pathway influences system behaviour. We investigate: (i) how the interaction between anti-γH2AX-TAT and γH2AX effects the kinetics of H2AX phosphorylation and DSB repair and (ii) model behaviour when the anti-γH2AX antibody is labelled with Auger electron-emitting (111)In and can thus instigate additional DNA damage. This work supports the conclusion that DSB kinetics are largely unaffected by the introduction of the anti-γH2AX antibody, a result that has been validated experimentally, and hence the hypothesis that the use of anti-γH2AX antibody to quantify DSBs does not violate the image tracer principle. Moreover, it provides a novel model of DNA damage

  8. Melting of duplex DNA in the absence of ATP by NS3 helicase domain through specific interaction with a single-strand/double-strand junction

    Science.gov (United States)

    Reynolds, Kimberly A.; Cameron, Craig E.; Raney, Kevin D.

    2016-01-01

    Helicases unwind double-stranded nucleic acids, remove secondary structures from single-stranded nucleic acids, and remove proteins bound to nucleic acids. For many helicases, the mechanisms for these different functions share the ability to translocate with a directional bias as a result of ATP binding and hydrolysis. The nonstructural protein 3 (NS3) is an essential enzyme expressed by the hepatitis C virus (HCV) and is known to catalyze the unwinding of both DNA and RNA substrates in a 3′-to-5′ direction. We investigated the role of nucleic acid binding in the unwinding mechanism by examining ATP-independent unwinding. We observed that even in the absence of ATP, NS3 helicase domain (NS3h) unwound duplexes only when they contained a 3′-tail (i.e., 3′-to-5′ directionality). Blunt-ended duplexes and 5′-tailed duplexes were not melted even in the presence of a large excess concentration of the protein. NS3h was found to diffuse rapidly along single-stranded DNA at a rate of 30 nt2·s−1. Upon encountering an appropriate single-strand/double-strand (ss/ds) junction, NS3h slowly melted the duplex under conditions with excess protein concentration relative to DNA concentration. When a biotin-streptavidin block was placed into the ssDNA region, no melting of DNA was observed, suggesting that NS3h must diffuse along the ssDNA, and that the streptavidin blocked the diffusion. We conclude that the specific interaction between NS3h and the ss/dsDNA junction, coupled with diffusion allows binding energy to melt duplex DNA with a directional bias. Alternatively, we found that the full-length NS3 protein did not exhibit strict directionality and was dependent on duplex DNA length. NS3 was able to unwind the duplex even in the presence of the biotin-streptavidin block. We propose a non-canonical model of unwinding for NS3 in which the enzyme binds directly to the duplex via protein-protein interactions to melt the substrate. PMID:26091150

  9. Labeling of double-stranded DNA by ROX-dideoxycytosine triphosphate using terminal deoxynucleotidyl transferase and separation by capillary electrophoresis

    Energy Technology Data Exchange (ETDEWEB)

    Figeys, D.; Renborg, A.; Dovichi, N.J. (Univ. of Alberta, Edmonton, Alberta (Canada))

    1994-12-01

    Terminal transferase is used to add a single fluorescently labeled dideoxynucleotide to double-stranded DNA prepared by restriction endonuclease action on a bacteriophage. The product is separated by capillary electrophoresis with both hydroxypropylmethylcellulose and non-cross-linked polyacrylamide. The reaction products generate single peaks for each fragment with hydroxypropylmethylcellulose. However, the higher resolution separation produced by non-cross-linked polyacrylamide shows that the product contains two components for each restriction digest fragment. This labeling technique should be useful in restriction fragment length polymorphism studies. 9 refs., 2 figs.

  10. CtIP-BRCA1 modulates the choice of DNA double-strand break repair pathway throughout the cell cycle

    OpenAIRE

    Yun, Maximina H.; Hiom, Kevin

    2009-01-01

    The repair of DNA double-strand breaks (DSB) is tightly regulated during the cell cycle. In G1 phase, the absence of a sister chromatid means that repair of DSB occurs through non-homologous end-joining (NHEJ) or microhomology-mediated end-joining (MMEJ)1. These pathways often involve loss of DNA sequences at the break site and are therefore error-prone. In late S and G2 phases, even though DNA end-joining pathways remain functional2, there is an increase in repair of DSB by homologous recomb...

  11. Do chromatin changes around a nascent double strand DNA break spread spherically into linearly non-adjacent chromatin?

    OpenAIRE

    Savic, Velibor

    2013-01-01

    In the last decade, a lot has been done in elucidating the sequence of events that occur at the nascent double strand DNA break. Nevertheless, the overall structure formed by the DNA damage response (DDR) factors around the break site, the repair focus, remains poorly understood. Although most of the data presented so far only address events that occur in chromatin in cis around the break, there are strong indications that in mammalian systems it may also occur in trans, analogous to the rece...

  12. Sgs1 helicase and two nucleases Dna2 and Exo1 resect DNA double strand break ends

    OpenAIRE

    Zhu, Zhu; Chung, Woo-Hyun; Shim, Eun Yong; Lee, Sang Eun; Ira, Grzegorz

    2008-01-01

    Formation of single-strand DNA (ssDNA) tails at a double-strand break (DSB) is a key step in homologous recombination and DNA damage signaling. The enzyme(s) producing ssDNA at DSBs in eukaryotes remains unknown. We monitored 5’-strand resection at inducible DSB ends and identified proteins required for two stages of resection: initiation and long-range 5’-strand resection. The Mre11-Rad50-Xrs2 complex (MRX) initiates 5’ degradation, whereas Sgs1 and Dna2 degrade 5’-strands exposing long 3’-s...

  13. Characterization of the nanostructure of complexes formed by single- or double-stranded oligonucleotides with a cationic surfactant.

    Science.gov (United States)

    Liu, Xiaoyang; Abbott, Nicholas L

    2010-12-02

    We report the use of dynamic light scattering (DLS), small-angle neutron scattering (SANS), and small-angle X-ray scattering (SAXS) to characterize the nanostructure of complexes formed by either single- or double-stranded oligonucleotides with a cationic surfactant (cetyltrimethylammonium bromide, CTAB) in aqueous solution (1 mM Li(2)SO(4)). For single-stranded oligonucleotides 5'-A(20)-3' and 5'-CCCCATTCTAGCAGCCCGGG-3', both the appearance of two Bragg peaks (at 0.14 and 0.28 Å(-1)) in SAXS spectra with a spacing of 1:2 and form factor fits to SANS spectra are consistent with the presence of multilamellar vesicles (with, on average, 6-9 layers with a periodicity of 45-48 Å). Some samples showed evidence of an additional Bragg peak (at 0.20 Å(-1)) associated with periodic packing (with a periodicity of 31 Å) of the oligonucleotides within the lamellae of the nanostructure. The nucleotide composition of the single-stranded oligonucleotides was also found to impact the number and size of the complexes formed with CTAB. In contrast to 5'-A(20)-3' and 5'-CCCCATTCTAGCAGCCCGGG-3', 5'-T(20)-3' did not change the state of aggregation of CTAB (globular micelles) over a wide range of oligonucleotide:CTAB charge ratios. These results support the proposition that hydrophobic interactions, as well as electrostatics, play a central role in the formation of complexes between cationic amphiphiles and single-stranded oligonucleotides and thus give rise to nanostructures that depend on nucleotide composition. In contrast to the single-stranded oligonucleotides, for double-stranded oligonucleotides mixed with CTAB, three Bragg peaks (0.13, 0.23, and 0.25 Å(-1)) in SAXS spectra with a spacing ratio of 1:√3:√4 and characteristic changes in SANS spectra indicate formation of a hexagonal nanostructure. Also, the composition of the double-stranded oligonucleotides did not measurably impact the nanostructure of complexes formed with CTAB, suggesting that electrostatic

  14. Ligation of double-stranded and single-stranded [Oligo(dT)] DNA by vaccinia virus DNA ligase

    OpenAIRE

    1996-01-01

    Vaccinia virus DNA ligase has been expressed in Escherichia coli, purified, and biochemically characterized. The enzyme ligates double-stranded (ds) DNA substrates with either cohesive or blunt-end termini and the latter reaction is stimulated by PEG. Vaccinia virus DNA ligase can also ligate oligo(dT) when annealed to either a poly(dA) or a poly(rA) backbone and, remarkably, free oligo(dT). This ligation of a single-stranded (ss) substrate is unique among eukaryotic DNA ligases. The enzyme r...

  15. Crystal Structure of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated Csn2 Protein Revealed Ca[superscript 2+]-dependent Double-stranded DNA Binding Activity

    Energy Technology Data Exchange (ETDEWEB)

    Nam, Ki Hyun; Kurinov, Igor; Ke, Ailong (Cornell); (NWU)

    2012-05-22

    Clustered regularly interspaced short palindromic repeats (CRISPR) and their associated protein genes (cas genes) are widespread in bacteria and archaea. They form a line of RNA-based immunity to eradicate invading bacteriophages and malicious plasmids. A key molecular event during this process is the acquisition of new spacers into the CRISPR loci to guide the selective degradation of the matching foreign genetic elements. Csn2 is a Nmeni subtype-specific cas gene required for new spacer acquisition. Here we characterize the Enterococcus faecalis Csn2 protein as a double-stranded (ds-) DNA-binding protein and report its 2.7 {angstrom} tetrameric ring structure. The inner circle of the Csn2 tetrameric ring is {approx}26 {angstrom} wide and populated with conserved lysine residues poised for nonspecific interactions with ds-DNA. Each Csn2 protomer contains an {alpha}/{beta} domain and an {alpha}-helical domain; significant hinge motion was observed between these two domains. Ca{sup 2+} was located at strategic positions in the oligomerization interface. We further showed that removal of Ca{sup 2+} ions altered the oligomerization state of Csn2, which in turn severely decreased its affinity for ds-DNA. In summary, our results provided the first insight into the function of the Csn2 protein in CRISPR adaptation by revealing that it is a ds-DNA-binding protein functioning at the quaternary structure level and regulated by Ca{sup 2+} ions.

  16. Crystal structure of clustered regularly interspaced short palindromic repeats (CRISPR)-associated Csn2 protein revealed Ca2+-dependent double-stranded DNA binding activity.

    Science.gov (United States)

    Nam, Ki Hyun; Kurinov, Igor; Ke, Ailong

    2011-09-02

    Clustered regularly interspaced short palindromic repeats (CRISPR) and their associated protein genes (cas genes) are widespread in bacteria and archaea. They form a line of RNA-based immunity to eradicate invading bacteriophages and malicious plasmids. A key molecular event during this process is the acquisition of new spacers into the CRISPR loci to guide the selective degradation of the matching foreign genetic elements. Csn2 is a Nmeni subtype-specific cas gene required for new spacer acquisition. Here we characterize the Enterococcus faecalis Csn2 protein as a double-stranded (ds-) DNA-binding protein and report its 2.7 Å tetrameric ring structure. The inner circle of the Csn2 tetrameric ring is ∼26 Å wide and populated with conserved lysine residues poised for nonspecific interactions with ds-DNA. Each Csn2 protomer contains an α/β domain and an α-helical domain; significant hinge motion was observed between these two domains. Ca(2+) was located at strategic positions in the oligomerization interface. We further showed that removal of Ca(2+) ions altered the oligomerization state of Csn2, which in turn severely decreased its affinity for ds-DNA. In summary, our results provided the first insight into the function of the Csn2 protein in CRISPR adaptation by revealing that it is a ds-DNA-binding protein functioning at the quaternary structure level and regulated by Ca(2+) ions.

  17. Depletion of Histone Demethylase Jarid1A Resulting in Histone Hyperacetylation and Radiation Sensitivity Does Not Affect DNA Double-Strand Break Repair.

    Directory of Open Access Journals (Sweden)

    Corina Penterling

    Full Text Available Histone demethylases have recently gained interest as potential targets in cancer treatment and several histone demethylases have been implicated in the DNA damage response. We investigated the effects of siRNA-mediated depletion of histone demethylase Jarid1A (KDM5A, RBP2, which demethylates transcription activating tri- and dimethylated lysine 4 at histone H3 (H3K4me3/me2, on growth characteristics and cellular response to radiation in several cancer cell lines. In unirradiated cells Jarid1A depletion lead to histone hyperacetylation while not affecting cell growth. In irradiated cells, depletion of Jarid1A significantly increased cellular radiosensitivity. Unexpectedly, the hyperacetylation phenotype did not lead to disturbed accumulation of DNA damage response and repair factors 53BP1, BRCA1, or Rad51 at damage sites, nor did it influence resolution of radiation-induced foci or rejoining of reporter constructs. We conclude that the radiation sensitivity observed following depletion of Jarid1A is not caused by a deficiency in repair of DNA double-strand breaks.

  18. The transcriptional histone acetyltransferase cofactor TRRAP associates with the MRN repair complex and plays a role in DNA double-strand break repair.

    Science.gov (United States)

    Robert, Flavie; Hardy, Sara; Nagy, Zita; Baldeyron, Céline; Murr, Rabih; Déry, Ugo; Masson, Jean-Yves; Papadopoulo, Dora; Herceg, Zdenko; Tora, Làszlò

    2006-01-01

    Transactivation-transformation domain-associated protein (TRRAP) is a component of several multiprotein histone acetyltransferase (HAT) complexes implicated in transcriptional regulation. TRRAP was shown to be required for the mitotic checkpoint and normal cell cycle progression. MRE11, RAD50, and NBS1 (product of the Nijmegan breakage syndrome gene) form the MRN complex that is involved in the detection, signaling, and repair of DNA double-strand breaks (DSBs). By using double immunopurification, mass spectrometry, and gel filtration, we describe the stable association of TRRAP with the MRN complex. The TRRAP-MRN complex is not associated with any detectable HAT activity, while the isolated other TRRAP complexes, containing either GCN5 or TIP60, are. TRRAP-depleted extracts show a reduced nonhomologous DNA end-joining activity in vitro. Importantly, small interfering RNA knockdown of TRRAP in HeLa cells or TRRAP knockout in mouse embryonic stem cells inhibit the DSB end-joining efficiency and the precise nonhomologous end-joining process, further suggesting a functional involvement of TRRAP in the DSB repair processes. Thus, TRRAP may function as a molecular link between DSB signaling, repair, and chromatin remodeling.

  19. The Double-Strand Break Landscape of Meiotic Chromosomes Is Shaped by the Paf1 Transcription Elongation Complex in Saccharomyces cerevisiae.

    Science.gov (United States)

    Gothwal, Santosh K; Patel, Neem J; Colletti, Meaghan M; Sasanuma, Hiroyuki; Shinohara, Miki; Hochwagen, Andreas; Shinohara, Akira

    2016-02-01

    Histone modification is a critical determinant of the frequency and location of meiotic double-strand breaks (DSBs), and thus recombination. Set1-dependent histone H3K4 methylation and Dot1-dependent H3K79 methylation play important roles in this process in budding yeast. Given that the RNA polymerase II associated factor 1 complex, Paf1C, promotes both types of methylation, we addressed the role of the Paf1C component, Rtf1, in the regulation of meiotic DSB formation. Similar to a set1 mutation, disruption of RTF1 decreased the occurrence of DSBs in the genome. However, the rtf1 set1 double mutant exhibited a larger reduction in the levels of DSBs than either of the single mutants, indicating independent contributions of Rtf1 and Set1 to DSB formation. Importantly, the distribution of DSBs along chromosomes in the rtf1 mutant changed in a manner that was different from the distributions observed in both set1 and set1 dot1 mutants, including enhanced DSB formation at some DSB-cold regions that are occupied by nucleosomes in wild-type cells. These observations suggest that Rtf1, and by extension the Paf1C, modulate the genomic DSB landscape independently of H3K4 methylation.

  20. [Presence of autocomplementary RNA with viral specificity in cells infected with herpes virus].

    Science.gov (United States)

    Béchet, J M; Montagnier, L; Latarjet, R

    1975-01-13

    RNA from cells infected with Herpes simplex virus contain a higher percentage of double-stranded RNA than non-infected cells. This percentage increases three-fold upon self-annealing. The complementary RNA sequences were shown to be virus-specific by the following criteria: (1) high melting temperature than double-stranded RNA from non infected cells; (2) higher density in caesium sulphate; (3) specific hybridization with viral DNA.

  1. Strategies underlying RNA silencing suppression by negative strand RNA viruses

    NARCIS (Netherlands)

    Hemmes, J.C.

    2007-01-01

    The research described in this thesis focused on the strategies of negative strand RNA viruses to counteract antiviral RNA silencing. In plants and insects, RNA silencing has been shown to act as a sequence specific antiviral defence mechanism that is characterised by the processing of double strand

  2. Atrazine Triggers DNA Damage Response and Induces DNA Double-Strand Breaks in MCF-10A Cells.

    Science.gov (United States)

    Huang, Peixin; Yang, John; Ning, Jie; Wang, Michael; Song, Qisheng

    2015-06-24

    Atrazine, a pre-emergent herbicide in the chloro-s-triazine family, has been widely used in crop lands and often detected in agriculture watersheds, which is considered as a potential threat to human health. Although atrazine and its metabolites showed an elevated incidence of mammary tumors in female Sprague-Dawley (SD) rats, no molecular evidence was found relevant to its carcinogenesis in humans. This study aims to determine whether atrazine could induce the expression of DNA damage response-related proteins in normal human breast epithelial cells (MCF-10A) and to examine the cytotoxicity of atrazine at a molecular level. Our results indicate that a short-term exposure of MCF-10A to an environmentally-detectable concentration of atrazine (0.1 µg/mL) significantly increased the expression of tumor necrosis factor receptor-1 (TNFR1) and phosphorylated Rad17 in the cells. Atrazine treatment increased H2AX phosphorylation (γH2AX) and the formation of γH2AX foci in the nuclei of MCF-10A cells. Atrazine also sequentially elevated DNA damage checkpoint proteins of ATM- and RAD3-related (ATR), ATRIP and phospho-Chk1, suggesting that atrazine could induce DNA double-strand breaks and trigger the DNA damage response ATR-Chk1 pathway in MCF-10A cells. Further investigations are needed to determine whether atrazine-triggered DNA double-strand breaks and DNA damage response ATR-Chk1 pathway occur in vivo.

  3. Dynamic DNA Nanotubes: Reversible Switching between Single and Double-Stranded Forms, and Effect of Base Deletions.

    Science.gov (United States)

    Rahbani, Janane F; Hariri, Amani A; Cosa, Gonzalo; Sleiman, Hanadi F

    2015-12-22

    DNA nanotubes hold great potential as drug delivery vehicles and as programmable templates for the organization of materials and biomolecules. Existing methods for their construction produce assemblies that are entirely double-stranded and rigid, and thus have limited intrinsic dynamic character, or they rely on chemically modified and ligated DNA structures. Here, we report a simple and efficient synthesis of DNA nanotubes from 11 short unmodified strands, and the study of their dynamic behavior by atomic force microscopy and in situ single molecule fluorescence microscopy. This method allows the programmable introduction of DNA structural changes within the repeat units of the tubes. We generate and study fully double-stranded nanotubes, and convert them to nanotubes with one, two and three single-stranded sides, using strand displacement strategies. The nanotubes can be reversibly switched between these forms without compromising their stability and micron-scale lengths. We then site-specifically introduce DNA strands that shorten two sides of the nanotubes, while keeping the length of the third side. The nanotubes undergo bending with increased length mismatch between their sides, until the distortion is significant enough to shorten them, as measured by AFM and single-molecule fluorescence photobleaching experiments. The method presented here produces dynamic and robust nanotubes that can potentially behave as actuators, and allows their site-specific addressability while using a minimal number of component strands.

  4. Simultaneous labeling of single- and double-strand DNA breaks by DNA breakage detection-FISH (DBD-FISH).

    Science.gov (United States)

    Fernández, José Luis; Cajigal, Dioleyda; Gosálvez, Jaime

    2011-01-01

    DNA Breakage Detection-Fluorescence In Situ Hybridization (DBD-FISH) permits simultaneous and selective labeling of single- and double-strand DNA breaks in individual cells, either in the whole genome or within specific DNA sequences. In this technique, cells are embedded into agarose microgels, lysed and subjected to electrophoresis under nondenaturing conditions. Subsequently, the produced "comets" are exposed to a controlled denaturation step which transforms DNA breaks into single-stranded DNA regions, detected by hybridization with whole genome fluorescent probes or the probes to specific DNA sequences. This makes possible a targeted analysis of various chromatin areas for the presence of DNA breaks. The migration length of the DBD-FISH signal is proportional to the number of double strand breaks, whereas its fluorescence intensity depends on numbers of single-strand breaks.The detailed protocol for detection of two types of DNA breaks produced by ionizing radiation is presented. The technique can be used to determine intragenomic and intercellular heterogeneity in the induction and repair of DNA damage.

  5. Zinc-finger directed double-strand breaks within CAG repeat tracts promote repeat instability in human cells.

    Science.gov (United States)

    Mittelman, David; Moye, Christopher; Morton, Jason; Sykoudis, Kristen; Lin, Yunfu; Carroll, Dana; Wilson, John H

    2009-06-16

    Expanded triplet repeats have been identified as the genetic basis for a growing number of neurological and skeletal disorders. To examine the contribution of double-strand break repair to CAG x CTG repeat instability in mammalian systems, we developed zinc finger nucleases (ZFNs) that recognize and cleave CAG repeat sequences. Engineered ZFNs use a tandem array of zinc fingers, fused to the FokI DNA cleavage domain, to direct double-strand breaks (DSBs) in a site-specific manner. We first determined that the ZFNs cleave CAG repeats in vitro. Then, using our previously described tissue culture assay for identifying modifiers of CAG repeat instability, we found that transfection of ZFN-expression vectors induced up to a 15-fold increase in changes to the CAG repeat in human and rodent cell lines, and that longer repeats were much more sensitive to cleavage than shorter ones. Analysis of individual colonies arising after treatment revealed a spectrum of events consistent with ZFN-induced DSBs and dominated by repeat contractions. We also found that expressing a dominant-negative form of RAD51 in combination with a ZFN, dramatically reduced the effect of the nuclease, suggesting that DSB-induced repeat instability is mediated, in part, through homology directed repair. These studies identify a ZFN as a useful reagent for characterizing the effects of DSBs on CAG repeats in cells.

  6. Single nucleotide-level mapping of DNA double-strand breaks in human HEK293T cells

    Directory of Open Access Journals (Sweden)

    Bernard J. Pope

    2017-03-01

    Full Text Available Constitutional biological processes involve the generation of DNA double-strand breaks (DSBs. The production of such breaks and their subsequent resolution are also highly relevant to neurodegenerative diseases and cancer, in which extensive DNA fragmentation has been described Stephens et al. (2011, Blondet et al. (2001. Tchurikov et al. Tchurikov et al. (2011, 2013 have reported previously that frequent sites of DSBs occur in chromosomal domains involved in the co-ordinated expression of genes. This group report that hot spots of DSBs in human HEK293T cells often coincide with H3K4me3 marks, associated with active transcription Kravatsky et al. (2015 and that frequent sites of DNA double-strand breakage are likely to be relevant to cancer genomics Tchurikov et al. (2013, 2016 . Recently, they applied a RAFT (rapid amplification of forum termini protocol that selects for blunt-ended DSB sites and mapped these to the human genome within defined co-ordinate ‘windows’. In this paper, we re-analyse public RAFT data to derive sites of DSBs at the single-nucleotide level across the built genome for human HEK293T cells (https://figshare.com/s/35220b2b79eaaaf64ed8. This refined mapping, combined with accessory ENCODE data tracks and ribosomal DNA-related sequence annotations, will likely be of value for the design of clinically relevant targeted assays such as those for cancer susceptibility, diagnosis, treatment-matching and prognostication.

  7. Contribution of DNA double-strand break repair gene XRCC3 genotypes to oral cancer susceptibility in Taiwan.

    Science.gov (United States)

    Tsai, Chia-Wen; Chang, Wen-Shin; Liu, Juhn-Cherng; Tsai, Ming-Hsui; Lin, Cheng-Chieh; Bau, Da-Tian

    2014-06-01

    The DNA repair gene X-ray repair cross complementing protein 3 (XRCC3) is thought to play a major role in double-strand break repair and in maintaining genomic stability. Very possibly, defective double-strand break repair of cells can lead to carcinogenesis. Therefore, a case-control study was performed to reveal the contribution of XRCC3 genotypes to individual oral cancer susceptibility. In this hospital-based research, the association of XRCC3 rs1799794, rs45603942, rs861530, rs3212057, rs1799796, rs861539, rs28903081 genotypes with oral cancer risk in a Taiwanese population was investigated. In total, 788 patients with oral cancer and 956 age- and gender-matched healthy controls were genotyped. The results showed that there was significant differential distribution among oral cancer and controls in the genotypic (p=0.001428) and allelic (p=0.0013) frequencies of XRCC3 rs861539. As for the other polymorphisms, there was no difference between case and control groups. In gene-lifestyle interaction analysis, we have provided the first evidence showing that there is an obvious joint effect of XRCC3 rs861539 genotype with individual areca chewing habits on oral cancer risk. In conclusion, the T allele of XRCC3 rs861539, which has an interaction with areca chewing habit in oral carcinogenesis, may be an early marker for oral cancer in Taiwanese.

  8. I-SceI-mediated double-strand DNA breaks stimulate efficient gene targeting in the industrial fungus Trichoderma reesei.

    Science.gov (United States)

    Ouedraogo, Jean Paul; Arentshorst, Mark; Nikolaev, Igor; Barends, Sharief; Ram, Arthur F J

    2015-12-01

    Targeted integration of expression cassettes for enzyme production in industrial microorganisms is desirable especially when enzyme variants are screened for improved enzymatic properties. However, currently used methods for targeted integration are inefficient and result in low transformation frequencies. In this study, we expressed the Saccharomyces cerevisiae I-SceI meganuclease to generate double-strand breaks at a defined locus in the Trichoderma reesei genome. We showed that the double-strand DNA breaks mediated by I-SceI can be efficiently repaired when an exogenous DNA cassette flanked by regions homologous to the I-SceI landing locus was added during transformation. Transformation efficiencies increased approximately sixfold compared to control transformation. Analysis of the transformants obtained via I-SceI-mediated gene targeting showed that about two thirds of the transformants resulted from a homologous recombination event at the predetermined locus. Counter selection of the transformants for the loss of the pyrG marker upon integration of the DNA cassette showed that almost all of the clones contained the cassette at the predetermined locus. Analysis of independently obtained transformants using targeted integration of a glucoamylase expression cassette demonstrated that glucoamylase production among the transformants was high and showing limited variation. In conclusion, the gene targeting system developed in this study significantly increases transformation efficiency as well as homologous recombination efficiency and omits the use of Δku70 strains. It is also suitable for high-throughput screening of enzyme variants or gene libraries in T. reesei.

  9. DNA double-strand breaks as potential indicators for the biological effects of ionising radiation exposure from cardiac CT and conventional coronary angiography: a randomised, controlled study

    Energy Technology Data Exchange (ETDEWEB)

    Geisel, Dominik; Zimmermann, Elke; Rief, Matthias; Greupner, Johannes; Hamm, Bernd [Charite Medical School, Department of Radiology, Berlin (Germany); Laule, Michael; Knebel, Fabian [Charite Medical School, Department of Cardiology, Berlin (Germany); Dewey, Marc [Charite Medical School, Department of Radiology, Berlin (Germany); Charite, Institut fuer Radiologie, Berlin (Germany)

    2012-08-15

    To prospectively compare induced DNA double-strand breaks by cardiac computed tomography (CT) and conventional coronary angiography (CCA). 56 patients with suspected coronary artery disease were randomised to undergo either CCA or cardiac CT. DNA double-strand breaks were assessed in fluorescence microscopy of blood lymphocytes as indicators of the biological effects of radiation exposure. Radiation doses were estimated using dose-length product (DLP) and dose-area product (DAP) with conversion factors for CT and CCA, respectively. On average there were 0.12 {+-} 0.06 induced double-strand breaks per lymphocyte for CT and 0.29 {+-} 0.18 for diagnostic CCA (P < 0.001). This relative biological effect of ionising radiation from CCA was 1.9 times higher (P < 0.001) than the effective dose estimated by conversion factors would have suggested. The correlation between the biological effects and the estimated radiation doses was excellent for CT (r = 0.951, P < 0.001) and moderate to good for CCA (r = 0.862, P < 0.001). One day after radiation, a complete repair of double-strand breaks to background levels was found in both groups. Conversion factors may underestimate the relative biological effects of ionising radiation from CCA. DNA double-strand break assessment may provide a strategy for individualised assessments of radiation. (orig.)

  10. Spectroscopic characterization of the interaction of phenosafranin and safranin O with double stranded, heat denatured and single stranded calf thymus DNA.

    Science.gov (United States)

    Saha, Ishita; Kumar, Gopinatha Suresh

    2011-01-01

    Interaction of phenosafranin and safranin O with double stranded, heat denatured and single stranded calf thymus DNA has been studied by fluorescence, absorbance and circular dichroic techniques. Binding to the double stranded and heat denatured DNA conformations induced strong quenching in the fluorescence spectra of both dyes. Linear Scatchard plots indicated the binding to be of one type and the affinity evaluated to be of the order of 10(5) M(-1) with double stranded and heat denatured DNAs. Fluorescence quenching was much weaker with the single stranded DNA and the binding affinity was one order lower. Ferrocyanide quenching studies revealed that the fluorescence emission of the dye molecules bound to the double stranded and heat denatured DNAs was quenched much less compared to that bound to the single stranded DNA. Further, there was significant emission polarization for the bound dyes and strong energy transfer from the DNA base pairs to the dye molecules indicating intercalative binding. Salt dependence of the binding phenomenon revealed that electrostatic forces have significant role in the binding process. The intercalation of these molecules to double stranded and heat denatured DNA and simple stacking to single strands was proved by these fluorescence techniques. Support to the fluorescence results have been derived from absorption and circular dichroic results. Phenosafranin was revealed to be a stronger binding species compared to safranin O.

  11. Elucidaton of DNA methylation changes in response to ionizng radiation induced double strand breaks

    Energy Technology Data Exchange (ETDEWEB)

    Herrlitz, Maren Linda

    2014-07-04

    would be an effect of overexpression or be indicative of a possible function in these nuclear subcompartments is yet to be elucidated. Additionally, by using flow cytometry analysis, exposure to IR and concomitant overexpression of TET2CD-GFP strongly induced 5hmC formation, therefore suggesting a function of TET2 in response to irradiation. Recruitment analysis showed that the TET2 catalytic domain was recruited to UV laser-induced but not X-rays- or heavy ion-induced damage sites. Endogenous TET2, which was analyzed in high TET2 expressing human fibroblasts, was recruited to damage sites after irradiation with heavy ions or X-rays. As 5hmC is the direct product of the catalytic activity of TET enzymes, local 5hmC formation and abundance at damage sites was investigated. It was observed that 5hmC accumulated at heavy ion- as well as X-ray-induced DNA double strand breaks (DSBs). In addition, investigating 5hmC foci over time after irradiation with X-rays revealed that 5hmC formation and kinetics is similar to that of γH2AX foci, whereby every 5hmC focus co-localized with γH2AX. However, this did not hold true for all γH2AX foci, whose total number was always higher than that of 5hmC. Furthermore, 5hmC (and γH2AX) foci formation was almost unaffected by the inhibition of DNA-PKcs' enzymatic activity. Conversely, 5hmC and γH2AX foci persistence was significantly delayed after DNA-PKcs inhibition. Results obtained in this thesis show that DNA methylation changes (5hmC formation) take place within the time frame of one replication cycle after exposure to IR and that these changes can be observed at sites of DSBs. 5hmC at DSBs might be formed by the oxidative function of TET2, which was shown to be recruited to DSBs. However, involvement of the other TET enzymes in 5hmC production cannot be excluded. Therefore, these results suggest a role of 5hmC in the response to IR induced DSBs, whereby the here presented data suggest that the fast, radiation induced

  12. Viral counterdefense on RNA silencing : analysis of RNA silencing suppressors from arthropod-borne negative strand RNA plant viruses

    NARCIS (Netherlands)

    Schnettler, E.

    2010-01-01

    This thesis describes that RNA silencing suppressor (RSS) proteins encoded by negative-stranded RNA plant viruses are able to interfere with different RNA silencing pathways in a variety of organisms by interacting with double stranded (ds)RNA molecules. These RSS proteins are able to counteract the

  13. Radiation Induced DNA Double Strand Break Studies of a Metal Sensitive Novel Bacterial Isolate from East Calcutta Wetland

    Directory of Open Access Journals (Sweden)

    Sanhita Chowdhury

    2009-01-01

    Full Text Available Problem statement: This study was an attempt to isolate anaerobic microbes with potential for DNA double strand break repair using methanogen specific medium (DSMZ 120 from East Calcutta Wetland in India. It also intended to verify the specificity of the medium for isolation of the desired family of microbe. Approach: Culture based technique was used to obtain the pure isolate that was further characterized in details. For double strand break repair studies, isolate was irradiated with different doses of 60Co gamma rays and its subsequent repair was observed using pulse field gel electrophoresis and asymmetric field inversion gel electrophoresis. Inhibitor was used to predict the mechanism of repair. Results: In this study we isolated and characterized a metal sensitive anaerobic microbial strain obtained using methanogen specific medium (DSMZ 120 from East Calcutta Wetland in India. The strain was one of the members of the group of uncultivated bacterium as evident from phylogenetic analysis, thus indicating the successful cultivation of an as yet uncultivable novel microbe (GenBank Acc. No. FJ 930097 and also the non-specific growth of microbes in prescribed medium. It was a Gram positive Bacilli, member of Fermicutes with optimum growth at 25°C and pH-7. The growth curve analysis showed a lag phase up to 24 h, log phase from 24-48 h, an early stationary phase from 96 h onwards. The strain could repair the DNA double strand break caused by irradiation with 60Co γ rays. The dose profile study revealed maximum repair at 60 Grays and thereafter a drop in repair ability with increase in irradiation dose. The time required for repair showed an essential incubation period of 4 h. The DNA polymerase inhibitor, Arabinose CTP inhibited the repair indicating the involvement of polymerase in the repair process and thus pointing towards homologous recombination as the underlying mechanism. Conclusion: In this study we were able to cultivate an as yet

  14. Coordination and processing of DNA ends during double-strand break repair: the role of the bacteriophage T4 Mre11/Rad50 (MR) complex.

    Science.gov (United States)

    Almond, Joshua R; Stohr, Bradley A; Panigrahi, Anil K; Albrecht, Dustin W; Nelson, Scott W; Kreuzer, Kenneth N

    2013-11-01

    The in vivo functions of the bacteriophage T4 Mre11/Rad50 (MR) complex (gp46/47) in double-strand-end processing, double-strand break repair, and recombination-dependent replication were investigated. The complex is essential for T4 growth, but we wanted to investigate the in vivo function during productive infections. We therefore generated a suppressed triple amber mutant in the Rad50 subunit to substantially reduce the level of complex and thereby reduce phage growth. Growth-limiting amounts of the complex caused a concordant decrease in phage genomic recombination-dependent replication. However, the efficiencies of double-strand break repair and of plasmid-based recombination-dependent replication remained relatively normal. Genetic analyses of linked markers indicated that double-strand ends were less protected from nuclease erosion in the depleted infection and also that end coordination during repair was compromised. We discuss models for why phage genomic recombination-dependent replication is more dependent on Mre11/Rad50 levels when compared to plasmid recombination-dependent replication. We also tested the importance of the conserved histidine residue in nuclease motif I of the T4 Mre11 protein. Substitution with multiple different amino acids (including serine) failed to support phage growth, completely blocked plasmid recombination-dependent replication, and led to the stabilization of double-strand ends. We also constructed and expressed an Mre11 mutant protein with the conserved histidine changed to serine. The mutant protein was found to be completely defective for nuclease activities, but retained the ability to bind the Rad50 subunit and double-stranded DNA. These results indicate that the nuclease activity of Mre11 is critical for phage growth and recombination-dependent replication during T4 infections.

  15. Escherichia coli radD (yejH) gene: a novel function involved in radiation resistance and double-strand break repair

    OpenAIRE

    Chen, Stefanie H.; Byrne, Rose T.; Wood, Elizabeth A; Cox, Michael M.

    2015-01-01

    A transposon insertion screen implicated the yejH gene in the repair of ionizing radiation-induced damage. The yejH gene, which exhibits significant homology to the human transcription-coupled DNA repair gene XPB, is involved in the repair of double strand DNA breaks. Deletion of yejH significantly sensitized cells to agents that cause double strand breaks (ionizing radiation, UV radiation, ciprofloxacin). In addition, deletion of both yejH and radA hypersensitized the cells to ionizing radia...

  16. Extensive ssDNA end formation at DNA double-strand breaks in non-homologous end-joining deficient cells during the S phase

    Directory of Open Access Journals (Sweden)

    Stenerlöw Bo

    2007-10-01

    Full Text Available Abstract Background Efficient and correct repair of DNA damage, especially DNA double-strand breaks, is critical for cellular survival. Defects in the DNA repair may lead to cell death or genomic instability and development of cancer. Non-homologous end-joining (NHEJ is the major repair pathway for DNA double-strand breaks in mammalian cells. The ability of other repair pathways, such as homologous recombination, to compensate for loss of NHEJ and the ways in which contributions of different pathways are regulated are far from fully understood. Results In this report we demonstrate that long single-stranded DNA (ssDNA ends are formed at radiation-induced DNA double-strand breaks in NHEJ deficient cells. At repair times ≥ 1 h, processing of unrejoined DNA double-strand breaks generated extensive ssDNA at the DNA ends in cells lacking the NHEJ protein complexes DNA-dependent protein kinase (DNA-PK or DNA Ligase IV/XRCC4. The ssDNA formation was cell cycle dependent, since no ssDNA ends were observed in G1-synchronized NHEJ deficient cells. Furthermore, in wild type cells irradiated in the presence of DNA-PKcs (catalytic subunit of DNA-PK inhibitors, or in DNA-PKcs deficient cells complemented with DNA-PKcs mutated in six autophosphorylation sites (ABCDE, no ssDNA was formed. The ssDNA generation also greatly influences DNA double-strand break quantification by pulsed-field gel electrophoresis, resulting in overestimation of the DNA double-strand break repair capability in NHEJ deficient cells when standard protocols for preparing naked DNA (i. e., lysis at 50°C are used. Conclusion We provide evidence that DNA Ligase IV/XRCC4 recruitment by DNA-PK to DNA double-strand breaks prevents the formation of long ssDNA ends at double-strand breaks during the S phase, indicating that NHEJ components may downregulate an alternative repair process where ssDNA ends are required.

  17. Local thermodynamics of the water molecules around single- and double-stranded DNA studied by grid inhomogeneous solvation theory

    Science.gov (United States)

    Nakano, Miki; Tateishi-Karimata, Hisae; Tanaka, Shigenori; Tama, Florence; Miyashita, Osamu; Nakano, Shu-ichi; Sugimoto, Naoki

    2016-09-01

    Thermodynamic properties of water molecules around single- and double-stranded DNAs (ssDNAs and dsDNAs) with different sequences were investigated using grid inhomogeneous solvation theory. Free energies of water molecules solvating the minor groove of dsDNAs are lower than those near ssDNAs, while water molecules should be released during the formation of dsDNA. Free energies of water molecules around dsDNA are lower than those around ssDNA even in the second and third hydration shells. Our findings will help to clarify the role of water molecules in the formation of dsDNA from ssDNAs, thus facilitating the designs of drugs or nanomaterials using DNA.

  18. Human RNF169 is a negative regulator of the ubiquitin-dependent response to DNA double-strand breaks

    DEFF Research Database (Denmark)

    Poulsen, Maria; Lukas, Claudia; Lukas, Jiri;

    2012-01-01

    Nonproteolytic ubiquitylation of chromatin surrounding deoxyribonucleic acid double-strand breaks (DSBs), mediated by the RNF8/RNF168 ubiquitin ligases, plays a key role in recruiting repair factors, including 53BP1 and BRCA1, to reestablish genome integrity. In this paper, we show that human RNF......169, an uncharacterized E3 ubiquitin ligase paralogous to RNF168, accumulated in DSB repair foci through recognition of RNF168-catalyzed ubiquitylation products by its motif interacting with ubiquitin domain. Unexpectedly, RNF169 was dispensable for chromatin ubiquitylation and ubiquitin......-dependent accumulation of repair factors at DSB sites. Instead, RNF169 functionally competed with 53BP1 and RAP80-BRCA1 for association with RNF168-modified chromatin independent of its catalytic activity, limiting the magnitude of their recruitment to DSB sites. By delaying accumulation of 53BP1 and RAP80 at damaged...

  19. The ubiquitin-selective segregase VCP/p97 orchestrates the response to DNA double-strand breaks

    DEFF Research Database (Denmark)

    Meerang, Mayura; Ritz, Danilo; Paliwal, Shreya;

    2011-01-01

    Unrepaired DNA double-strand breaks (DSBs) cause genetic instability that leads to malignant transformation or cell death. Cells respond to DSBs with the ordered recruitment of signalling and repair proteins to the site of lesion. Protein modification with ubiquitin is crucial for the signalling...... complexes and efficient DSB repair after exposure to ionizing radiation. p97 is recruited to DNA lesions by its ubiquitin adaptor UFD1-NPL4 and Lys-48-linked ubiquitin (K48-Ub) chains, whose formation is regulated by RNF8. p97 subsequently removes K48-Ub conjugates from sites of DNA damage to orchestrate...... proper association of 53BP1, BRCA1 and RAD51, three factors critical for DNA repair and genome surveillance mechanisms. Impairment of p97 activity decreases the level of DSB repair and cell survival after exposure to ionizing radiation. These findings identify the p97-UFD1-NPL4 complex as an essential...

  20. A new powerful method for site-specific transgene stabilization based on chromosomal double-strand break repair.

    Directory of Open Access Journals (Sweden)

    Artem Tkachuk

    Full Text Available Transgenic insects are a promising tool in sterile insect techniques and population replacement strategies. Such transgenic insects can be created using nonautonomous transposons, which cannot be transferred without a transposase source. In biocontrol procedures where large numbers of insects are released, there is increased risk of transgene remobilization caused by external transposase sources that can alter the characteristics of the transgenic organisms lead horizontal transgene transfer to other species. Here we describe a novel, effective method for transgene stabilization based on the introduction of directed double-strand breaks (DSB into a genome-integrated sequence and their subsequent repair by the single-strand annealing (SSA pathway. Due to the construct's organization, the repair pathway is predictable, such that all transposon and marker sequences can be deleted, while preserving integration of exogenous DNA in the genome. The exceptional conservation of DNA repair pathways makes this method suitable for a broad range of organisms.

  1. Study of Interaction between Red-tide Toxin, Domoic Acid and Double -stranded DNA by Capillary Zone Electrophoresis

    Institute of Scientific and Technical Information of China (English)

    Da Zhi LI; Xin Ya HE; Hui WANG; Li SUN; Bing Cheng LIN

    2004-01-01

    The interactions between amnesic red-tide toxin, domoic acid (DA) and 14mer double-stranded DNA (dsDNA with three kinds of sequences) were studied by capillary zone electrophoresis (CZE). For the dsDNA with a sequence of 5'-CCCCCTATACCCGC-3', the amount of free dsDNA decreases with the increase of added DA; and the signal of DA-dsDNA complex was observed. Meanwhile, the other two dsDNAs, 5'-(C)12GC-3' and 5'-(AT)7-3', the existence of DA could not lead to the change of dsDNA signal and indicated that there is no interaction between DA and these two dsDNAs.

  2. Simple Elastic Network Models for Exhaustive Analysis of Long Double-Stranded DNA Dynamics with Sequence Geometry Dependence.

    Directory of Open Access Journals (Sweden)

    Shuhei Isami

    Full Text Available Simple elastic network models of DNA were developed to reveal the structure-dynamics relationships for several nucleotide sequences. First, we propose a simple all-atom elastic network model of DNA that can explain the profiles of temperature factors for several crystal structures of DNA. Second, we propose a coarse-grained elastic network model of DNA, where each nucleotide is described only by one node. This model could effectively reproduce the detailed dynamics obtained with the all-atom elastic network model according to the sequence-dependent geometry. Through normal-mode analysis for the coarse-grained elastic network model, we exhaustively analyzed the dynamic features of a large number of long DNA sequences, approximately ∼150 bp in length. These analyses revealed positive correlations between the nucleosome-forming abilities and the inter-strand fluctuation strength of double-stranded DNA for several DNA sequences.

  3. Subtelomeric I-SceI-Mediated Double-Strand Breaks Are Repaired by Homologous Recombination in Trypanosoma cruzi

    Science.gov (United States)

    Chiurillo, Miguel A.; Moraes Barros, Roberto R.; Souza, Renata T.; Marini, Marjorie M.; Antonio, Cristiane R.; Cortez, Danielle R.; Curto, María Á.; Lorenzi, Hernán A.; Schijman, Alejandro G.; Ramirez, José L.; da Silveira, José F.

    2016-01-01

    Trypanosoma cruzi chromosome ends are enriched in surface protein genes and pseudogenes (e.g., trans-sialidases) surrounded by repetitive sequences. It has been proposed that the extensive sequence variability among members of these protein families could play a role in parasite infectivity and evasion of host immune response. In previous reports we showed evidence suggesting that sequences located in these regions are subjected to recombination. To support this hypothesis we introduced a double-strand break (DSB) at a specific target site in a T. cruzi subtelomeric region cloned into an artificial chromosome (pTAC). This construct was used to transfect T. cruzi epimastigotes expressing the I-SceI meganuclease. Examination of the repaired sequences showed that DNA repair occurred only through homologous recombination (HR) with endogenous subtelomeric sequences. Our findings suggest that DSBs in subtelomeric repetitive sequences followed by HR between them may contribute to increased variability in T. cruzi multigene families. PMID:28066363

  4. Simple Elastic Network Models for Exhaustive Analysis of Long Double-Stranded DNA Dynamics with Sequence Geometry Dependence

    CERN Document Server

    Isami, Shuhei; Nishimori, Hiraku; Awazu, Akinori

    2015-01-01

    Simple elastic network models of DNA were developed to reveal the structure-dynamics relationships for several nucleotide sequences. First, we propose a simple all-atom elastic network model of DNA that can explain the profiles of temperature factors for several crystal structures of DNA. Second, we propose a coarse-grained elastic network model of DNA, where each nucleotide is described only by one node. This model could effectively reproduce the detailed dynamics obtained with the all-atom elastic network model according to the sequence-dependent geometry. Through normal-mode analysis for the coarse-grained elastic network model, we exhaustively analyzed the dynamic features of a large number of long DNA sequences, approximately $\\sim 150$ bp in length. These analyses revealed positive correlations between the nucleosome-forming abilities and the inter-strand fluctuation strength of double-stranded DNA for several DNA sequences.

  5. A New Powerful Method for Site-Specific Transgene Stabilization Based on Chromosomal Double-Strand Break Repair

    Science.gov (United States)

    Kravchuk, Oksana; Savitsky, Mikhail

    2011-01-01

    Transgenic insects are a promising tool in sterile insect techniques and population replacement strategies. Such transgenic insects can be created using nonautonomous transposons, which cannot be transferred without a transposase source. In biocontrol procedures where large numbers of insects are released, there is increased risk of transgene remobilization caused by external transposase sources that can alter the characteristics of the transgenic organisms lead horizontal transgene transfer to other species. Here we describe a novel, effective method for transgene stabilization based on the introduction of directed double-strand breaks (DSB) into a genome-integrated sequence and their subsequent repair by the single-strand annealing (SSA) pathway. Due to the construct's organization, the repair pathway is predictable, such that all transposon and marker sequences can be deleted, while preserving integration of exogenous DNA in the genome. The exceptional conservation of DNA repair pathways makes this method suitable for a broad range of organisms. PMID:22022613

  6. Endonuclease-based Method for Detecting the Sequence Specific DNA Binding Protein on Double-stranded DNA Microarray

    Institute of Scientific and Technical Information of China (English)

    Yun Fei BAI; Qin Yu GE; Tong Xiang LI; Jin Ke WANG; Quan Jun LIU; Zu Hong LU

    2005-01-01

    The double-stranded DNA (dsDNA) probe contains two different protein binding sites.One is for DNA- binding proteins to be detected and the other is for a DNA restriction enzyme.The two sites were arranged together with no base interval. The working principle of the capturing dsDNA probe is described as follows: the capturing probe can be cut with the DNA restriction enzyme (such as EcoR I) to cause a sticky terminal, if the probe is not bound with a target protein, and the sticky terminal can be extended and labeled with Cy3-dUTP by DNA polymerase. When the probe is bound with a target protein, the probe is not capable to be cut by the restriction enzyme because of space obstruction. The amount of the target DNA binding proteins can be measured according to the variations of fluorescent signals of the corresponding probes.

  7. Subtelomeric I-SceI-Mediated Double-Strand Breaks Are Repaired by Homologous Recombination in Trypanosoma cruzi.

    Science.gov (United States)

    Chiurillo, Miguel A; Moraes Barros, Roberto R; Souza, Renata T; Marini, Marjorie M; Antonio, Cristiane R; Cortez, Danielle R; Curto, María Á; Lorenzi, Hernán A; Schijman, Alejandro G; Ramirez, José L; da Silveira, José F

    2016-01-01

    Trypanosoma cruzi chromosome ends are enriched in surface protein genes and pseudogenes (e.g., trans-sialidases) surrounded by repetitive sequences. It has been proposed that the extensive sequence variability among members of these protein families could play a role in parasite infectivity and evasion of host immune response. In previous reports we showed evidence suggesting that sequences located in these regions are subjected to recombination. To support this hypothesis we introduced a double-strand break (DSB) at a specific target site in a T. cruzi subtelomeric region cloned into an artificial chromosome (pTAC). This construct was used to transfect T. cruzi epimastigotes expressing the I-SceI meganuclease. Examination of the repaired sequences showed that DNA repair occurred only through homologous recombination (HR) with endogenous subtelomeric sequences. Our findings suggest that DSBs in subtelomeric repetitive sequences followed by HR between them may contribute to increased variability in T. cruzi multigene families.

  8. Effect of pH and Salt on Adsorption of Double-Stranded DNA on Graphene Oxide.

    Science.gov (United States)

    Kim, Seyeon; Park, Chanoong; Gang, Jongback

    2015-10-01

    Graphene oxide (GO) has a large surface-to-volume ratio and hydrophobic hexagonal rings that can interact with biomolecules. Single-stranded DNA adsorbs strongly to the surface of GO via hydrophobic interactions. GO has been used in optical biosensors and biomedical platforms for the detection of DNA, proteins, and small molecules. This study was designed to measure the adsorption of double-stranded DNA (dsDNA) onto GO according to DNA length, salt concentration, and pH of the reaction. Results showed that dsDNA molecules were adsorbed progressively as the pH changed from 6.0 to 4.0. At high pH, dsDNA adsorption was enhanced by the presence of MgCl2 rather than NaCl. Desorption of DNA from GO, with triton X-100 led to the rapid release of DNA from GO in the presence of MgCl2.

  9. Double-strand breaks in heterochromatin move outside of a dynamic HP1a domain to complete recombinational repair.

    Science.gov (United States)

    Chiolo, Irene; Minoda, Aki; Colmenares, Serafin U; Polyzos, Aris; Costes, Sylvain V; Karpen, Gary H

    2011-03-04

    Double-strand breaks (DSBs) in heterochromatic repetitive DNAs pose significant threats to genome integrity, but information about how such lesions are processed and repaired is sparse. We observe dramatic expansion and dynamic protrusions of the heterochromatin domain in response to ionizing radiation (IR) in Drosophila cells. We also find that heterochromatic DSBs are repaired by homologous recombination (HR) but with striking differences from euchromatin. Proteins involved in early HR events (resection) are rapidly recruited to DSBs within heterochromatin. In contrast, Rad51, which mediates strand invasion, only associates with DSBs that relocalize outside of the domain. Heterochromatin expansion and relocalization of foci require checkpoint and resection proteins. Finally, the Smc5/6 complex is enriched in heterochromatin and is required to exclude Rad51 from the domain and prevent abnormal recombination. We propose that the spatial and temporal control of DSB repair in heterochromatin safeguards genome stability by preventing aberrant exchanges between repeats.

  10. The structure-specific endonuclease Mus81 contributes to replication restart by generating double-strand DNA breaks.

    Science.gov (United States)

    Hanada, Katsuhiro; Budzowska, Magda; Davies, Sally L; van Drunen, Ellen; Onizawa, Hideo; Beverloo, H Berna; Maas, Alex; Essers, Jeroen; Hickson, Ian D; Kanaar, Roland

    2007-11-01

    Faithful duplication of the genome requires structure-specific endonucleases such as the RuvABC complex in Escherichia coli. These enzymes help to resolve problems at replication forks that have been disrupted by DNA damage in the template. Much less is known about the identities of these enzymes in mammalian cells. Mus81 is the catalytic component of a eukaryotic structure-specific endonuclease that preferentially cleaves branched DNA substrates reminiscent of replication and recombination intermediates. Here we explore the mechanisms by which Mus81 maintains chromosomal stability. We found that Mus81 is involved in the formation of double-strand DNA breaks in response to the inhibition of replication. Moreover, in the absence of chromosome processing by Mus81, recovery of stalled DNA replication forks is attenuated and chromosomal aberrations arise. We suggest that Mus81 suppresses chromosomal instability by converting potentially detrimental replication-associated DNA structures into intermediates that are more amenable to DNA repair.

  11. Atrazine Triggers DNA Damage Response and Induces DNA Double-Strand Breaks in MCF-10A Cells

    Directory of Open Access Journals (Sweden)

    Peixin Huang

    2015-06-01

    Full Text Available Atrazine, a pre-emergent herbicide in the chloro-s-triazine family, has been widely used in crop lands and often detected in agriculture watersheds, which is considered as a potential threat to human health. Although atrazine and its metabolites showed an elevated incidence of mammary tumors in female Sprague–Dawley (SD rats, no molecular evidence was found relevant to its carcinogenesis in humans. This study aims to determine whether atrazine could induce the expression of DNA damage response-related proteins in normal human breast epithelial cells (MCF-10A and to examine the cytotoxicity of atrazine at a molecular level. Our results indicate that a short-term exposure of MCF-10A to an environmentally-detectable concentration of atrazine (0.1 µg/mL significantly increased the expression of tumor necrosis factor receptor-1 (TNFR1 and phosphorylated Rad17 in the cells. Atrazine treatment increased H2AX phosphorylation (γH2AX and the formation of γH2AX foci in the nuclei of MCF-10A cells. Atrazine also sequentially elevated DNA damage checkpoint proteins of ATM- and RAD3-related (ATR, ATRIP and phospho-Chk1, suggesting that atrazine could induce DNA double-strand breaks and trigger the DNA damage response ATR-Chk1 pathway in MCF-10A cells. Further investigations are needed to determine whether atrazine-triggered DNA double-strand breaks and DNA damage response ATR-Chk1 pathway occur in vivo.

  12. Deficiency of double-strand DNA break repair does not impair Mycobacterium tuberculosis virulence in multiple animal models of infection.

    Science.gov (United States)

    Heaton, Brook E; Barkan, Daniel; Bongiorno, Paola; Karakousis, Petros C; Glickman, Michael S

    2014-08-01

    Mycobacterium tuberculosis persistence within its human host requires mechanisms to resist the effector molecules of host immunity, which exert their bactericidal effects through damaging pathogen proteins, membranes, and DNA. Substantial evidence indicates that bacterial pathogens, including M. tuberculosis, require DNA repair systems to repair the DNA damage inflicted by the host during infection, but the role of double-strand DNA break (DSB) repair systems is unclear. Double-strand DNA breaks are the most cytotoxic form of DNA damage and must be repaired for chromosome replication to proceed. M. tuberculosis elaborates three genetically distinct DSB repair systems: homologous recombination (HR), nonhomologous end joining (NHEJ), and single-strand annealing (SSA). NHEJ, which repairs DSBs in quiescent cells, may be particularly relevant to M. tuberculosis latency. However, very little information is available about the phenotype of DSB repair-deficient M. tuberculosis in animal models of infection. Here we tested M. tuberculosis strains lacking NHEJ (a Δku ΔligD strain), HR (a ΔrecA strain), or both (a ΔrecA Δku strain) in C57BL/6J mice, C3HeB/FeJ mice, guinea pigs, and a mouse hollow-fiber model of infection. We found no difference in bacterial load, histopathology, or host mortality between wild-type and DSB repair mutant strains in any model of infection. These results suggest that the animal models tested do not inflict DSBs on the mycobacterial chromosome, that other repair pathways can compensate for the loss of NHEJ and HR, or that DSB repair is not required for M. tuberculosis pathogenesis.

  13. Repair on the go: E. coli maintains a high proliferation rate while repairing a chronic DNA double-strand break.

    Directory of Open Access Journals (Sweden)

    Elise Darmon

    Full Text Available DNA damage checkpoints exist to promote cell survival and the faithful inheritance of genetic information. It is thought that one function of such checkpoints is to ensure that cell division does not occur before DNA damage is repaired. However, in unicellular organisms, rapid cell multiplication confers a powerful selective advantage, leading to a dilemma. Is the activation of a DNA damage checkpoint compatible with rapid cell multiplication? By uncoupling the initiation of DNA replication from cell division, the Escherichia coli cell cycle offers a solution to this dilemma. Here, we show that a DNA double-strand break, which occurs once per replication cycle, induces the SOS response. This SOS induction is needed for cell survival due to a requirement for an elevated level of expression of the RecA protein. Cell division is delayed, leading to an increase in average cell length but with no detectable consequence on mutagenesis and little effect on growth rate and viability. The increase in cell length caused by chronic DNA double-strand break repair comprises three components: two types of increase in the unit cell size, one independent of SfiA and SlmA, the other dependent of the presence of SfiA and the absence of SlmA, and a filamentation component that is dependent on the presence of either SfiA or SlmA. These results imply that chronic checkpoint induction in E. coli is compatible with rapid cell multiplication. Therefore, under conditions of chronic low-level DNA damage, the SOS checkpoint operates seamlessly in a cell cycle where the initiation of DNA replication is uncoupled from cell division.

  14. C. elegans germ cells switch between distinct modes of double-strand break repair during meiotic prophase progression.

    Directory of Open Access Journals (Sweden)

    Michiko Hayashi

    2007-11-01

    Full Text Available Chromosome inheritance during sexual reproduction relies on deliberate induction of double-strand DNA breaks (DSBs and repair of a subset of these breaks as interhomolog crossovers (COs. Here we provide a direct demonstration, based on our analysis of rad-50 mutants, that the meiotic program in Caenorhabditis elegans involves both acquisition and loss of a specialized mode of double-strand break repair (DSBR. In premeiotic germ cells, RAD-50 is not required to load strand-exchange protein RAD-51 at sites of spontaneous or ionizing radiation (IR-induced DSBs. A specialized meiotic DSBR mode is engaged at the onset of meiotic prophase, coincident with assembly of meiotic chromosome axis structures. This meiotic DSBR mode is characterized both by dependence on RAD-50 for rapid accumulation of RAD-51 at DSB sites and by competence for converting DSBs into interhomolog COs. At the mid-pachytene to late pachytene transition, germ cells undergo an abrupt release from the meiotic DSBR mode, characterized by reversion to RAD-50-independent loading of RAD-51 and loss of competence to convert DSBs into interhomolog COs. This transition in DSBR mode is dependent on MAP kinase-triggered prophase progression and coincides temporally with a major remodeling of chromosome architecture. We propose that at least two developmentally programmed switches in DSBR mode, likely conferred by changes in chromosome architecture, operate in the C. elegans germ line to allow formation of meiotic crossovers without jeopardizing genomic integrity. Our data further suggest that meiotic cohesin component REC-8 may play a role in limiting the activity of SPO-11 in generating meiotic DSBs and that RAD-50 may function in counteracting this inhibition.

  15. Binding of synthetic double-stranded DNA by serum from patients with systemic lupus erythematosus: correlation with renal histology.

    Science.gov (United States)

    Steinman, C R; Grishman, E; Spiera, H; Deesomochok, U

    1977-03-01

    Detection of antibody to double-stranded DNA by direct binding assays has proved useful in clinical management of patients with systemic lupus erythematosus (SLE). Recent confusion regarding specificity of these antibodies for SLE appears to be due, at least in part, to contamination of natural DNA preparations with nondouble-stranded DNA antigens. Measurement of binding of a synthetic, self-complementary DNA copolymer (dAT) rather than of natural DNA (KB) has been shown to obviate some of these difficulties, apparently because of freedom of dAT from nondouble-stranded DNA antigens. Among the advantages found in this way was a higher degree of specificity of antibodies to double-stranded DNA for clinically-judged active lupus nephritis than had been suspected. Since activity of nephritis is difficult to assess clinically, histologic data were sought to confirm these observations. Thirty-two kidney specimens were examined by light and/or electron microscopy. The degree of histologic activity and the amount and location of glomerular electron-dense deposits were semiquantitated blindly. The binding of both dAT and KB DNA was measured by the ammonium sulfate method. Correlation with the amount of electron-defense deposits was highly significant for dAT binding and somewhat less so for KB DNA binding as determined by both parametric and nonparametric statistical methods. Significant correlation with histologic activity was found for dAT but not KB DNA binding. These results are consistent with previous data and suggest that dAT binding may provide a useful, noninvasive means of clinically assessing both nephritis activity and the intensity of glomerular immune-complex deposition as reflected by the amount of electron-dense deposits. If it can be confirmed that the latter provides long-term prognostic information, then dAT binding (and perhaps its reponse to therapy) may also prove of value in this regard.

  16. Human RECQ1 interacts with Ku70/80 and modulates DNA end-joining of double-strand breaks.

    Directory of Open Access Journals (Sweden)

    Swetha Parvathaneni

    Full Text Available Genomic instability is a known precursor to cancer and aging. The RecQ helicases are a highly conserved family of DNA-unwinding enzymes that play key roles in maintaining genome stability in all living organisms. Human RecQ homologs include RECQ1, BLM, WRN, RECQ4, and RECQ5β, three of which have been linked to diseases with elevated risk of cancer and growth defects (Bloom Syndrome and Rothmund-Thomson Syndrome or premature aging (Werner Syndrome. RECQ1, the first RecQ helicase discovered and the most abundant in human cells, is the least well understood of the five human RecQ homologs. We have previously described that knockout of RECQ1 in mice or knockdown of its expression in human cells results in elevated frequency of spontaneous sister chromatid exchanges, chromosomal instability, increased load of DNA damage and heightened sensitivity to ionizing radiation. We have now obtained evidence implicating RECQ1 in the nonhomologous end-joining pathway of DNA double-strand break repair. We show that RECQ1 interacts directly with the Ku70/80 subunit of the DNA-PK complex, and depletion of RECQ1 results in reduced end-joining in cell free extracts. In vitro, RECQ1 binds and unwinds the Ku70/80-bound partial duplex DNA substrate efficiently. Linear DNA is co-bound by RECQ1 and Ku70/80, and DNA binding by Ku70/80 is modulated by RECQ1. Collectively, these results provide the first evidence for an interaction of RECQ1 with Ku70/80 and a role of the human RecQ helicase in double-strand break repair through nonhomologous end-joining.

  17. Artemis is required to improve the accuracy of repair of double-strand breaks with 5'-blocked termini generated from non-DSB-clustered lesions.

    Science.gov (United States)

    Malyarchuk, Svitlana; Castore, Reneau; Shi, Runhua; Harrison, Lynn

    2013-05-01

    Clustered DNA lesions are defined as ≥2 damage events within 20 bp. Oxidised bases, abasic (AP) sites, single-strand breaks and double-strand breaks (DSBs) exist in radiation-induced clusters, and these lesions are more difficult to repair and can be more mutagenic than single lesions. Understanding clustered lesion repair is therefore important for the design of complementary treatments to enhance radiotherapy. Non-DSB-clustered lesions consisting of opposing AP sites can be converted to DSBs by base excision repair, and non-homologous end-joining (NHEJ) plays a role in repairing these DSBs. Artemis is an endonuclease that removes blocking groups from DSB termini during NHEJ. Hence, we hypothesised that Artemis plays a role in the processing of DSBs or complex DSBs generated from non-DSB-clustered lesions. We examined the repair of clusters containing two or three lesions in wild-type (WT) or Artemis-deficient (ART(-/-)) mouse fibroblasts using a reporter plasmid. Each cluster contained two opposing tetrahydrofurans (an AP site analogue), which AP endonuclease can convert to a DSB with blocked 5' termini. Loss of Artemis did not decrease plasmid survival, but did result in more mutagenic repair with plasmids containing larger deletions. This increase in deletions did not occur with ClaI-linearised plasmid. Since Mre11 has been implicated in deletional NHEJ, we used small interfering RNA to reduce Mre11 in WT and ART(-/-) cells, but decreasing Mre11 did not change the size of deletions in the repair products. This work implicates Artemis in limiting the deletions introduced during repair of 5'-blocked termini DSBs generated from non-DSB-clustered lesions. Decreasing repair accuracy without decreasing repair capacity could result in mutated cells surviving irradiation. Inhibiting Artemis in normal cells could promote carcinogenesis, while in tumour cells enhanced mutagenic repair following irradiation could promote tumour recurrence.

  18. DNA-PK triggers histone ubiquitination and signaling in response to DNA double-strand breaks produced during the repair of transcription-blocking topoisomerase I lesions.

    Science.gov (United States)

    Cristini, Agnese; Park, Joon-Hyung; Capranico, Giovanni; Legube, Gaëlle; Favre, Gilles; Sordet, Olivier

    2016-02-18

    Although defective repair of DNA double-strand breaks (DSBs) leads to neurodegenerative diseases, the processes underlying their production and signaling in non-replicating cells are largely unknown. Stabilized topoisomerase I cleavage complexes (Top1cc) by natural compounds or common DNA alterations are transcription-blocking lesions whose repair depends primarily on Top1 proteolysis and excision by tyrosyl-DNA phosphodiesterase-1 (TDP1). We previously reported that stabilized Top1cc produce transcription-dependent DSBs that activate ATM in neurons. Here, we use camptothecin (CPT)-treated serum-starved quiescent cells to induce transcription-blocking Top1cc and show that those DSBs are generated during Top1cc repair from Top1 peptide-linked DNA single-strand breaks generated after Top1 proteolysis and before excision by TDP1. Following DSB induction, ATM activates DNA-PK whose inhibition suppresses H2AX and H2A ubiquitination and the later assembly of activated ATM into nuclear foci. Inhibition of DNA-PK also reduces Top1 ubiquitination and proteolysis as well as resumption of RNA synthesis suggesting that DSB signaling further enhances Top1cc repair. Finally, we show that co-transcriptional DSBs kill quiescent cells. Together, these new findings reveal that DSB production and signaling by transcription-blocking Top1 lesions impact on non-replicating cell fate and provide insights on the molecular pathogenesis of neurodegenerative diseases such as SCAN1 and AT syndromes, which are caused by TDP1 and ATM deficiency, respectively.

  19. Identification and Characterization of Second-Generation Invader Locked Nucleic Acids (LNAs) for Mixed-Sequence Recognition of Double-Stranded DNA

    DEFF Research Database (Denmark)

    Sau, Sujay P; Madsen, Andreas S; Podbevsek, Peter;

    2013-01-01

    The development of synthetic agents that recognize double-stranded DNA (dsDNA) is a long-standing goal that is inspired by the promise for tools that detect, regulate, and modify genes. Progress has been made with triplex-forming oligonucleotides, peptide nucleic acids, and polyamides, but substa...

  20. Correlation between slowly repairable double-strand breaks and thermal radiosensitization in the human HeLa S3 cell line

    NARCIS (Netherlands)

    Kampinga, HH; Hiemstra, YS; Konings, AWT; Dikomey, E

    1997-01-01

    The effect of heat on double-strand breaks (dsb) repair was compared with thermal radiosensitization using HeLa S3 cells. Cells were exposed to a combined treatment of X-irradiation followed by heat (44 degrees C, 0.5 h) separated by time intervals up to 8h. DNA dsb were measured by PFGE and surviva

  1. The Caenorhabditis elegans homolog of Gen1/Yen1 resolvases links DNA damage signaling to DNA double-strand break repair.

    Directory of Open Access Journals (Sweden)

    Aymeric P Bailly

    2010-07-01

    Full Text Available DNA double-strand breaks (DSBs can be repaired by homologous recombination (HR, which can involve Holliday junction (HJ intermediates that are ultimately resolved by nucleolytic enzymes. An N-terminal fragment of human GEN1 has recently been shown to act as a Holliday junction resolvase, but little is known about the role of GEN-1 in vivo. Holliday junction resolution signifies the completion of DNA repair, a step that may be coupled to signaling proteins that regulate cell cycle progression in response to DNA damage. Using forward genetic approaches, we identified a Caenorhabditis elegans dual function DNA double-strand break repair and DNA damage signaling protein orthologous to the human GEN1 Holliday junction resolving enzyme. GEN-1 has biochemical activities related to the human enzyme and facilitates repair of DNA double-strand breaks, but is not essential for DNA double-strand break repair during meiotic recombination. Mutational analysis reveals that the DNA damage-signaling function of GEN-1 is separable from its role in DNA repair. GEN-1 promotes germ cell cycle arrest and apoptosis via a pathway that acts in parallel to the canonical DNA damage response pathway mediated by RPA loading, CHK1 activation, and CEP-1/p53-mediated apoptosis induction. Furthermore, GEN-1 acts redundantly with the 9-1-1 complex to ensure genome stability. Our study suggests that GEN-1 might act as a dual function Holliday junction resolvase that may coordinate DNA damage signaling with a late step in DNA double-strand break repair.

  2. Evidence of RNAi in humans from systemically administered siRNA via targeted nanoparticles

    OpenAIRE

    Davis, Mark E.; Zuckerman, Jonathan E.; Choi, Chung Hang J.; Seligson, David; Tolcher, Anthony; Alabi, Christopher A.; Yen, Yun; Heidel, Jeremy D.; Ribas, Antoni

    2010-01-01

    Therapeutics that are designed to engage RNA interference (RNAi) pathways have the potential to provide new, major ways of imparting therapy to patients. Long, double-stranded RNAs were first shown to mediate RNAi in Caenorhabditis elegans, and the potential use of RNAi for human therapy has been demonstrated by the finding that small interfering RNAs (siRNAs; approximately 21-base-pair double-stranded RNA) can elicit RNAi in mammalian cells without producing an interferon response. We are at...

  3. DMPD: Transcriptional signaling by double-stranded RNA: role of TLR3. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available csml) Open .csml file with CIOPlayer Open .csml file with CIOPlayer - ※CIO Playerのご利用上の注意 Open .csml file with CIO Open .csml file with CIO - ※CIOのご利用上の注意 ...

  4. A New Strategy of Insect Pest Control:Down-regulating Cotton Boliworm Gene Expression by Engineering Plant Double Stranded RNA

    Institute of Scientific and Technical Information of China (English)

    MAO Ying-bo; XUE Xue-yi; WANG Ling-jiang; CHEN Xiao-ya

    2008-01-01

    @@ Cotton bollworm (Helicoverpa armigera ) is an important agricultural pest that causes severeyield loss to crops,particularly to cotton.Transgenic Bt crops have been successful in protectingplants,however,Bt proteins are toxic to all lepidopteran insects but have little effects to sucking pests,such as aphids.Furthermore,the continuous use of Bt crops increases insect resistance.

  5. Beyond repair foci: DNA double-strand break repair in euchromatic and heterochromatic compartments analyzed by transmission electron microscopy.

    Directory of Open Access Journals (Sweden)

    Yvonne Lorat

    Full Text Available PURPOSE: DNA double-strand breaks (DSBs generated by ionizing radiation pose a serious threat to the preservation of genetic and epigenetic information. The known importance of local chromatin configuration in DSB repair raises the question of whether breaks in different chromatin environments are recognized and repaired by the same repair machinery and with similar efficiency. An essential step in DSB processing by non-homologous end joining is the high-affinity binding of Ku70-Ku80 and DNA-PKcs to double-stranded DNA ends that holds the ends in physical proximity for subsequent repair. METHODS AND MATERIALS: Using transmission electron microscopy to localize gold-labeled pKu70 and pDNA-PKcs within nuclear ultrastructure, we monitored the formation and repair of actual DSBs within euchromatin (electron-lucent and heterochromatin (electron-dense in cortical neurons of irradiated mouse brain. RESULTS: While DNA lesions in euchromatin (characterized by two pKu70-gold beads, reflecting the Ku70-Ku80 heterodimer are promptly sensed and rejoined, DNA packaging in heterochromatin appears to retard DSB processing, due to the time needed to unravel higher-order chromatin structures. Complex pKu70-clusters formed in heterochromatin (consisting of 4 or ≥ 6 gold beads may represent multiple breaks in close proximity caused by ionizing radiation of highly-compacted DNA. All pKu70-clusters disappeared within 72 hours post-irradiation, indicating efficient DSB rejoining. However, persistent 53BP1 clusters in heterochromatin (comprising ≥ 10 gold beads, occasionally co-localizing with γH2AX, but not pKu70 or pDNA-PKcs, may reflect incomplete or incorrect restoration of chromatin structure rather than persistently unrepaired DNA damage. DISCUSSION: Higher-order organization of chromatin determines the accessibility of DNA lesions to repair complexes, defining how readily DSBs are detected and processed. DNA lesions in heterochromatin appear to be more

  6. Rtt107 phosphorylation promotes localisation to DNA double-stranded breaks (DSBs and recombinational repair between sister chromatids.

    Directory of Open Access Journals (Sweden)

    Pranav Ullal

    Full Text Available Efficient repair of DNA double-stranded breaks (DSB requires a coordinated response at the site of lesion. Nucleolytic resection commits repair towards homologous recombination, which preferentially occurs between sister chromatids. DSB resection promotes recruitment of the Mec1 checkpoint kinase to the break. Rtt107 is a target of Mec1 and serves as a scaffold during repair. Rtt107 plays an important role during rescue of damaged replication forks, however whether Rtt107 contributes to the repair of DSBs is unknown. Here we show that Rtt107 is recruited to DSBs induced by the HO endonuclease. Rtt107 phosphorylation by Mec1 and its interaction with the Smc5-Smc6 complex are both required for Rtt107 loading to breaks, while Rtt107 regulators Slx4 and Rtt101 are not. We demonstrate that Rtt107 has an effect on the efficiency of sister chromatid recombination (SCR and propose that its recruitment to DSBs, together with the Smc5-Smc6 complex is important for repair through the SCR pathway.

  7. Do Exogenous DNA Double-Strand Breaks Change Incomplete Synapsis and Chiasma Localization in the Grasshopper Stethophyma grossum?

    Science.gov (United States)

    2016-01-01

    Meiotic recombination occurs as a programmed event that initiates by the formation of DNA double-strand breaks (DSBs) that give rise to the formation of crossovers that are observed as chiasmata. Chiasmata are essential for the accurate chromosome segregation and the generation of new combinations of parental alleles. Some treatments that provoke exogenous DSBs also lead to alterations in the recombination pattern of some species in which full homologous synapsis is achieved at pachytene. We have carried out a similar approach in males of the grasshopper Stethophyma grossum, whose homologues show incomplete synapsis and proximal chiasma localization. After irradiating males with γ rays we have studied the distribution of both the histone variant γ-H2AX and the recombinase RAD51. These proteins are cytological markers of DSBs at early prophase I. We have inferred synaptonemal complex (SC) formation via identification of SMC3 and RAD 21 cohesin subunits. Whereas thick and thin SMC3 filaments would correspond to synapsed and unsynapsed regions, the presence of RAD21 is only restricted to synapsed regions. Results show that irradiated spermatocytes maintain restricted synapsis between homologues. However, the frequency and distribution of chiasmata in metaphase I bivalents is slightly changed and quadrivalents were also observed. These results could be related to the singular nuclear polarization displayed by the spermatocytes of this species. PMID:28005992

  8. Establishment of a markerless mutation delivery system in Bacillus subtilis stimulated by a double-strand break in the chromosome.

    Directory of Open Access Journals (Sweden)

    Ting Shi

    Full Text Available Bacillus subtilis has been a model for gram-positive bacteria and it has long been exploited for industrial and biotechnological applications. However, the availability of facile genetic tools for physiological analysis has generally lagged substantially behind traditional genetic models such as Escherichia coli and Saccharomyces cerevisiae. In this work, we have developed an efficient, precise and scarless method for rapid multiple genetic modifications without altering the chromosome of B. subtilis. This method employs upp gene as a counter-selectable marker, double-strand break (DSB repair caused by exogenous endonuclease I-SceI and comK overexpression for fast preparation of competent cell. Foreign dsDNA can be simply and efficiently integrated into the chromosome by double-crossover homologous recombination. The DSB repair is a potent inducement for stimulating the second intramolecular homologous recombination, which not only enhances the frequency of resolution by one to two orders of magnitude, but also selects for the resolved product. This method has been successfully and reiteratively used in B. subtilis to deliver point mutations, to generate in-frame deletions, and to construct large-scale deletions. Experimental results proved that it allowed repeated use of the selectable marker gene for multiple modifications and could be a useful technique for B. subtilis.

  9. Induction of DNA double-strand breaks in primary gingival fibroblasts by exposure to dental resin composites.

    Science.gov (United States)

    Urcan, Ebru; Scherthan, Harry; Styllou, Marianthi; Haertel, Uschi; Hickel, Reinhard; Reichl, Franz-Xaver

    2010-03-01

    Dental resin composites and their reactive monomers/co-monomers have been shown to elicit cytotoxic responses in human gingival fibroblasts (HGF), and their metabolic radical intermediates have the potential to attack the DNA backbone, which may induce DNA double-strand breaks (DSBs). In this study we have tested the cytotoxicity and induction of DSBs by the most common composite resin monomers/co-monomers: BisGMA, HEMA, TEGDMA, and UDMA in gingival fibroblasts using the sensitive gamma-H2AX DNA repair focus assay. Our results show increasing monomer cytotoxicities in the order of BisGMA>UDMA>TEGDMA>HEMA, an order that was also observed for their capacity to induce DSBs. BisGMA at the EC50 concentration of 0.09 mm evoked the highest rate of gamma-H2AX foci-formation that was 11-fold higher DNA DSBs as compared to the negative controls that ranged between 0.25 and 0.5gamma-H2AX foci/HGF cell. Our results for the first time show that exposure to dental resin monomers can induce DSBs in primary human oral cavity cells, which underscores their genotoxic capacity.

  10. RecA Binding to a Single Double-Stranded DNA Molecule: A Possible Role of DNA Conformational Fluctuations

    Science.gov (United States)

    Leger, J. F.; Robert, J.; Bourdieu, L.; Chatenay, D.; Marko, J. F.

    1998-10-01

    Most genetic regulatory mechanisms involve protein-DNA interactions. In these processes, the classical Watson-Crick DNA structure sometimes is distorted severely, which in turn enables the precise recognition of the specific sites by the protein. Despite its key importance, very little is known about such deformation processes. To address this general question, we have studied a model system, namely, RecA binding to double-stranded DNA. Results from micromanipulation experiments indicate that RecA binds strongly to stretched DNA; based on this observation, we propose that spontaneous thermal stretching fluctuations may play a role in the binding of RecA to DNA. This has fundamental implications for the protein-DNA binding mechanism, which must therefore rely in part on a combination of flexibility and thermal fluctuations of the DNA structure. We also show that this mechanism is sequence sensitive. Theoretical simulations support this interpretation of our experimental results, and it is argued that this is of broad relevance to DNA-protein interactions.

  11. Double Strand Breaks and Cell-Cycle Arrest Induced by the Cyanobacterial Toxin Cylindrospermopsin in HepG2 Cells

    Directory of Open Access Journals (Sweden)

    Bojana Žegura

    2013-08-01

    Full Text Available The newly emerging cyanobacterial cytotoxin cylindrospermopsin (CYN is increasingly found in surface freshwaters, worldwide. It poses a potential threat to humans after chronic exposure as it was shown to be genotoxic in a range of test systems and is potentially carcinogenic. However, the mechanisms of CYN toxicity and genotoxicity are not well understood. In the present study CYN induced formation of DNA double strand breaks (DSBs, after prolonged exposure (72 h, in human hepatoma cells, HepG2. CYN (0.1–0.5 µg/mL, 24–96 h induced morphological changes and reduced cell viability in a dose and time dependent manner. No significant increase in lactate dehydrogenase (LDH leakage could be observed after CYN exposure, indicating that the reduction in cell number was due to decreased cell proliferation and not due to cytotoxicity. This was confirmed by imunocytochemical analysis of the cell-proliferation marker Ki67. Analysis of the cell-cycle using flow-cytometry showed that CYN has an impact on the cell cycle, indicating G0/G1 arrest after 24 h and S-phase arrest after longer exposure (72 and 96 h. Our results provide new evidence that CYN is a direct acting genotoxin, causing DSBs, and these facts need to be considered in the human health risk assessment.

  12. Synthetic lethal targeting of DNA double strand break repair deficient cells by human apurinic/apyrimidinic endonuclease (APE1) inhibitors

    Science.gov (United States)

    Sultana, Rebeka; McNeill, Daniel R.; Abbotts, Rachel; Mohammed, Mohammed Z.; Zdzienicka, Małgorzata Z.; Qutob, Haitham; Seedhouse, Claire; Laughton, Charles A.; Fischer, Peter M.; Patel, Poulam M.; Wilson, David M.; Madhusudan, Srinivasan

    2013-01-01

    An apurinic/apyrimidinic (AP) site is an obligatory cytotoxic intermediate in DNA Base Excision Repair (BER) that is processed by human AP endonuclease 1 (APE1). APE1 is essential for BER and an emerging drug target in cancer. We have isolated novel small molecule inhibitors of APE1. In the current study we have investigated the ability of APE1 inhibitors to induce synthetic lethality in a panel of DNA double strand break (DSB) repair deficient and proficient cells; a) Chinese hamster (CH) cells: BRCA2 deficient (V-C8), ATM deficient (V-E5), wild type (V79) and BRCA2 revertant (V-C8(Rev1)). b) Human cancer cells: BRCA1 deficient (MDA-MB-436), BRCA1 proficient (MCF-7), BRCA2 deficient (CAPAN-1 and HeLa SilenciX cells), BRCA2 proficient (PANC1 and control SilenciX cells). We also tested synthetic lethality (SL) in CH ovary cells expressing a dominant–negative form of APE1 (E8 cells) using ATM inhibitors and DNA-PKcs inhibitors (DSB inhibitors). APE1 inhibitors are synthetically lethal in BRCA and ATM deficient cells. APE1 inhibition resulted in accumulation of DNA DSBs and G2/M cell cycle arrest. Synthetic lethality was also demonstrated in CH cells expressing a dominant–negative form of APE1 treated with ATM or DNA-PKcs inhibitors. We conclude that APE1 is a promising synthetic lethality target in cancer. PMID:22377908

  13. Non-redundant Functions of ATM and DNA-PKcs in Response to DNA Double-Strand Breaks

    Directory of Open Access Journals (Sweden)

    Pierre Caron

    2015-11-01

    Full Text Available DNA double-strand breaks (DSBs elicit the so-called DNA damage response (DDR, largely relying on ataxia telangiectasia mutated (ATM and DNA-dependent protein kinase (DNA-PKcs, two members of the PI3K-like kinase family, whose respective functions during the sequential steps of the DDR remains controversial. Using the DIvA system (DSB inducible via AsiSI combined with high-resolution mapping and advanced microscopy, we uncovered that both ATM and DNA-PKcs spread in cis on a confined region surrounding DSBs, independently of the pathway used for repair. However, once recruited, these kinases exhibit non-overlapping functions on end joining and γH2AX domain establishment. More specifically, we found that ATM is required to ensure the association of multiple DSBs within “repair foci.” Our results suggest that ATM acts not only on chromatin marks but also on higher-order chromatin organization to ensure repair accuracy and survival.

  14. The importance of becoming double-stranded: Innate immunity and the kinetic model of HIV-1 central plus strand synthesis

    Energy Technology Data Exchange (ETDEWEB)

    Poeschla, Eric, E-mail: poeschla.eric@mayo.edu

    2013-06-20

    Central initiation of plus strand synthesis is a conserved feature of lentiviruses and certain other retroelements. This complication of the standard reverse transcription mechanism produces a transient “central DNA flap” in the viral cDNA, which has been proposed to mediate its subsequent nuclear import. This model has assumed that the important feature is the flapped DNA structure itself rather than the process that produces it. Recently, an alternative kinetic model was proposed. It posits that central plus strand synthesis functions to accelerate conversion to the double-stranded state, thereby helping HIV-1 to evade single-strand DNA-targeting antiviral restrictions such as APOBEC3 proteins, and perhaps to avoid innate immune sensor mechanisms. The model is consistent with evidence that lentiviruses must often synthesize their cDNAs when dNTP concentrations are limiting and with data linking reverse transcription and uncoating. There may be additional kinetic advantages for the artificial genomes of lentiviral gene therapy vectors. - Highlights: • Two main functional models for HIV central plus strand synthesis have been proposed. • In one, a transient central DNA flap in the viral cDNA mediates HIV-1 nuclear import. • In the other, multiple kinetic consequences are emphasized. • One is defense against APOBEC3G, which deaminates single-stranded DNA. • Future questions pertain to antiviral restriction, uncoating and nuclear import.

  15. Molecular Basis for DNA Double-Strand Break Annealing and Primer Extension by an NHEJ DNA Polymerase

    Directory of Open Access Journals (Sweden)

    Nigel C. Brissett

    2013-11-01

    Full Text Available Nonhomologous end-joining (NHEJ is one of the major DNA double-strand break (DSB repair pathways. The mechanisms by which breaks are competently brought together and extended during NHEJ is poorly understood. As polymerases extend DNA in a 5′-3′ direction by nucleotide addition to a primer, it is unclear how NHEJ polymerases fill in break termini containing 3′ overhangs that lack a primer strand. Here, we describe, at the molecular level, how prokaryotic NHEJ polymerases configure a primer-template substrate by annealing the 3′ overhanging strands from opposing breaks, forming a gapped intermediate that can be extended in trans. We identify structural elements that facilitate docking of the 3′ ends in the active sites of adjacent polymerases and reveal how the termini act as primers for extension of the annealed break, thus explaining how such DSBs are extended in trans. This study clarifies how polymerases couple break-synapsis to catalysis, providing a molecular mechanism to explain how primer extension is achieved on DNA breaks.

  16. Real Estate in the DNA Damage Response: Ubiquitin and SUMO Ligases Home in on DNA Double-Strand Breaks.

    Science.gov (United States)

    Dantuma, Nico P; Pfeiffer, Annika

    2016-01-01

    Ubiquitin and the ubiquitin-like modifier SUMO are intimately connected with the cellular response to various types of DNA damage. A striking feature is the local accumulation of these proteinaceous post-translational modifications in the direct vicinity to DNA double-strand breaks, which plays a critical role in the formation of ionizing radiation-induced foci. The functional significance of these modifications is the coordinated recruitment and removal of proteins involved in DNA damage signaling and repair in a timely manner. The central orchestrators of these processes are the ubiquitin and SUMO ligases that are responsible for accurately tagging a broad array of chromatin and chromatin-associated proteins thereby changing their behavior or destination. Despite many differences in the mode of action of these enzymes, they share some striking features that are of direct relevance for their function in the DNA damage response. In this review, we outline the molecular mechanisms that are responsible for the recruitment of ubiquitin and SUMO ligases and discuss the importance of chromatin proximity in this process.

  17. Correlativity study between expression of DNA double-strand break repair protein and radiosensitivity of tumor cells

    Institute of Scientific and Technical Information of China (English)

    Liang ZHUANG; Shiying YU; Xiaoyuan HUANG; Yang CAO; Huihua XIONG

    2009-01-01

    DNA double-strand break (DSB) is generally regarded as the most lethal of all DNA lesions after radiation. KuS0, DNA-PK catalytic subunit (DNA-PKcs) and ataxia telangiectasia mutated (ATM) proteins are major DSB repair proteins. In this study, survival fraction at 2Gy (SF2) values of eight human tumor cell lines (including four human cervical carcinoma cell lines HeLa, SiHa, C33A, Caski, three human breast carcinoma cell lines MCF-7, MDA-MB-231, MDA-MB-453, and one human lung carcinoma cell line A549) were acquired by clone formation assay, and western blot was applied to detect the expressions of Ku80, DNA-PKcs and ATM protein. The correlativity of protein expression with SF2 value was analyzed by Pearson linear correlation analysis. We found that the expression of the same protein in different cell lines and the expression of three proteins in the same cell line had a significant difference. The SF2 values were also different in eight tumor cell lines and there was a positive correlativity between the expression of DNA-PKcs and SF2 (r=0.723, P =0.043), but Ku80 and ATM expression had no correlation with SF2 (P>0.05). These findings suggest that the expression level of DNA-PKcs protein can be an indicator for predicting the radiosensitivity of tumor cells.

  18. Induction by gamma irradiation of double-strand breaks of Escherichia coli chromosomes and their role in cell lethality

    Energy Technology Data Exchange (ETDEWEB)

    Bresler, S.E.; Noskin, L.A.; Suslov, A.V.

    1984-04-01

    Viscoelastometric measurements of DNA from ..gamma..-irradiated bacteria were used to identify the induction of double-strand breaks (DSBs) in the chromosome of Escherichia coli. It is shown by means of inhibitors of repair endonucleases and different repair mutants that most DSBs in DNA of E. coli, ..gamma..-irradiated in buffer, arise from enzymatic incision of primary ..gamma..-damages; therefore, previous conclusions regarding DSB repair must be reconsidered. Based on these results, much of the repairable damage is single-strand breaks, and this damage can initiate formation of gaps and ultimately, when repair is insufficient, generation of enzymatically caused DSBs. After extensive repair, the first residual DSB in the E. coli chromosome is generated at approx.160 Gray (Gy), which corresponds to the D/sub 37/ dose. We propose that DSBs induced directly by ..gamma..-irradiation are not repaired in wild-type strains. In a recently isolated ..gamma..-resistant strain, E. coli Gam/sup r/444, the dose required for observation of DSB after postirradiation incubation is 1000 Gy, which corresponds to the D/sub 37/ of this strain. The resistance is proposed to be due to an ability to repair genuine DSBs.

  19. Ubiquitin-specific protease 5 is required for the efficient repair of DNA double-strand breaks.

    Directory of Open Access Journals (Sweden)

    Satoshi Nakajima

    Full Text Available During the DNA damage response (DDR, ubiquitination plays an important role in the recruitment and regulation of repair proteins. However, little is known about elimination of the ubiquitination signal after repair is completed. Here we show that the ubiquitin-specific protease 5 (USP5, a deubiquitinating enzyme, is involved in the elimination of the ubiquitin signal from damaged sites and is required for efficient DNA double-strand break (DSB repair. Depletion of USP5 sensitizes cells to DNA damaging agents, produces DSBs, causes delayed disappearance of γH2AX foci after Bleocin treatment, and influences DSB repair efficiency in the homologous recombination pathway but not in the non-homologous end joining pathway. USP5 co-localizes to DSBs induced by laser micro-irradiation in a RAD18-dependent manner. Importantly, polyubiquitin chains at sites of DNA damage remained for longer periods in USP5-depleted cells. Our results show that disassembly of polyubiquitin chains by USP5 at sites of damage is important for efficient DSB repair.

  20. Molecular Process Producing Oncogene Fusion in Lung Cancer Cells by Illegitimate Repair of DNA Double-Strand Breaks

    Directory of Open Access Journals (Sweden)

    Yoshitaka Seki

    2015-09-01

    Full Text Available Constitutive activation of oncogenes by fusion to partner genes, caused by chromosome translocation and inversion, is a critical genetic event driving lung carcinogenesis. Fusions of the tyrosine kinase genes ALK (anaplastic lymphoma kinase, ROS1 (c-ros oncogene 1, or RET (rearranged during transfection occur in 1%–5% of lung adenocarcinomas (LADCs and their products constitute therapeutic targets for kinase inhibitory drugs. Interestingly, ALK, RET, and ROS1 fusions occur preferentially in LADCs of never- and light-smokers, suggesting that the molecular mechanisms that cause these rearrangements are smoking-independent. In this study, using previously reported next generation LADC genome sequencing data of the breakpoint junction structures of chromosome rearrangements that cause oncogenic fusions in human cancer cells, we employed the structures of breakpoint junctions of ALK, RET, and ROS1 fusions in 41 LADC cases as “traces” to deduce the molecular processes of chromosome rearrangements caused by DNA double-strand breaks (DSBs and illegitimate joining. We found that gene fusion was produced by illegitimate repair of DSBs at unspecified sites in genomic regions of a few kb through DNA synthesis-dependent or -independent end-joining pathways, according to DSB type. This information will assist in the understanding of how oncogene fusions are generated and which etiological factors trigger them.

  1. Preventing damage limitation: targeting DNA-PKcs and DNA double strand break repair pathways for ovarian cancer therapy

    Directory of Open Access Journals (Sweden)

    Daniela A Dungl

    2015-10-01

    Full Text Available Platinum-based chemotherapy is the cornerstone of ovarian cancer treatment, and its efficacy is dependent on the generation of DNA damage, with subsequent induction of apoptosis. Inappropriate or aberrant activation of the DNA damage response network is are associated with resistance to platinum, and defects in DNA repair pathways play critical roles in determining patient response to chemotherapy. In ovarian cancer, tumour cell defects in homologous recombination - a repair pathway activated in response to DNA double strand breaks (DSB - are most commonly associated with platinum sensitive disease. However, despite initial sensitivity, the emergence of resistance is frequent. Here, we review strategies for directly interfering with DNA repair pathways, with particular focus on direct inhibition of non-homologous end joining (NHEJ, another DSB repair pathway. DNA-PKcs is a core component of NHEJ and it has shown considerable promise as a chemosensitization target in numerous cancer types, including ovarian cancer where it functions to promote platinum-induced survival signalling, via AKT activation. The development of pharmacological inhibitors of DNA-PKcs is on-going, and clinic-ready agents offer real hope to patients with chemoresistant disease.

  2. The Arabidopsis thaliana DSB formation (AtDFO) gene is required for meiotic double-strand break formation.

    Science.gov (United States)

    Zhang, Cheng; Song, Yao; Cheng, Zhi-hao; Wang, Ying-xiang; Zhu, Jun; Ma, Hong; Xu, Ling; Yang, Zhong-Nan

    2012-10-01

    DNA double-strand break (DSB) formation is the initial event for meiotic recombination catalyzed by the conserved Spo11 protein. In Arabidopsis, several proteins have been reported to be involved in DSB formation. Here, we report an Arabidopsis DSB forming (DFO) gene in Arabidopsis that is involved in DSB formation. The dfo mutant exhibits reduced fertility, producing polyads with an abnormal number of microspores, unlike the tetrads in the wild type. The dfo meiocytes were defective in homologous chromosome synapsis and segregation. Genetic analysis revealed that the homologous recombination of Atdfo-1 is severely affected in meiotic prophase I. DFO encodes a protein without any known conserved domain. There was no homologue identified outside the plant kingdom, indicating that AtDFO is a plant-specific protein. AtMRE11 has been reported to be responsible for processing SPO11-generated DSBs. The Atmre11 mutant displays chromosome fragmentation during meiosis. However, the Atdfo Atmre11 double mutant had no such chromosome fragmentation, indicating that AtDFO is required for DSB formation.

  3. Xbp1-mediated histone H4 deacetylation contributes to DNA double-strand break repair in yeast

    Institute of Scientific and Technical Information of China (English)

    Ran Tao; Hua Chen; Chan Gao; Pcng Xue; Fuquan Yang; Jing-Dong J Han; Bing Zhou; Ye-Guang Chen

    2011-01-01

    Xbp1 has been shown to regulate the cell cycle as a transcriptional repressor in budding yeast Saccharomyces cerevisiae.In this study,we demonstrated that Xbp1 regulates DNA double-strand break (DSB) repair in S.cerevisiae.Xbp1 physically and genetically interacts with the histone deacetylase Rpd3 complex.Chromatin immunoprecipitation revealed that Xbp1 is required for efficient deacetylation of histone H4 flanking DSBs by the Rpd3 complex.Deletion of XBP1 leads to the delayed deacetylation of histone H4,which is coupled with increased nucleosome displacement,increased DNA end resection and decreased non-homologous end-joining (NHEJ).In response to DNA damage,Xbp1 is upregulated in a Mec1-Rad9-Rad53 checkpoint pathway-dependent manner and undergoes dephosphorylation.Cdk1,a central regulator of S.cerevisiae cell cycle,is responsible for Xbp1 phosphorylation at residues Ser146,Ser271 and Ser551.Substitution of these serine residues with alanine not only increases the association of Xbp1 with the Rpd3 complex and its recruitment to a DSB,but also promotes DSB repair.Together,our findings reveal a role for Xbp1 in DSB repair via NHEJ through regulation of histone H4 acetylation and nucleosome displacement in a positive feedback manner.

  4. Coordination of Double Strand Break Repair and Meiotic Progression in Yeast by a Mek1- Ndt80 Negative Feedback Loop.

    Science.gov (United States)

    Prugar, Evelyn; Burnett, Cameron; Chen, Xiangyu; Hollingsworth, Nancy M

    2017-03-01

    During meiosis, homologous chromosomes are physically connected by crossovers and sister chromatid cohesion. Interhomolog crossovers are generated by the highly regulated repair of programmed double strand breaks (DSBs). The meiosis-specific kinase, Mek1, is critical for this regulation. Mek1 down-regulates the mitotic recombinase Rad51, indirectly promoting interhomolog strand invasion by the meiosis-specific recombinase, Dmc1. Mek1 also promotes the formation of crossovers that are distributed throughout the genome by interference and is the effector kinase for a meiosis-specific checkpoint that delays entry into Meiosis I until DSBs have been repaired. The target of this checkpoint is a meiosis-specific transcription factor, Ndt80, which is necessary to express the polo-like kinase, CDC5, and the cyclin, CLB1, thereby allowing completion of recombination and meiotic progression. This work shows that Mek1 and Ndt80 negatively feedback on each other such that when DSB levels are high, Ndt80 is inactive due to high levels of Mek1 activity. As DSBs are repaired, chromosomes synapse and Mek1 activity is reduced below a threshold that allows activation of Ndt80. Ndt80 transcription of CDC5 results in degradation of Red1, a meiosis-specific protein required for Mek1 activation, thereby abolishing Mek1 activity completely. Elimination of Mek1 kinase activity allows Rad51-mediated repair of any remaining DSBs. In this way, cells do not enter Meiosis I until recombination is complete and all DSBs are repaired.

  5. Improving DNA double-strand repair inhibitor KU55933 therapeutic index in cancer radiotherapy using nanoparticle drug delivery

    Science.gov (United States)

    Tian, Xi; Lara, Haydee; Wagner, Kyle T.; Saripalli, Srinivas; Hyder, Syed Nabeel; Foote, Michael; Sethi, Manish; Wang, Edina; Caster, Joseph M.; Zhang, Longzhen; Wang, Andrew Z.

    2015-11-01

    Radiotherapy is a key component of cancer treatment. Because of its importance, there has been high interest in developing agents and strategies to further improve the therapeutic index of radiotherapy. DNA double-strand repair inhibitors (DSBRIs) are among the most promising agents to improve radiotherapy. However, their clinical translation has been limited by their potential toxicity to normal tissue. Recent advances in nanomedicine offer an opportunity to overcome this limitation. In this study, we aim to demonstrate the proof of principle by developing and evaluating nanoparticle (NP) formulations of KU55933, a DSBRI. We engineered a NP formulation of KU55933 using nanoprecipitation method with different lipid polymer nanoparticle formulation. NP KU55933 using PLGA formulation has the best loading efficacy as well as prolonged drug release profile. We demonstrated that NP KU55933 is a potent radiosensitizer in vitro using clonogenic assay and is more effective as a radiosensitizer than free KU55933 in vivo using mouse xenograft models of non-small cell lung cancer (NSCLC). Western blots and immunofluorescence showed NP KU55933 exhibited more prolonged inhibition of DNA repair pathway. In addition, NP KU55933 leads to lower skin toxicity than KU55933. Our study supports further investigations using NP to deliver DSBRIs to improve cancer radiotherapy treatment.

  6. Crystal Structure of E. coli RecE Protein Reveals a Toroidal Tetramer for Processing Double-Stranded DNA Breaks

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Jinjin; Xing, Xu; Herr, Andrew B.; Bell, Charles E.; (OSU); (UCIN)

    2009-07-21

    Escherichia coli RecE protein is part of the classical RecET recombination system that has recently been used in powerful new methods for genetic engineering. RecE binds to free double-stranded DNA (dsDNA) ends and processively digests the 5{prime}-ended strand to form 5{prime}-mononucleotides and a 3{prime}-overhang that is a substrate for single strand annealing promoted by RecT. Here, we report the crystal structure of the C-terminal nuclease domain of RecE at 2.8 {angstrom} resolution. RecE forms a toroidal tetramer with a central tapered channel that is wide enough to bind dsDNA at one end, but is partially plugged at the other end by the C-terminal segment of the protein. Four narrow tunnels, one within each subunit of the tetramer, lead from the central channel to the four active sites, which lie about 15 {angstrom} from the channel. The structure, combined with mutational studies, suggests a mechanism in which dsDNA enters through the open end of the central channel, the 5{prime}-ended strand passes through a tunnel to access one of the four active sites, and the 3{prime}-ended strand passes through the plugged end of the channel at the back of the tetramer.

  7. Increased repair of {gamma}-induced DNA double-strand breaks at lower dose-rate in CHO cells

    Energy Technology Data Exchange (ETDEWEB)

    Boucher, D.; Hindo, J.; Averbeck, D. [Centre Universitaire d' Orsay, Inst. Curie-Section de Recherche, Orsay CEDEX (France)]. E-mail: dietrich.averbeck@curie.u-psud.fr

    2004-02-01

    DNA double-strand breaks (DSBs) are highly cell damaging. We asked whether for a given dose a longer irradiation time would be advantageous for the repair of DSBs. Varying the {gamma}-irradiation dose and its delivery time (0.05 Gy/min low dose-rate (LDR) compared with 3.5 Gy/min high dose-rate), confluent Chinese hamster ovary cells (CHO-K1) and Ku80 mutant cells (xrs-6) deficient in nonhomologous end-joining (NHEJ) were irradiated in agarose plugs at room temperature using a cesium-137 {gamma}-ray source. We used pulsed-field gel electrophoresis (PFGE) to measure DSBs in terms of the fraction of activity released (FAR). At LDR, one third of DSBs were repaired in CHO-K1 but not in xrs-6 cells, indicating the involvement of NHEJ in the repair of {gamma}-induced DSBs at a prolonged irradiation incubation time. To improve DSB measurements, we introduced in our PFGE protocol an antioxidant at the cell lysis step, thus avoiding free-radical side reactions on DNA and spurious DSBs. Addition of the metal chelator deferoxamine (DFO) decreased more efficiently the basal DSB level than did reduced glutathione (GSH), showing that measuring DSBs in the absence of DFO reduces precision and underestimates the role of NHEJ in the dose-rate effect on DSB yield. (author)

  8. The peptide antibiotic microcin B17 induces double-strand cleavage of DNA mediated by E. coli DNA gyrase.

    Science.gov (United States)

    Vizán, J L; Hernández-Chico, C; del Castillo, I; Moreno, F

    1991-02-01

    Microcin B17 (MccB17) is a bactericidal peptide antibiotic which inhibits DNA replication. Two Escherichia coli MccB17 resistant mutants were isolated and the mutations were shown to map to 83 min of the genetic map. Cloning of the mutations and Tn5 insertional analysis demonstrated that they were located inside gyrB. The approximate location of the mutations within gyrB was determined by constructing hybrid genes, as a previous step to sequencing. Both mutations were shown to consist of a single AT----GC transition at position 2251 of the gene, which produces a Trp751----Arg substitution in the amino acid sequence of the GyrB polypeptide. The inhibitory effect of MccB17 on replicative cell-free extracts was assayed. In this in vitro system, interaction of MccB17 with a component of the extracts induced double-strand cleavage of plasmid DNA. In vivo treatment with MccB17 also induced a well-defined cleavage pattern on chromosomal DNA. These effects were not observed with a MccB17-resistant, gyrB mutant. Altogether, our results indicate that MccB17 blocks DNA gyrase by trapping an enzyme-DNA cleavable complex. Thus, the mode of action of this peptide antibiotic resembles that of quinolones and a variety of antitumour drugs currently used in cancer chemotherapy. MccB17 is the first peptide shown to inhibit a type II DNA topoisomerase.

  9. Adaptation of the neutral bacterial comet assay to assess antimicrobial-mediated DNA double-strand breaks in Escherichia coli

    Science.gov (United States)

    SOLANKY, DIPESH; HAYDEL, SHELLEY E.

    2012-01-01

    This study aimed to determine the mechanism of action of a natural antibacterial clay mineral mixture, designated CB, by investigating the induction of DNA double-strand breaks (DSBs) in Escherichia coli. To quantify DNA damage upon exposure to soluble antimicrobial compounds, we modified a bacterial neutral comet assay, which primarily associates the general length of an electrophoresed chromosome, or comet, with the degree of DSB-associated DNA damage. To appropriately account for antimicrobial-mediated strand fragmentation, suitable control reactions consisting of exposures to water, ethanol, kanamycin, and bleomycin were developed and optimized for the assay. Bacterial exposure to the CB clay resulted in significantly longer comet lengths, compared to water and kanamycin exposures, suggesting that the induction of DNA DSBs contributes to the killing activity of this antibacterial clay mineral mixture. The comet assay protocol described herein provides a general technique for evaluating soluble antimicrobial-derived DNA damage and for comparing DNA fragmentation between experimental and control assays. PMID:22940101

  10. Small Rad51 and Dmc1 Complexes Often Co-occupy Both Ends of a Meiotic DNA Double Strand Break.

    Directory of Open Access Journals (Sweden)

    M Scott Brown

    2015-12-01

    Full Text Available The Eukaryotic RecA-like proteins Rad51 and Dmc1 cooperate during meiosis to promote recombination between homologous chromosomes by repairing programmed DNA double strand breaks (DSBs. Previous studies showed that Rad51 and Dmc1 form partially overlapping co-foci. Here we show these Rad51-Dmc1 co-foci are often arranged in pairs separated by distances of up to 400 nm. Paired co-foci remain prevalent when DSBs are dramatically reduced or when strand exchange or synapsis is blocked. Super-resolution dSTORM microscopy reveals that individual foci observed by conventional light microscopy are often composed of two or more substructures. The data support a model in which the two tracts of ssDNA formed by a single DSB separate from one another by distances of up to 400 nm, with both tracts often bound by one or more short (about 100 nt Rad51 filaments and also by one or more short Dmc1 filaments.

  11. Haploid meiosis in Arabidopsis: double-strand breaks are formed and repaired but without synapsis and crossovers.

    Science.gov (United States)

    Cifuentes, Marta; Rivard, Maud; Pereira, Lucie; Chelysheva, Liudmila; Mercier, Raphael

    2013-01-01

    Two hallmark features of meiosis are i) the formation of crossovers (COs) between homologs and ii) the production of genetically-unique haploid spores that will fuse to restore the somatic ploidy level upon fertilization. In this study we analysed meiosis in haploid Arabidopsis thaliana plants and a range of haploid mutants to understand how meiosis progresses without a homolog. Extremely low chiasma frequency and very limited synapsis occurred in wild-type haploids. The resulting univalents segregated in two uneven groups at the first division, and sister chromatids segregated to opposite poles at the second division, leading to the production of unbalanced spores. DNA double-strand breaks that initiate meiotic recombination were formed, but in half the number compared to diploid meiosis. They were repaired in a RAD51- and REC8-dependent manner, but independently of DMC1, presumably using the sister chromatid as a template. Additionally, turning meiosis into mitosis (MiMe genotype) in haploids resulted in the production of balanced haploid gametes and restoration of fertility. The variability of the effect on meiosis of the absence of homologous chromosomes in different organisms is then discussed.

  12. Unveiling the pentagonal nature of perfectly aligned single-and double-strand Si nano-ribbons on Ag(110)

    Science.gov (United States)

    Cerdá, Jorge I.; Sławińska, Jagoda; Le Lay, Guy; Marele, Antonela C.; Gómez-Rodríguez, José M.; Dávila, María E.

    2016-10-01

    Carbon and silicon pentagonal low-dimensional structures attract a great interest as they may lead to new exotic phenomena such as topologically protected phases or increased spin-orbit effects. However, no pure pentagonal phase has yet been realized for any of them. Here we unveil through extensive density functional theory calculations and scanning tunnelling microscope simulations, confronted to key experimental facts, the hidden pentagonal nature of single- and double-strand chiral Si nano-ribbons perfectly aligned on Ag(110) surfaces whose structure has remained elusive for over a decade. Our study reveals an unprecedented one-dimensional Si atomic arrangement solely comprising almost perfect alternating pentagons residing in the missing row troughs of the reconstructed surface. We additionally characterize the precursor structure of the nano-ribbons, which consists of a Si cluster (nano-dot) occupying a silver di-vacancy in a quasi-hexagonal configuration. The system thus materializes a paradigmatic shift from a silicene-like packing to a pentagonal one.

  13. Haploid meiosis in Arabidopsis: double-strand breaks are formed and repaired but without synapsis and crossovers.

    Directory of Open Access Journals (Sweden)

    Marta Cifuentes

    Full Text Available Two hallmark features of meiosis are i the formation of crossovers (COs between homologs and ii the production of genetically-unique haploid spores that will fuse to restore the somatic ploidy level upon fertilization. In this study we analysed meiosis in haploid Arabidopsis thaliana plants and a range of haploid mutants to understand how meiosis progresses without a homolog. Extremely low chiasma frequency and very limited synapsis occurred in wild-type haploids. The resulting univalents segregated in two uneven groups at the first division, and sister chromatids segregated to opposite poles at the second division, leading to the production of unbalanced spores. DNA double-strand breaks that initiate meiotic recombination were formed, but in half the number compared to diploid meiosis. They were repaired in a RAD51- and REC8-dependent manner, but independently of DMC1, presumably using the sister chromatid as a template. Additionally, turning meiosis into mitosis (MiMe genotype in haploids resulted in the production of balanced haploid gametes and restoration of fertility. The variability of the effect on meiosis of the absence of homologous chromosomes in different organisms is then discussed.

  14. Recombinational repair of radiation-induced double-strand breaks occurs in the absence of extensive resection

    Science.gov (United States)

    Westmoreland, James W.; Resnick, Michael A.

    2016-01-01

    Recombinational repair provides accurate chromosomal restitution after double-strand break (DSB) induction. While all DSB recombination repair models include 5′-3′ resection, there are no studies that directly assess the resection needed for repair between sister chromatids in G-2 arrested cells of random, radiation-induced ‘dirty’ DSBs. Using our Pulse Field Gel Electrophoresis-shift approach, we determined resection at IR-DSBs in WT and mutants lacking exonuclease1 or Sgs1 helicase. Lack of either reduced resection length by half, without decreased DSB repair or survival. In the exo1Δ sgs1Δ double mutant, resection was barely detectable, yet it only took an additional hour to achieve a level of repair comparable to WT and there was only a 2-fold dose-modifying effect on survival. Results with a Dnl4 deletion strain showed that remaining repair was not due to endjoining. Thus, similar to what has been shown for a single, clean HO-induced DSB, a severe reduction in resection tract length has only a modest effect on repair of multiple, dirty DSBs in G2-arrested cells. Significantly, this study provides the first opportunity to directly relate resection length at DSBs to the capability for global recombination repair between sister chromatids. PMID:26503252

  15. Tandem repeat modification during double-strand break repair induced by an engineered TAL effector nuclease in zebrafish genome.

    Directory of Open Access Journals (Sweden)

    Wanxu Huang

    Full Text Available Tandem repeats (TRs are abundant and widely distributed in eukaryotic genomes. TRs are thought to have various functions in gene transcription, DNA methylation, nucleosome position and chromatin organization. Variation of repeat units in the genome is observed in association with a number of diseases, such as Fragile X Syndrome, Huntington's disease and Friedreich's ataxia. However, the underlying mechanisms involved are poorly understood, largely owing to the technical limitations in modification of TRs at definite sites in the genome in vivo. Transcription activator-like effector nucleases (TALENs are widely used in recent years in gene targeting for their specific binding to target sequences when engineered in vitro. Here, we show that the repair of a double-strand break (DSB induced by TALENs adjacent to a TR can produce serial types of mutations in the TR region. Sequencing analysis revealed that there are three types of mutations induced by the DSB repair, including indels only within the TR region or within the flanking TALEN target region or simutaneously within both regions. Therefore, desired TR mutant types can be conveniently obtained by using engineered TALENs. These results demonstrate that TALENs can serve as a convenient tool for modifying TRs in the genome in studying the functions of TRs.

  16. Inhibition of proteasomal degradation of rpn4 impairs nonhomologous end-joining repair of DNA double-strand breaks.

    Directory of Open Access Journals (Sweden)

    Donghong Ju

    Full Text Available BACKGROUND: The proteasome homeostasis in Saccharomyces cerevisiae is regulated by a negative feedback circuit in which the transcription factor Rpn4 induces the proteasome genes and is rapidly degraded by the assembled proteasome. The integrity of the Rpn4-proteasome feedback loop is critical for cell viability under stressed conditions. We have demonstrated that inhibition of Rpn4 degradation sensitizes cells to DNA damage, particularly in response to high doses of DNA damaging agents. The underlying mechanism, however, remains unclear. METHODOLOGY/PRINCIPAL FINDINGS: Using yeast genetics and biochemical approach we show that inhibition of Rpn4 degradation displays a synthetic growth defect with deletion of the MEC1 checkpoint gene and sensitizes several checkpoint mutants to DNA damage. In addition, inhibition of Rpn4 degradation leads to a defect in repair of double-strand breaks (DSBs by nonhomologous end-joining (NHEJ. The expression levels of several key NHEJ genes are downregulated and the recruitment of Yku70 to a DSB is reduced by inhibition of Rpn4 degradation. We find that Rpn4 and the proteasome are recruited to a DSB, suggesting their direct participation in NHEJ. Inhibition of Rpn4 degradation may result in a concomitant delay of release of Rpn4 and the proteasome from a DSB. CONCLUSION/SIGNIFICANCE: This study provides the first evidence for the role of proteasomal degradation of Rpn4 in NHEJ.

  17. Transcription of a donor enhances its use during double-strand break-induced gene conversion in human cells.

    Science.gov (United States)

    Schildkraut, Ezra; Miller, Cheryl A; Nickoloff, Jac A

    2006-04-01

    Homologous recombination (HR) mediates accurate repair of double-strand breaks (DSBs) but carries the risk of large-scale genetic change, including loss of heterozygosity, deletions, inversions, and translocations. Nearly one-third of the human genome consists of repetitive sequences, and DSB repair by HR often requires choices among several homologous repair templates, including homologous chromosomes, sister chromatids, and linked or unlinked repeats. Donor preference during DSB-induced gene conversion was analyzed by using several HR substrates with three copies of neo targeted to a human chromosome. Repair of I-SceI nuclease-induced DSBs in one neo (the recipient) required a choice between two donor neo genes. When both donors were downstream, there was no significant bias for proximal or distal donors. When donors flanked the recipient, we observed a marked (85%) preference for the downstream donor. Reversing the HR substrate in the chromosome eliminated this preference, indicating that donor choice is influenced by factors extrinsic to the HR substrate. Prior indirect evidence suggested that transcription might increase donor use. We tested this question directly and found that increased transcription of a donor enhances its use during gene conversion. A preference for transcribed donors would minimize the use of nontranscribed (i.e., pseudogene) templates during repair and thus help maintain genome stability.

  18. Dynamics of yeast histone H2A and H2B phosphorylation in response to a double-strand break.

    Science.gov (United States)

    Lee, Cheng-Sheng; Lee, Kihoon; Legube, Gaëlle; Haber, James E

    2014-01-01

    In budding yeast, a single double-strand break (DSB) triggers extensive Tel1 (ATM)- and Mec1 (ATR)-dependent phosphorylation of histone H2A around the DSB, to form γ-H2AX. We describe Mec1- and Tel1-dependent phosphorylation of histone H2B at T129. γ-H2B formation is impaired by γ-H2AX and its binding partner Rad9. High-density microarray analyses show similar γ-H2AX and γ-H2B distributions, but γ-H2B is absent near telomeres. Both γ-H2AX and γ-H2B are strongly diminished over highly transcribed regions. When transcription of GAL7, GAL10 and GAL1 genes is turned off, γ-H2AX is restored within 5 min, in a Mec1-dependent manner; after reinduction of these genes, γ-H2AX is rapidly lost. Moreover, when a DSB is induced near CEN2, γ-H2AX spreads to all other pericentromeric regions, again depending on Mec1. Our data provide new insights in the function and establishment of phosphorylation events occurring on chromatin after DSB induction.

  19. The mutagenic potential of a single DNA double-strand break in a mammalian chromosome is not influenced by transcription.

    Science.gov (United States)

    Allen, Chris; Miller, Cheryl A; Nickoloff, Jac A

    2003-10-07

    In eukaryotes, DNA double-strand breaks (DSBs) are repaired by competing HR and non-homologous end-joining (NHEJ) pathways. DSB repair by HR is highly accurate, while NHEJ can result in deletions and insertions. Transcription enhances certain DNA repair pathways and spontaneous homologous recombination (HR). As a means to promote accurate repair in active genes, we thought it possible that the balance between HR and NHEJ would be shifted toward HR in highly transcribed regions. We tested this idea by examining products of DSB repair in integrated neo-direct repeats under conditions of low-level constitutive, or high-level induced transcription regulated by the dexamethasone (Dex)-responsive mouse mammary tumor virus (MMTV) promoter. DSBs were introduced into one copy of neo by expressing I-SceI nuclease, and DSB repair products were isolated and characterized with an efficient, non-selective assay. We found that transcription does not significantly change the relative frequencies of HR and NHEJ, the relative frequencies of sequence capture and gross chromosomal rearrangement, nor the average size of deletions. About one-third of DSB repair products showed large-scale rearrangements, indicating that a single DSB in a mammalian chromosome has significant mutagenic potential.

  20. c-Myc directly regulates the transcription of the NBS1 gene involved in DNA double-strand break repair.

    Science.gov (United States)

    Chiang, Yu-Chi; Teng, Shu-Chun; Su, Yi-Ning; Hsieh, Fon-Jou; Wu, Kou-Juey

    2003-05-23

    The c-myc proto-oncogene encodes a ubiquitous transcription factor involved in the control of cell growth and implicated in inducing tumorigenesis. Understanding the function of c-Myc and its role in cancer depends upon the identification of c-Myc target genes. Nijmegen breakage syndrome (NBS) is a chromosomal-instability syndrome associated with cancer predisposition, radiosensitivity, and chromosomal instability. The NBS gene product, NBS1 (p95 or nibrin), is a part of the hMre11 complex, a central player associated with double-strand break (DSB) repair. NBS1 contains domains characteristic for proteins involved in DNA repair, recombination, and replication. Here we show that c-Myc directly activates NBS1. c-Myc-mediated induction of NBS1 gene transcription occurs in different tissues, is independent of cell proliferation, and is mediated by a c-Myc binding site in the intron 1 region of NBS1 gene. Overexpression of NBS1 in Rat1a cells increased cell proliferation. These results indicate that NBS1 is a direct transcriptional target of c-Myc and links the function of c-Myc to the regulation of DNA DSB repair pathway operating during DNA replication.

  1. RSC facilitates Rad59-dependent homologous recombination between sister chromatids by promoting cohesin loading at DNA double-strand breaks.

    Science.gov (United States)

    Oum, Ji-Hyun; Seong, Changhyun; Kwon, Youngho; Ji, Jae-Hoon; Sid, Amy; Ramakrishnan, Sreejith; Ira, Grzegorz; Malkova, Anna; Sung, Patrick; Lee, Sang Eun; Shim, Eun Yong

    2011-10-01

    Homologous recombination repairs DNA double-strand breaks by searching for, invading, and copying information from a homologous template, typically the homologous chromosome or sister chromatid. Tight wrapping of DNA around histone octamers, however, impedes access of repair proteins to DNA damage. To facilitate DNA repair, modifications of histones and energy-dependent remodeling of chromatin are required, but the precise mechanisms by which chromatin modification and remodeling enzymes contribute to homologous DNA repair are unknown. Here we have systematically assessed the role of budding yeast RSC (remodel structure of chromatin), an abundant, ATP-dependent chromatin-remodeling complex, in the cellular response to spontaneous and induced DNA damage. RSC physically interacts with the recombination protein Rad59 and functions in homologous recombination. Multiple recombination assays revealed that RSC is uniquely required for recombination between sister chromatids by virtue of its ability to recruit cohesin at DNA breaks and thereby promoting sister chromatid cohesion. This study provides molecular insights into how chromatin remodeling contributes to DNA repair and maintenance of chromatin fidelity in the face of DNA damage.

  2. Genetic requirements for the single-strand annealing pathway of double-strand break repair in Saccharomyces cerevisiae

    Energy Technology Data Exchange (ETDEWEB)

    Ivanov, E.L.; Sugawara, N.; Haber, J.E. [Brandeis Univ., Waltham, MA (United States)] [and others

    1996-03-01

    HO endonuclease-induced double-strand breaks (DSBs) within a direct duplication of Escherichia coli lacZ genes are repaired either by gene conversion or by single-strand annealing (SSA), with >80% being SSA. Previously it was demonstrated that the RAD52 gene is required for DSB-induced SSA. In the present study, the effects of other genes belonging to the RAD52 epistasis group were analyzed. We show that RAD51, RAD54, RAD55, and RAD57 genes are not required for SSA irrespective of whether recombination occurred in plasmid or chromosomal DNA. In both plasmid and chromosomal constructs with homologous sequences in direct orientation, the proportion of SSA events over gene conversion was significantly elevated in the mutant strains. However, gene conversion was not affected when the two lacZ sequences were in inverted orientation. These results suggest that there is a competition between SSA and gene conversion processes that favors SSA in the absence of RAD51, RAD54, RAD55 and RAD57. Mutations in RAD50 and XRS2 genes do not prevent the completion, but markedly retard the kinetics, of DSB repair by both mechanisms in the lacZ direct repeat plasmid, a result resembling the effects of these genes during mating-type (MAT) switching. 43 refs., 8 figs., 3 tabs.

  3. Unveiling the pentagonal nature of perfectly aligned single-and double-strand Si nano-ribbons on Ag(110)

    Science.gov (United States)

    Cerdá, Jorge I.; Sławińska, Jagoda; Le Lay, Guy; Marele, Antonela C.; Gómez-Rodríguez, José M.; Dávila, María E.

    2016-01-01

    Carbon and silicon pentagonal low-dimensional structures attract a great interest as they may lead to new exotic phenomena such as topologically protected phases or increased spin–orbit effects. However, no pure pentagonal phase has yet been realized for any of them. Here we unveil through extensive density functional theory calculations and scanning tunnelling microscope simulations, confronted to key experimental facts, the hidden pentagonal nature of single- and double-strand chiral Si nano-ribbons perfectly aligned on Ag(110) surfaces whose structure has remained elusive for over a decade. Our study reveals an unprecedented one-dimensional Si atomic arrangement solely comprising almost perfect alternating pentagons residing in the missing row troughs of the reconstructed surface. We additionally characterize the precursor structure of the nano-ribbons, which consists of a Si cluster (nano-dot) occupying a silver di-vacancy in a quasi-hexagonal configuration. The system thus materializes a paradigmatic shift from a silicene-like packing to a pentagonal one. PMID:27708263

  4. Interleukin-9 Is Associated with Elevated Anti-Double-Stranded DNA Antibodies in Lupus-Prone Mice.

    Science.gov (United States)

    Yang, Ji; Li, Qiao; Yang, Xue; Li, Ming

    2015-04-15

    Interleukin (IL)-9, which is produced mainly by CD4(+) T cells, is implicated in mast cell-related allergic diseases, although its involvement in systemic lupus erythematosus (SLE) pathogenesis remains unclear. Thus, we investigated the presence of IL-9 in lupus-prone MRL/Mp-lpr/lpr (MRL/lpr) mice and examined the role of IL-9 in lupus pathogenesis. Increased levels of IL-9(+) lymphocytes were detected in the spleens and kidneys of MRL/lpr mice and increased IL-9 levels in the spleen correlated with PNA(+) germinal center (GC) cell expansion. The percentage of CD4(+)IL-9(+) (Th9) cells was increased in MRL/lpr mice and serum IL-9 levels were elevated and closely related to the production of antibodies against double-stranded DNA (dsDNA). IL-9 appears to promote B-cell proliferation and immunoglobulin production, which could be blocked by inhibition of signal transducer and activator of transcription 3 (STAT3). Treatment with neutralizing anti-IL-9 antibody in vivo decreased serum anti-dsDNA-antibody titers and alleviated lupus nephritis in MRL/lpr mice. Our findings indicate expansion of Th9 cells in lupus-prone MRL/lpr mice and the correlation of IL-9 with B-cell proliferation and autoantibody production. These findings suggest that IL-9 is a potential therapeutic target for SLE.

  5. Non-histone chromosomal proteins HMG1 and 2 enhance ligation reaction of DNA double-strand breaks.

    Science.gov (United States)

    Nagaki, S; Yamamoto, M; Yumoto, Y; Shirakawa, H; Yoshida, M; Teraoka, H

    1998-05-08

    DNA ligase IV in a complex with XRCC4 is responsible for DNA end-joining in repair of DNA double-strand breaks (DSB) and V(D)J recombination. We found that non-histone chromosomal high mobility group (HMG) proteins 1 and 2 enhanced the ligation of linearized pUC119 DNA with DNA ligase IV from rat liver nuclear extract. Intra-molecular and inter-molecular ligations of cohesive-ended and blunt-ended DNA were markedly stimulated by HMG1 and 2. Recombinant HMG2-domain A, B, and (A + B) polypeptides were similarly, but non-identically, effective for the stimulation of DSB ligation reaction. Ligation of single-strand breaks (nicks) was only slightly activated by the HMG proteins. The DNA end-binding Ku protein singly or in combination with the catalytic component of DNA-dependent protein kinase (DNA-PK) as the DNA-PK holoenzyme was ineffective for the ligation of linearized pUC119 DNA. Although the stimulatory effect of HMG1 and 2 on ligation of DSB in vitro was not specific to DNA ligase IV, these results suggest that HMG1 and 2 are involved in the final ligation step in DNA end-joining processes of DSB repair and V(D)J recombination.

  6. Dual roles for DNA polymerase theta in alternative end-joining repair of double-strand breaks in Drosophila.

    Directory of Open Access Journals (Sweden)

    Sze Ham Chan

    2010-07-01

    Full Text Available DNA double-strand breaks are repaired by multiple mechanisms that are roughly grouped into the categories of homology-directed repair and non-homologous end joining. End-joining repair can be further classified as either classical non-homologous end joining, which requires DNA ligase 4, or "alternative" end joining, which does not. Alternative end joining has been associated with genomic deletions and translocations, but its molecular mechanism(s are largely uncharacterized. Here, we report that Drosophila melanogaster DNA polymerase theta (pol theta, encoded by the mus308 gene and previously implicated in DNA interstrand crosslink repair, plays a crucial role in DNA ligase 4-independent alternative end joining. In the absence of pol theta, end joining is impaired and residual repair often creates large deletions flanking the break site. Analysis of break repair junctions from flies with mus308 separation-of-function alleles suggests that pol theta promotes the use of long microhomologies during alternative end joining and increases the likelihood of complex insertion events. Our results establish pol theta as a key protein in alternative end joining in Drosophila and suggest a potential mechanistic link between alternative end joining and interstrand crosslink repair.

  7. Transcription-associated processes cause DNA double-strand breaks and translocations in neural stem/progenitor cells.

    Science.gov (United States)

    Schwer, Bjoern; Wei, Pei-Chi; Chang, Amelia N; Kao, Jennifer; Du, Zhou; Meyers, Robin M; Alt, Frederick W

    2016-02-23

    High-throughput, genome-wide translocation sequencing (HTGTS) studies of activated B cells have revealed that DNA double-strand breaks (DSBs) capable of translocating to defined bait DSBs are enriched around the transcription start sites (TSSs) of active genes. We used the HTGTS approach to investigate whether a similar phenomenon occurs in primary neural stem/progenitor cells (NSPCs). We report that breakpoint junctions indeed are enriched around TSSs that were determined to be active by global run-on sequencing analyses of NSPCs. Comparative analyses of transcription profiles in NSPCs and B cells revealed that the great majority of TSS-proximal junctions occurred in genes commonly expressed in both cell types, possibly because this common set has higher transcription levels on average than genes transcribed in only one or the other cell type. In the latter context, among all actively transcribed genes containing translocation junctions in NSPCs, those with junctions located within 2 kb of the TSS show a significantly higher transcription rate on average than genes with junctions in the gene body located at distances greater than 2 kb from the TSS. Finally, analysis of repair junction signatures of TSS-associated translocations in wild-type versus classical nonhomologous end-joining (C-NHEJ)-deficient NSPCs reveals that both C-NHEJ and alternative end-joining pathways can generate translocations by joining TSS-proximal DSBs to DSBs on other chromosomes. Our studies show that the generation of transcription-associated DSBs is conserved across divergent cell types.

  8. More efficient repair of DNA double-strand breaks in skeletal muscle stem cells compared to their committed progeny

    Directory of Open Access Journals (Sweden)

    Leyla Vahidi Ferdousi

    2014-11-01

    Full Text Available The loss of genome integrity in adult stem cells results in accelerated tissue aging and is possibly cancerogenic. Adult stem cells in different tissues appear to react robustly to DNA damage. We report that adult skeletal stem (satellite cells do not primarily respond to radiation-induced DNA double-strand breaks (DSBs via differentiation and exhibit less apoptosis compared to other myogenic cells. Satellite cells repair these DNA lesions more efficiently than their committed progeny. Importantly, non-proliferating satellite cells and post-mitotic nuclei in the fiber exhibit dramatically distinct repair efficiencies. Altogether, reduction of the repair capacity appears to be more a function of differentiation than of the proliferation status of the muscle cell. Notably, satellite cells retain a high efficiency of DSB repair also when isolated from the natural niche. Finally, we show that repair of DSB substrates is not only very efficient but, surprisingly, also very accurate in satellite cells and that accurate repair depends on the key non-homologous end-joining factor DNA-PKcs.

  9. Targeted Modification of Gene Function Exploiting Homology-Directed Repair of TALEN-Mediated Double-Strand Breaks in Barley.

    Science.gov (United States)

    Budhagatapalli, Nagaveni; Rutten, Twan; Gurushidze, Maia; Kumlehn, Jochen; Hensel, Goetz

    2015-07-06

    Transcription activator-like effector nucleases open up new opportunities for targeted mutagenesis in eukaryotic genomes. Similar to zinc-finger nucleases, sequence-specific DNA-binding domains can be fused with effector domains like the nucleolytically active part of FokI to induce double-strand breaks and thereby modify the host genome on a predefined target site via nonhomologous end joining. More sophisticated applications of programmable endonucleases involve the use of a DNA repair template facilitating homology-directed repair (HDR) so as to create predefined rather than random DNA sequence modifications. The aim of this study was to demonstrate the feasibility of editing the barley genome by precisely modifying a defined target DNA sequence resulting in a predicted alteration of gene function. We used gfp-specific transcription activator-like effector nucleases along with a repair template that, via HDR, facilitates conversion of gfp into yfp, which is associated with a single amino acid exchange in the gene product. As a result of co-bombardment of leaf epidermis, we detected yellow fluorescent protein accumulation in about three of 100 mutated cells. The creation of a functional yfp gene via HDR was unambiguously confirmed by sequencing of the respective genomic site. In addition to the allele conversion accomplished in planta, a readily screenable marker system is introduced that might be useful for optimization approaches in the field of genome editing.

  10. Post-irradiation chemical processing of DNA damage generates double-strand breaks in cells already engaged in repair

    Science.gov (United States)

    Singh, Satyendra K.; Wang, Minli; Staudt, Christian; Iliakis, George

    2011-01-01

    In cells exposed to ionizing radiation (IR), double-strand breaks (DSBs) form within clustered-damage sites from lesions disrupting the DNA sugar–phosphate backbone. It is commonly assumed that these DSBs form promptly and are immediately detected and processed by the cellular DNA damage response (DDR) apparatus. This assumption is questioned by the observation that after irradiation of naked DNA, a fraction of DSBs forms minutes to hours after exposure as a result of temperature dependent, chemical processing of labile sugar lesions. Excess DSBs also form when IR-exposed cells are processed at 50°C, but have been hitherto considered method-related artifact. Thus, it remains unknown whether DSBs actually develop in cells after IR exposure from chemically labile damage. Here, we show that irradiation of ‘naked’ or chromatin-organized mammalian DNA produces lesions, which evolve to DSBs and add to those promptly induced, after 8–24 h in vitro incubation at 37°C or 50°C. The conversion is more efficient in chromatin-associated DNA, completed within 1 h in cells and delayed in a reducing environment. We conclude that IR generates sugar lesions within clustered-damage sites contributing to DSB formation only after chemical processing, which occurs efficiently at 37°C. This subset of delayed DSBs may challenge DDR, may affect the perceived repair kinetics and requires further characterization. PMID:21745815

  11. Novel Smad proteins localize to IR-induced double-strand breaks: interplay between TGFβ and ATM pathways

    Science.gov (United States)

    Wang, Minli; Saha, Janapriya; Hada, Megumi; Anderson, Jennifer A.; Pluth, Janice M.; O’Neill, Peter; Cucinotta, Francis A.

    2013-01-01

    Cellular damage from ionizing radiation (IR) is in part due to DNA damage and reactive oxygen species, which activate DNA damage response (DDR) and cytokine signaling pathways, including the ataxia telangiectasia mutated (ATM) and transforming growth factor (TGF)β/Smad pathways. Using classic double-strand breaks (DSBs) markers, we studied the roles of Smad proteins in DDR and the crosstalk between TGFβ and ATM pathways. We observed co-localization of phospho-Smad2 (pSmad2) and Smad7 with DSB repair proteins following low and high linear energy transfer (LET) radiation in human fibroblasts and epithelial cells. The decays of both foci were similar to that of γH2AX foci. Irradiation with high LET particles induced pSmad2 and Smad7 foci tracks indicating the particle trajectory through cells. pSmad2 foci were absent in S phase cells, while Smad7 foci were present in all phases of cell cycle. pSmad2 (but not Smad7) foci were completely abolished when ATM was depleted or inactivated. In contrast, a TGFβ receptor 1 (TGFβR1) inhibitor abrogated Smad7, but not pSmad2 foci at DSBs sites. In summary, we suggest that Smad2 and Smad7 contribute to IR-induced DSB signaling in an ATM or TGFβR1-dependent manner, respectively. PMID:23221633

  12. Co-Localization of Somatic and Meiotic Double Strand Breaks Near the Myc Oncogene on Mouse Chromosome 15

    Science.gov (United States)

    Ng, Siemon H.; Maas, Sarah A.; Petkov, Petko M.; Mills, Kevin D.; Paigen, Kenneth

    2010-01-01

    Both somatic and meiotic recombinations involve the repair of DNA double strand breaks (DSBs) that occur at preferred locations in the genome. Improper repair of DSBs during either mitosis or meiosis can lead to mutations, chromosomal aberration such as translocations, cancer and/or cell death. Currently, no model exists that explains the locations of either spontaneous somatic DSBs or programmed meiotic DSBs or relates them to each other. One common class of tumorigenic translocations arising from DSBs is chromosomal rearrangements near the Myc oncogene. Myc translocations have been associated with Burkitt lymphoma in humans, plasmacytoma in mice and immunocytoma in rats. Comparing the locations of somatic and meiotic DSBs near the mouse Myc oncogene, we demonstrated that the placement of these DSBs is not random and that both events clustered in the same short discrete region of the genome. Our work shows that both somatic and meiotic DSBs tend to occur in proximity to each other within the Myc region, suggesting that they share common originating features. It is likely that some regions of the genome are more susceptible to both somatic and meiotic DSBs, and the locations of meiotic hotspots may be an indicator of genomic regions more susceptible to DNA damage. PMID:19603522

  13. Bi-directional routing of DNA mismatch repair protein human exonuclease 1 to replication foci and DNA double strand breaks

    DEFF Research Database (Denmark)

    Liberti, Sascha E; Andersen, Sofie Dabros; Wang, Jing

    2011-01-01

    Human exonuclease 1 (hEXO1) is implicated in DNA metabolism, including replication, recombination and repair, substantiated by its interactions with PCNA, DNA helicases BLM and WRN, and several DNA mismatch repair (MMR) proteins. We investigated the sub-nuclear localization of hEXO1 during S......-phase progression and in response to laser-induced DNA double strand breaks (DSBs). We show that hEXO1 and PCNA co-localize in replication foci. This apparent interaction is sustained throughout S-phase. We also demonstrate that hEXO1 is rapidly recruited to DNA DSBs. We have identified a PCNA interacting protein...... (PIP-box) region on hEXO1 located in its COOH-terminal ((788)QIKLNELW(795)). This motif is essential for PCNA binding and co-localization during S-phase. Recruitment of hEXO1 to DNA DSB sites is dependent on the MMR protein hMLH1. We show that two distinct hMLH1 interaction regions of hEXO1 (residues...

  14. Enrichment of Cdk1-cyclins at DNA double-strand breaks stimulates Fun30 phosphorylation and DNA end resection.

    Science.gov (United States)

    Chen, Xuefeng; Niu, Hengyao; Yu, Yang; Wang, Jingjing; Zhu, Shuangyi; Zhou, Jianjie; Papusha, Alma; Cui, Dandan; Pan, Xuewen; Kwon, Youngho; Sung, Patrick; Ira, Grzegorz

    2016-04-07

    DNA double-strand breaks (DSBs) are one of the most cytotoxic types of DNA lesion challenging genome integrity. The activity of cyclin-dependent kinase Cdk1 is essential for DSB repair by homologous recombination and for DNA damage signaling. Here we identify the Fun30 chromatin remodeler as a new target of Cdk1. Fun30 is phosphorylated by Cdk1 on Serine 28 to stimulate its functions in DNA damage response including resection of DSB ends. Importantly, Cdk1-dependent phosphorylation of Fun30-S28 increases upon DNA damage and requires the recruitment of Fun30 to DSBs, suggesting that phosphorylation increases in situ at the DNA damage. Consistently, we find that Cdk1 and multiple cyclins become highly enriched at DSBs and that the recruitment of Cdk1 and cyclins Clb2 and Clb5 ensures optimal Fun30 phosphorylation and checkpoint activation. We propose that the enrichment of Cdk1-cyclin complexes at DSBs serves as a mechanism for enhanced targeting and modulating of the activity of DNA damage response proteins.

  15. ADAR1 forms a complex with Dicer to promote microRNA processing and RNA-induced gene silencing.

    NARCIS (Netherlands)

    Ota, H.; Sakurai, M.; Gupta, R; Valente, L.; Wulff, B.E.; Ariyoshi, K.; Iizasa, H.; Davuluri, R.V.; Nishikura, K.

    2013-01-01

    Adenosine deaminases acting on RNA (ADARs) are involved in RNA editing that converts adenosine residues to inosine specifically in double-stranded RNAs. In this study, we investigated the interaction of the RNA editing mechanism with the RNA interference (RNAi) machinery and found that ADAR1 forms a

  16. Dose-ranging evaluation of intravitreal siRNA PF-04523655 for diabetic macular edema (the DEGAS study)

    DEFF Research Database (Denmark)

    Nguyen, Quan Dong; Schachar, Ronald A; Nduaka, Chudy I;

    2012-01-01

    To evaluate the safety and efficacy of three doses of PF-04523655, a 19-nucleotide methylated double stranded siRNA targeting the RTP801 gene, for the treatment of diabetic macular edema (DME) compared to focal/grid laser photocoagulation.......To evaluate the safety and efficacy of three doses of PF-04523655, a 19-nucleotide methylated double stranded siRNA targeting the RTP801 gene, for the treatment of diabetic macular edema (DME) compared to focal/grid laser photocoagulation....

  17. Surface shapes and surrounding environment analysis of single- and double-stranded DNA-binding proteins in protein-DNA interface.

    Science.gov (United States)

    Wang, Wei; Liu, Juan; Sun, Lin

    2016-07-01

    Protein-DNA bindings are critical to many biological processes. However, the structural mechanisms underlying these interactions are not fully understood. Here, we analyzed the residues shape (peak, flat, or valley) and the surrounding environment of double-stranded DNA-binding proteins (DSBs) and single-stranded DNA-binding proteins (SSBs) in protein-DNA interfaces. In the results, we found that the interface shapes, hydrogen bonds, and the surrounding environment present significant differences between the two kinds of proteins. Built on the investigation results, we constructed a random forest (RF) classifier to distinguish DSBs and SSBs with satisfying performance. In conclusion, we present a novel methodology to characterize protein interfaces, which will deepen our understanding of the specificity of proteins binding to ssDNA (single-stranded DNA) or dsDNA (double-stranded DNA). Proteins 2016; 84:979-989. © 2016 Wiley Periodicals, Inc.

  18. FBH1 co-operates with MUS81 in inducing DNA double-strand breaks and cell death following replication stress

    DEFF Research Database (Denmark)

    Fugger, Kasper; Chu, Wai Kit; Haahr, Peter;

    2013-01-01

    The molecular events occurring following the disruption of DNA replication forks are poorly characterized, despite extensive use of replication inhibitors such as hydroxyurea in the treatment of malignancies. Here, we identify a key role for the FBH1 helicase in mediating DNA double-strand break...... formation following replication inhibition. We show that FBH1-deficient cells are resistant to killing by hydroxyurea, and exhibit impaired activation of the pro-apoptotic factor p53, consistent with decreased DNA double-strand break formation. Similar findings were obtained in murine ES cells carrying...... disrupted alleles of Fbh1. We also show that FBH1 through its helicase activity co-operates with the MUS81 nuclease in promoting the endonucleolytic DNA cleavage following prolonged replication stress. Accordingly, MUS81 and EME1-depleted cells show increased resistance to the cytotoxic effects...

  19. To Nick or Not to Nick: Comparison of I-SceI Single- and Double-Strand Break-Induced Recombination in Yeast and Human Cells

    OpenAIRE

    2014-01-01

    Genetic modification of a chromosomal locus to replace an existing dysfunctional allele with a corrected sequence can be accomplished through targeted gene correction using the cell's homologous recombination (HR) machinery. Gene targeting is stimulated by generation of a DNA double-strand break (DSB) at or near the site of correction, but repair of the break via non-homologous end-joining without using the homologous template can lead to deleterious genomic changes such as in/del mutations, ...

  20. Temperature-induced melting of double-stranded DNA in the absence and presence of covalently bonded antitumour drugs: insight from molecular dynamics simulations

    OpenAIRE

    Bueren-Calabuig, J. A.; Giraudon, C.; Galmarini, C M; Egly, J M; Gago, F.

    2011-01-01

    The difference in melting temperature of a double-stranded (ds) DNA molecule in the absence and presence of bound ligands can provide experimental information about the stabilization brought about by ligand binding. By simulating the dynamic behaviour of a duplex of sequence 5′-d(TAATAACGGATTATT)·5′-d(AATAATCCGTTATTA) in 0.1 M NaCl aqueous solution at 400 K, we have characterized in atomic detail its complete thermal denaturation profile in

  1. A strategy of tumor treatment in mice with doxorubicin-cyclophosphamide combination based on dendritic cell activation by human double-stranded DNA preparation

    OpenAIRE

    Alyamkina, Ekaterina A; Nikolin, Valeriy P; Popova, Nelly A.; Dolgova, Evgenia V; Proskurina, Anastasia S; Orishchenko, Konstantin E.; Efremov, Yaroslav R.; Chernykh, Elena R.; Ostanin, Alexandr A.; Sidorov, Sergey V; Ponomarenko, Dmitriy M.; Zagrebelniy, Stanislav N; Bogachev, Sergey S.; Shurdov, Mikhail A

    2010-01-01

    Background Immunization of mice with tumor homogenate after combined treatment with cyclophosphamide (CP) and double-stranded DNA (dsDNA) preparation is effective at inhibition of growth of tumor challenged after the treatment. It was assumed that this inhibition might be due to activation of the antigen-presenting cells. The purpose was to develop improved antitumor strategy using mice. We studied the combined action of cytostatics doxorubicin (Dox) plus CP with subsequent dsDNA preparation ...

  2. Linear induction of DNA double-strand breakage with X-ray dose, as determined from DNA fragment size distribution

    Energy Technology Data Exchange (ETDEWEB)

    Erixon, K.; Cedervall, B. [Karolinksa Institutet, Stockholm (Sweden)

    1995-05-01

    Pulsed-field gel electrophoresis has been applied to separate DNA from mouse L1210 cells exposed to X-ray doses of 1 to 50 Gy. Simultaneous separation of marker chromosomes in the range 0.1 to 12.6 Mbp allowed calculation of the size distribution of the radiation-induced fragments. The distribution was consistent with a random induction of double-strand breaks (DSBs). A theoretical relationship between the size distribution of such fragments and the average number of induced breaks was used to calculate the yield and dose response. The DNA distribution was determined by both radiolabeling and fluorescence staining. Two independent methods were use to evaluate the radiation-induced yield of DSBs, both assuming that all DNA is broken at random. In the first method we compared the theoretical and experimental fraction of DNA that is below a given size limit. By this method we estimated the yield to be 0.006-0.007 DSB/GY per million base pairs using the radiolabel and 0.004-0.008 DSB/Gy per million base pairs by fluorescence staining. The dose response was linear in both cases. In the second method we looked only at the size distribution in the resolving part of the gel and compared it to the theoretical distribution. By this method a value of approximately 0.012 DSB/Gy/Mb was found, using fluorescence as a measure of DNA distribution. In a normal diploid mammalian genome of size 60000 Mbp, this is equivalent to a yield of 25-50 DSBs/Gy or 70 DSBs/GY, respectively. The second approach, which looks only at the smaller fragments, may overestimate the yield, while the first approach suffers from uncertainties about the fraction of DNA irreversibly trapped in the well. The assay has the capacity to detect a dose of less than 1 Gy. 58 refs., 10 figs.

  3. MeHg Developing Exposure Causes DNA Double-Strand Breaks and Elicits Cell Cycle Arrest in Spinal Cord Cells

    Science.gov (United States)

    Ferreira, Fabiana F.; Ammar, Dib; Bourckhardt, Gilian F.; Kobus-Bianchini, Karoline; Müller, Yara M. R.; Nazari, Evelise M.

    2015-01-01

    The neurotoxicity caused by methylmercury (MeHg) is well documented; however, the developmental neurotoxicity in spinal cord is still not fully understood. Here we investigated whether MeHg affects the spinal cord layers development. Chicken embryos at E3 were treated in ovo with 0.1 μg MeHg/50 μL saline solution and analyzed at E10. Thus, we performed immunostaining using anti-γ-H2A.X to recognize DNA double-strand breaks and antiphosphohistone H3, anti-p21, and anti-cyclin E to identify cells in proliferation and cell cycle proteins. Also, to identify neuronal cells, we used anti-NeuN and anti-βIII-tubulin antibodies. After the MeHg treatment, we observed the increase on γ-H2A.X in response to DNA damage. MeHg caused a decrease in the proliferating cells and in the thickness of spinal cord layers. Moreover, we verified that MeHg induced an increase in the number of p21-positive cells but did not change the cyclin E-positive cells. A significantly high number of TUNEL-positive cells indicating DNA fragmentation were observed in MeHg-treated embryos. Regarding the neuronal differentiation, MeHg induced a decrease in NeuN expression and did not change the expression of βIII-tubulin. These results showed that in ovo MeHg exposure alters spinal cord development by disturbing the cell proliferation and death, also interfering in early neuronal differentiation. PMID:26793240

  4. Mapping meiotic single-strand DNA reveals a new landscape of DNA double-strand breaks in Saccharomyces cerevisiae.

    Directory of Open Access Journals (Sweden)

    Cyril Buhler

    2007-12-01

    Full Text Available DNA double-strand breaks (DSBs, which are formed by the Spo11 protein, initiate meiotic recombination. Previous DSB-mapping studies have used rad50S or sae2Delta mutants, which are defective in break processing, to accumulate Spo11-linked DSBs, and report large (> or = 50 kb "DSB-hot" regions that are separated by "DSB-cold" domains of similar size. Substantial recombination occurs in some DSB-cold regions, suggesting that DSB patterns are not normal in rad50S or sae2Delta mutants. We therefore developed a novel method to map genome-wide, single-strand DNA (ssDNA-associated DSBs that accumulate in processing-capable, repair-defective dmc1Delta and dmc1Delta rad51Delta mutants. DSBs were observed at known hot spots, but also in most previously identified "DSB-cold" regions, including near centromeres and telomeres. Although approximately 40% of the genome is DSB-cold in rad50S mutants, analysis of meiotic ssDNA from dmc1Delta shows that most of these regions have substantial DSB activity. Southern blot assays of DSBs in selected regions in dmc1Delta, rad50S, and wild-type cells confirm these findings. Thus, DSBs are distributed much more uniformly than was previously believed. Comparisons of DSB signals in dmc1, dmc1 rad51, and dmc1 spo11 mutant strains identify Dmc1 as a critical strand-exchange activity genome-wide, and confirm previous conclusions that Spo11-induced lesions initiate all meiotic recombination.

  5. Reduced Activity of Double-Strand Break Repair Genes in Prostate Cancer Patients With Late Normal Tissue Radiation Toxicity

    Energy Technology Data Exchange (ETDEWEB)

    Oorschot, Bregje van, E-mail: b.vanoorschot@amc.uva.nl [Laboratory for Experimental Oncology and Radiobiology (LEXOR), Center for Molecular Medicine (CEMM), Department of Radiation Oncology, Academic Medical Center, University of Amsterdam, Amsterdam (Netherlands); Hovingh, Suzanne E. [Laboratory for Experimental Oncology and Radiobiology (LEXOR), Center for Molecular Medicine (CEMM), Department of Radiation Oncology, Academic Medical Center, University of Amsterdam, Amsterdam (Netherlands); Moerland, Perry D. [Bioinformatics Laboratory, Department of Clinical Epidemiology, Biostatistics and Bioinformatics, Academic Medical Center, University of Amsterdam, Amsterdam (Netherlands); Medema, Jan Paul; Stalpers, Lukas J.A. [Laboratory for Experimental Oncology and Radiobiology (LEXOR), Center for Molecular Medicine (CEMM), Department of Radiation Oncology, Academic Medical Center, University of Amsterdam, Amsterdam (Netherlands); Vrieling, Harry [Department of Toxicogenetics, Leiden University Medical Center, Leiden (Netherlands); Franken, Nicolaas A.P. [Laboratory for Experimental Oncology and Radiobiology (LEXOR), Center for Molecular Medicine (CEMM), Department of Radiation Oncology, Academic Medical Center, University of Amsterdam, Amsterdam (Netherlands)

    2014-03-01

    Purpose: To investigate clinical parameters and DNA damage response as possible risk factors for radiation toxicity in the setting of prostate cancer. Methods and Materials: Clinical parameters of 61 prostate cancer patients, 34 with (overresponding, OR) and 27 without (non-responding, NR) severe late radiation toxicity were assembled. In addition, for a matched subset the DNA damage repair kinetics (γ-H2AX assay) and expression profiles of DNA repair genes were determined in ex vivo irradiated lymphocytes. Results: Examination of clinical data indicated none of the considered clinical parameters to be correlated with the susceptibility of patients to develop late radiation toxicity. Although frequencies of γ-H2AX foci induced immediately after irradiation were similar (P=.32), significantly higher numbers of γ-H2AX foci were found 24 hours after irradiation in OR compared with NR patients (P=.03). Patient-specific γ-H2AX foci decay ratios were significantly higher in NR patients than in OR patients (P<.0001). Consequently, NR patients seem to repair DNA double-strand breaks (DSBs) more efficiently than OR patients. Moreover, gene expression analysis indicated several genes of the homologous recombination pathway to be stronger induced in NR compared with OR patients (P<.05). A similar trend was observed in genes of the nonhomologous end-joining repair pathway (P=.09). This is congruent with more proficient repair of DNA DSBs in patients without late radiation toxicity. Conclusions: Both gene expression profiling and DNA DSB repair kinetics data imply that less-efficient repair of radiation-induced DSBs may contribute to the development of late normal tissue damage. Induction levels of DSB repair genes (eg, RAD51) may potentially be used to assess the risk for late radiation toxicity.

  6. Impact of histone H4 lysine 20 methylation on 53BP1 responses to chromosomal double strand breaks.

    Directory of Open Access Journals (Sweden)

    Andrea J Hartlerode

    Full Text Available Recruitment of 53BP1 to chromatin flanking double strand breaks (DSBs requires γH2AX/MDC1/RNF8-dependent ubiquitination of chromatin and interaction of 53BP1 with histone H4 methylated on lysine 20 (H4K20me. Several histone methyltransferases have been implicated in 53BP1 recruitment, but their quantitative contributions to the 53BP1 response are unclear. We have developed a multi-photon laser (MPL system to target DSBs to subfemtoliter nuclear volumes and used this to mathematically model DSB response kinetics of MDC1 and of 53BP1. In contrast to MDC1, which revealed first order kinetics, the 53BP1 MPL-DSB response is best fitted by a Gompertz growth function. The 53BP1 MPL response shows the expected dependency on MDC1 and RNF8. We determined the impact of altered H4K20 methylation on 53BP1 MPL response kinetics in mouse embryonic fibroblasts (MEFs lacking key H4K20 histone methyltransferases. This revealed no major requirement for the known H4K20 dimethylases Suv4-20h1 and Suv4-20h2 in 53BP1 recruitment or DSB repair function, but a key role for the H4K20 monomethylase, PR-SET7. The histone methyltransferase MMSET/WHSC1 has recently been implicated in 53BP1 DSB recruitment. We found that WHSC1 homozygous mutant MEFs reveal an alteration in balance of H4K20 methylation patterns; however, 53BP1 DSB responses in these cells appear normal.

  7. DNA repair in modeled microgravity: Double strand break rejoining activity in human lymphocytes irradiated with {gamma}-rays

    Energy Technology Data Exchange (ETDEWEB)

    Mognato, Maddalena, E-mail: maddalena.mognato@unipd.it [Dipartimento di Biologia, Universita di Padova, via U. Bassi 58 B, 35121 Padova (Italy); Girardi, Cristina; Fabris, Sonia [Dipartimento di Biologia, Universita di Padova, via U. Bassi 58 B, 35121 Padova (Italy); Celotti, Lucia [Dipartimento di Biologia, Universita di Padova, via U. Bassi 58 B, 35121 Padova (Italy); Laboratori Nazionali di Legnaro, INFN, Padova (Italy)

    2009-04-26

    Cell response to ionising radiation depends, besides on genetic and physiological features of the biological systems, on environmental conditions occurring during DNA repair. Many data showed that microgravity, experienced by astronauts during space flights or modeled on Earth, causes apoptosis, cytoskeletal alteration, cell growth inhibition, increased frequency of mutations and chromosome aberrations. In this study, we analysed the progression of the rejoining of double strand breaks (DSBs) in human peripheral blood lymphocytes (PBLs) irradiated with {gamma}-rays and incubated in static condition (1g) or in modeled microgravity (MMG). {gamma}-H2AX foci formation and disappearance, monitored during the repair incubation, showed that the kinetics of DSBs rejoining was different in the two gravity conditions. The fraction of foci-positive cells decreased slower in MMG than in 1g at 6 and 24 h after irradiation (P < 0.01) and the mean number of {gamma}-H2AX foci per nucleus was significantly higher in MMG than in 1g at the same time-points (P < 0.001). In the same samples we determined apoptotic level and the rate of DSB rejoining during post-irradiation incubation. A significant induction of apoptosis was observed in MMG at 24 h after irradiation (P < 0.001), whereas at shorter times the level of apoptosis was slightly higher in MMG respect to 1g. In accordance with the kinetics of {gamma}-H2AX foci, the slower rejoining of radiation-induced DSBs in MMG was observed by DNA fragmentation analyses during the repair incubation; the data of pulsed-field gel electrophoresis assay showed that the fraction of DNA released in the gel was significantly higher in PBL incubated in MMG after irradiation with respect to cells maintained in 1g. Our results provide evidences that MMG incubation during DNA repair delayed the rate of radiation-induced DSB rejoining, and increased, as a consequence, the genotoxic effects of ionising radiation.

  8. Hepatorenal correction in murine glycogen storage disease type I with a double-stranded adeno-associated virus vector.

    LENUS (Irish Health Repository)

    Luo, Xiaoyan

    2011-11-01

    Glycogen storage disease type Ia (GSD-Ia) is caused by the deficiency of glucose-6-phosphatase (G6Pase). Long-term complications of GSD-Ia include life-threatening hypoglycemia and proteinuria progressing to renal failure. A double-stranded (ds) adeno-associated virus serotype 2 (AAV2) vector encoding human G6Pase was pseudotyped with four serotypes, AAV2, AAV7, AAV8, and AAV9, and we evaluated efficacy in 12-day-old G6pase (-\\/-) mice. Hypoglycemia during fasting (plasma glucose <100 mg\\/dl) was prevented for >6 months by the dsAAV2\\/7, dsAAV2\\/8, and dsAAV2\\/9 vectors. Prolonged fasting for 8 hours revealed normalization of blood glucose following dsAAV2\\/9 vector administration at the higher dose. The glycogen content of kidney was reduced by >65% with both the dsAAV2\\/7 and dsAAV2\\/9 vectors, and renal glycogen content was stably reduced between 7 and 12 months of age for the dsAAV2\\/9 vector-treated mice. Every vector-treated group had significantly reduced glycogen content in the liver, in comparison with untreated G6pase (-\\/-) mice. G6Pase was expressed in many renal epithelial cells of with the dsAAV2\\/9 vector for up to 12 months. Albuminuria and renal fibrosis were reduced by the dsAAV2\\/9 vector. Hepatorenal correction in G6pase (-\\/-) mice demonstrates the potential of AAV vectors for the correction of inherited diseases of metabolism.

  9. Approach to the classical radiation biology. Ionizing radiation effects and repair mechanism of DNA double strand breaks

    Energy Technology Data Exchange (ETDEWEB)

    Utsumi, Hiroshi [Kyoto Univ., Kumatori, Osaka (Japan). Research Reactor Inst

    2000-09-01

    Split-dose recovery has been observed under a variety of experimental conditions in many cell systems and believed to be the recovery of sublethal damage (SLD). It is considered to be one of the most widespread and important cellular responses in clinical radiotherapy. To study the molecular mechanism of this recovery, we analyzed the knockout mutants KU70{sup -/-}, RAD54{sup -/-}, and KU70{sup -/-}/ RAD54{sup -/-} of the chicken B-cell line, DT40. Rad54 participates in the homologous recombinational (HR) repair of DNA double-strand breaks (DSB), while Ku proteins are involved in non-homologous end-joining (NHEJ). Split-dose recovery was observed in the parent DT40 and KU70{sup -/-} cells. Moreover the split-dose survival enhancement had all of the characteristics of SLD recovery that had been demonstrated earlier: e.g., the reappearance of the shoulder of the survival curve with dose fractionation; repair at 25degC; and inhibition by the antibiotic actinomycin D. These results strongly suggest that SLD recovery is due to DSB repair via or mediated by HR, and that these breaks constitute SLD. The tonicity-sensitive potentially lethal damage (PLD) recovery was also found only in DT40 and KU70 {sup -/-} cells. Delayed-plating PLD recovery may be controlled by NHEJ repair that works through the cell cycle. These results lead to the conclusion that the repair of DSBs could explain the classical operational recovery phenomena. We have also investigated RBE/LET using those mutants. (author)

  10. ATM alters the otherwise robust chromatin mobility at sites of DNA double-strand breaks (DSBs in human cells.

    Directory of Open Access Journals (Sweden)

    Annabelle Becker

    Full Text Available Ionizing radiation induces DNA double strand breaks (DSBs which can lead to the formation of chromosome rearrangements through error prone repair. In mammalian cells the positional stability of chromatin contributes to the maintenance of genome integrity. DSBs exhibit only a small, submicron scale diffusive mobility, but a slight increase in the mobility of chromatin domains by the induction of DSBs might influence repair fidelity and the formation of translocations. The radiation-induced local DNA decondensation in the vicinity of DSBs is one factor potentially enhancing the mobility of DSB-containing chromatin domains. Therefore in this study we focus on the influence of different chromatin modifying proteins, known to be activated by the DNA damage response, on the mobility of DSBs. IRIF (ionizing radiation induced foci in U2OS cells stably expressing 53BP1-GFP were used as a surrogate marker of DSBs. Low angle charged particle irradiation, known to trigger a pronounced DNA decondensation, was used for the defined induction of linear tracks of IRIF. Our results show that movement of IRIF is independent of the investigated chromatin modifying proteins like ACF1 or PARP1 and PARG. Also depletion of proteins that tether DNA strands like MRE11 and cohesin did not alter IRIF dynamics significantly. Inhibition of ATM, a key component of DNA damage response signaling, resulted in a pronounced confinement of DSB mobility, which might be attributed to a diminished radiation induced decondensation. This confinement following ATM inhibition was confirmed using X-rays, proving that this effect is not restricted to densely ionizing radiation. In conclusion, repair sites of DSBs exhibit a limited mobility on a small spatial scale that is mainly unaffected by depletion of single remodeling or DNA tethering proteins. However, it relies on functional ATM kinase which is considered to influence the chromatin structure after irradiation.

  11. MeHg Developing Exposure Causes DNA Double-Strand Breaks and Elicits Cell Cycle Arrest in Spinal Cord Cells

    Directory of Open Access Journals (Sweden)

    Fabiana F. Ferreira

    2015-01-01

    Full Text Available The neurotoxicity caused by methylmercury (MeHg is well documented; however, the developmental neurotoxicity in spinal cord is still not fully understood. Here we investigated whether MeHg affects the spinal cord layers development. Chicken embryos at E3 were treated in ovo with 0.1 μg MeHg/50 μL saline solution and analyzed at E10. Thus, we performed immunostaining using anti-γ-H2A.X to recognize DNA double-strand breaks and antiphosphohistone H3, anti-p21, and anti-cyclin E to identify cells in proliferation and cell cycle proteins. Also, to identify neuronal cells, we used anti-NeuN and anti-βIII-tubulin antibodies. After the MeHg treatment, we observed the increase on γ-H2A.X in response to DNA damage. MeHg caused a decrease in the proliferating cells and in the thickness of spinal cord layers. Moreover, we verified that MeHg induced an increase in the number of p21-positive cells but did not change the cyclin E-positive cells. A significantly high number of TUNEL-positive cells indicating DNA fragmentation were observed in MeHg-treated embryos. Regarding the neuronal differentiation, MeHg induced a decrease in NeuN expression and did not change the expression of βIII-tubulin. These results showed that in ovo MeHg exposure alters spinal cord development by disturbing the cell proliferation and death, also interfering in early neuronal differentiation.

  12. Development of novel visual-plus quantitative analysis systems for studying DNA double-strand break repairs in zebrafish.

    Science.gov (United States)

    Liu, Jingang; Gong, Lu; Chang, Changqing; Liu, Cong; Peng, Jinrong; Chen, Jun

    2012-09-20

    The use of reporter systems to analyze DNA double-strand break (DSB) repairs, based on the enhanced green fluorescent protein (EGFP) and meganuclease such as I-Sce I, is usually carried out with cell lines. In this study, we developed three visual-plus quantitative assay systems for homologous recombination (HR), non-homologous end joining (NHEJ) and single-strand annealing (SSA) DSB repair pathways at the organismal level in zebrafish embryos. To initiate DNA DSB repair, we used two I-Sce I recognition sites in opposite orientation rather than the usual single site. The NHEJ, HR and SSA repair pathways were separately triggered by the injection of three corresponding I-Sce I-cut constructions, and the repair of DNA lesion caused by I-Sce I could be tracked by EGFP expression in the embryos. Apart from monitoring the intensity of green fluorescence, the repair frequencies could also be precisely measured by quantitative real-time polymerase chain reaction (qPCR). Analysis of DNA sequences at the DSB sites showed that NHEJ was predominant among these three repair pathways in zebrafish embryos. Furthermore, while HR and SSA reporter systems could be effectively decreased by the knockdown of rad51 and rad52, respectively, NHEJ could only be impaired by the knockdown of ligaseIV (lig4) when the NHEJ construct was cut by I-Sce I in vivo. More interestingly, blocking NHEJ with lig4-MO increased the frequency of HR, but decreased the frequency of SSA. Our studies demonstrate that the major mechanisms used to repair DNA DSBs are conserved from zebrafish to mammal, and zebrafish provides an excellent model for studying and manipulating DNA DSB repair at the organismal level.

  13. Chromosome thripsis by DNA double strand break clusters causes enhanced cell lethality, chromosomal translocations and 53BP1-recruitment.

    Science.gov (United States)

    Schipler, Agnes; Mladenova, Veronika; Soni, Aashish; Nikolov, Vladimir; Saha, Janapriya; Mladenov, Emil; Iliakis, George

    2016-09-19

    Chromosome translocations are hallmark of cancer and of radiation-induced cell killing, reflecting joining of incongruent DNA-ends that alter the genome. Translocation-formation requires DNA end-joining mechanisms and incompletely characterized, permissive chromatin conditions. We show that chromatin destabilization by clusters of DNA double-strand-breaks (DSBs) generated by the I-SceI meganuclease at multiple, appropriately engineered genomic sites, compromises c-NHEJ and markedly increases cell killing and translocation-formation compared to single-DSBs. Translocation-formation from DSB-clusters utilizes Parp1 activity, implicating alt-EJ in their formation. Immunofluorescence experiments show that single-DSBs and DSB-clusters uniformly provoke the formation of single γ-H2AX foci, suggesting similar activation of early DNA damage response (DDR). Live-cell imaging also shows similar single-focus recruitment of the early-response protein MDC1, to single-DSBs and DSB-clusters. Notably, the late DDR protein, 53BP1 shows in live-cell imaging strikingly stronger recruitment to DSB-clusters as compared to single-DSBs. This is the first report that chromatin thripsis, in the form of engineered DSB-clusters, compromises first-line DSB-repair pathways, allowing alt-EJ to function as rescuing-backup. DSB-cluster-formation is indirectly linked to the increased biological effectiveness of high ionization-density radiations, such as the alpha-particles emitted by radon gas or the heavy-ions utilized in cancer therapy. Our observations provide the first direct mechanistic explanation for this long-known effect.

  14. Development of Novel Visual-Plus Quantitative Analysis Systems for Studying DNA Double-Strand Break Repairs in Zebrafish

    Institute of Scientific and Technical Information of China (English)

    Jingang Liu; Lu Gong; Changqing Chang; Cong Liu; Jinrong Peng; Jun Chen

    2012-01-01

    The use of reporter systems to analyze DNA double-strand break (DSB) repairs,based on the enhanced green fluorescent protein (EGFP) and meganuclease such as I-Sce Ⅰ,is usually carried out with cell lines.In this study,we developed three visual-plus quantitative assay systems for homologous recombination (HR),non-homologous end joining (NHEJ) and single-strand annealing (SSA) DSB repair pathways at the organismal level in zebrafish embryos.To initiate DNA DSB repair,we used two I-Sce Ⅰ recognition sites in opposite orientation rather than the usual single site.The NHEJ,HR and SSA repair pathways were separately triggered by the injection of three corresponding I-Sce I-cut constructions,and the repair of DNA lesion caused by I-Sce Ⅰ could be tracked by EGFP expression in the embryos.Apart from monitoring the intensity of green fluorescence,the repair frequencies could also be precisely measured by quantitative real-time polymerase chain reaction (qPCR).Analysis of DNA sequences at the DSB sites showed that NHEJ was predominant among these three repair pathways in zebrafish embryos.Furthermore,while HR and SSA reporter systems could be effectively decreased by the knockdown of rad51 and rad52,respectively,NHEJ could only be impaired by the knockdown of ligaseⅣ (lig4) when the NHEJ construct was cut by I-Sce Ⅰ in vivo.More interestingly,blocking NHEJ with lig4-MO increased the frequency of HR,but decreased the frequency of SSA.Our studies demonstrate that the major mechanisms used to repair DNA DSBs are conserved from zebrafish to mammal,and zebrafish provides an excellent model for studying and manipulating DNA DSB repair at the organismal level.

  15. Arabidopsis DNA polymerase lambda mutant is mildly sensitive to DNA double strand breaks but defective in integration ofa transgene.

    Directory of Open Access Journals (Sweden)

    Tomoyuki eFurukawa

    2015-05-01

    Full Text Available The DNA double-strand break (DSB is a critical type of damage, and can be induced by both endogenous sources (e.g. errors of oxidative metabolism, transposable elements, programmed meiotic breaks, or perturbation of the DNA replication fork and exogenous sources (e.g. ionizing radiation or radiomimetic chemicals. Although higher plants, like mammals, are thought to preferentially repair DSBs via nonhomologous end joining (NHEJ, much remains unclear about plant DSB repair pathways. Our reverse genetic approach suggests that DNA polymerase λ is involved in DSB repair in Arabidopsis. The Arabidopsis T-DNA insertion mutant (atpolλ-1 displayed sensitivity to both gamma-irradiation and treatment with radiomimetic reagents, but not to other DNA damaging treatments. The atpolλ-1 mutant showed a moderate sensitivity to DSBs, while Arabidopsis Ku70 and DNA ligase 4 mutants (atku70-3 and atlig4-2, both of which play critical roles in NHEJ, exhibited a hypersensitivity to these treatments. The atpolλ-1/atlig4-2 double mutant exhibited a higher sensitivity to DSBs than each single mutant, but the atku70/atpolλ-1 showed similar sensitivity to the atku70-3 mutant. We showed that transcription of the DNA ligase 1, DNA ligase 6, and Wee1 genes was quickly induced by BLM in several NHEJ deficient mutants in contrast to wild-type. Finally, the T-DNA transformation efficiency dropped in NHEJ deficient mutants and the lowest transformation efficiency was scored in the atpolλ-1/atlig4-2 double mutant. These results imply that AtPolλ is involved in both DSB repair and DNA damage response pathway.

  16. DNA ligase 1 deficient plants display severe growth defects and delayed repair of both DNA single and double strand breaks

    Directory of Open Access Journals (Sweden)

    Bray Clifford M

    2009-06-01

    Full Text Available Abstract Background DNA ligase enzymes catalyse the joining of adjacent polynucleotides and as such play important roles in DNA replication and repair pathways. Eukaryotes possess multiple DNA ligases with distinct roles in DNA metabolism, with clear differences in the functions of DNA ligase orthologues between animals, yeast and plants. DNA ligase 1, present in all eukaryotes, plays critical roles in both DNA repair and replication and is indispensable for cell viability. Results Knockout mutants of atlig1 are lethal. Therefore, RNAi lines with reduced levels of AtLIG1 were generated to allow the roles and importance of Arabidopsis DNA ligase 1 in DNA metabolism to be elucidated. Viable plants were fertile but displayed a severely stunted and stressed growth phenotype. Cell size was reduced in the silenced lines, whilst flow cytometry analysis revealed an increase of cells in S-phase in atlig1-RNAi lines relative to wild type plants. Comet assay analysis of isolated nuclei showed atlig1-RNAi lines displayed slower repair of single strand breaks (SSBs and also double strand breaks (DSBs, implicating AtLIG1 in repair of both these lesions. Conclusion Reduced levels of Arabidopsis DNA ligase 1 in the silenced lines are sufficient to support plant development but result in retarded growth and reduced cell size, which may reflect roles for AtLIG1 in both replication and repair. The finding that DNA ligase 1 plays an important role in DSB repair in addition to its known function in SSB repair, demonstrates the existence of a previously uncharacterised novel pathway, independent of the conserved NHEJ. These results indicate that DNA ligase 1 functions in both DNA replication and in repair of both ss and dsDNA strand breaks in higher plants.

  17. Microbial pathogens trigger host DNA double-strand breaks whose abundance is reduced by plant defense responses.

    Directory of Open Access Journals (Sweden)

    Junqi Song

    2014-04-01

    Full Text Available Immune responses and DNA damage repair are two fundamental processes that have been characterized extensively, but the links between them remain largely unknown. We report that multiple bacterial, fungal and oomycete plant pathogen species induce double-strand breaks (DSBs in host plant DNA. DNA damage detected by histone γ-H2AX abundance or DNA comet assays arose hours before the disease-associated necrosis caused by virulent Pseudomonas syringae pv. tomato. Necrosis-inducing paraquat did not cause detectable DSBs at similar stages after application. Non-pathogenic E. coli and Pseudomonas fluorescens bacteria also did not induce DSBs. Elevation of reactive oxygen species (ROS is common during plant immune responses, ROS are known DNA damaging agents, and the infection-induced host ROS burst has been implicated as a cause of host DNA damage in animal studies. However, we found that DSB formation in Arabidopsis in response to P. syringae infection still occurs in the absence of the infection-associated oxidative burst mediated by AtrbohD and AtrbohF. Plant MAMP receptor stimulation or application of defense-activating salicylic acid or jasmonic acid failed to induce a detectable level of DSBs in the absence of introduced pathogens, further suggesting that pathogen activities beyond host defense activation cause infection-induced DNA damage. The abundance of infection-induced DSBs was reduced by salicylic acid and NPR1-mediated defenses, and by certain R gene-mediated defenses. Infection-induced formation of γ-H2AX still occurred in Arabidopsis atr/atm double mutants, suggesting the presence of an alternative mediator of pathogen-induced H2AX phosphorylation. In summary, pathogenic microorganisms can induce plant DNA damage. Plant defense mechanisms help to suppress rather than promote this damage, thereby contributing to the maintenance of genome integrity in somatic tissues.

  18. CRISPR/Cas9-Induced Double-Strand Break Repair in Arabidopsis Nonhomologous End-Joining Mutants

    Directory of Open Access Journals (Sweden)

    Hexi Shen

    2017-01-01

    Full Text Available Double-strand breaks (DSBs are one of the most harmful DNA lesions. Cells utilize two main pathways for DSB repair: homologous recombination (HR and nonhomologous end-joining (NHEJ. NHEJ can be subdivided into the KU-dependent classical NHEJ (c-NHEJ and the more error-prone KU-independent backup-NHEJ (b-NHEJ pathways, involving the poly (ADP-ribose polymerases (PARPs. However, in the absence of these factors, cells still seem able to adequately maintain genome integrity, suggesting the presence of other b-NHEJ repair factors or pathways independent from KU and PARPs. The outcome of DSB repair by NHEJ pathways can be investigated by using artificial sequence-specific nucleases such as CRISPR/Cas9 to induce DSBs at a target of interest. Here, we used CRISPR/Cas9 for DSB induction at the Arabidopsis cruciferin 3 (CRU3 and protoporphyrinogen oxidase (PPO genes. DSB repair outcomes via NHEJ were analyzed using footprint analysis in wild-type plants and plants deficient in key factors of c-NHEJ (ku80, b-NHEJ (parp1 parp2, or both (ku80 parp1 parp2. We found that larger deletions of >20 bp predominated after DSB repair in ku80 and ku80 parp1 parp2 mutants, corroborating with a role of KU in preventing DSB end resection. Deletion lengths did not significantly differ between ku80 and ku80 parp1 parp2 mutants, suggesting that a KU- and PARP-independent b-NHEJ mechanism becomes active in these mutants. Furthermore, microhomologies and templated insertions were observed at the repair junctions in the wild type and all mutants. Since these characteristics are hallmarks of polymerase θ-mediated DSB repair, we suggest a possible role for this recently discovered polymerase in DSB repair in plants.

  19. Dynamics of dsRNA mycoviruses in black Aspergillus population.

    NARCIS (Netherlands)

    Diepeningen, van A.D.; Debets, A.J.M.; Hoekstra, R.F.

    2006-01-01

    Approximately 10% of all examined 668 representatives of black Aspergillus species, independent of worldwide location, were infected with double-stranded RNA (dsRNA) mycoviruses. These isometric viruses (25-40 nm diameter) contained a variety of often multiple segments of different dsRNA sizes rangi

  20. Thermodynamics and kinetic studies in the binding interaction of cyclic naphthalene diimide derivatives with double stranded DNAs.

    Science.gov (United States)

    Islam, Md Monirul; Fujii, Satoshi; Sato, Shinobu; Okauchi, Tatsuo; Takenaka, Shigeori

    2015-08-01

    Previously, we reported our investigations of the interaction between a cyclic naphthalene diimide derivative (cNDI 1) and double stranded DNA (dsDNA) (Bioorg. Med. Chem.2014, 22, 2593). Here, we report the synthesis of the novel cNDI 2, which has shorter linker chains than cNDI 1. We performed comparative investigations of the interactions of both cNDI 1 and cNDI 2 with different types of dsDNA, including analysis of their thermodynamics and kinetics. Interactions between the cNDIs and calf thymus DNA (CT-DNA), poly[d(A-T)]2, or poly[d(G-C)]2 were explored by physicochemical and biochemical methods, including UV-Vis spectroscopy, circular dichroism (CD) spectroscopy, stopped-flow kinetics, and a topoisomerase I assay. Upon addition of cNDIs to CT-DNA, the existence of an induced CD signal at approximately the wavelength of the naphthalene diimide chromophore and unwinding of the DNA duplex, as detected by the topoisomerase I assay, revealed that cNDIs bound to the DNA duplex. As indicated by the steric constraint in the formation of the complex, bis-threading intercalation was the more favorable binding mode. UV-Vis spectroscopic titration of the cNDIs with DNA duplexes showed affinities on the order of 10(5)-10(6)M(-1), with a stoichiometry of one cNDI molecule per four DNA base pairs. Thermodynamic parameters (ΔG, ΔH, and ΔS) based on the van't Hoff equation indicated that exothermic and entropy-dependent hydrophobic interactions played a major role in the reaction. Stopped-flow association and dissociation analysis showed that cNDI interactions with poly[d(G-C)]2 were more stable and had a slower dissociation rate than their interactions with poly[d(A-T)]2 and CT-DNA. Measurement of ionic strength indicated that electrostatic attraction is also an important component of the interaction between cNDIs and CT-DNA. Because of its longer linker chain, cNDI 1 showed higher binding selectivity, a more entropically favorable interaction, and much slower dissociation

  1. Rapid MCNP simulation of DNA double strand break (DSB) relative biological effectiveness (RBE) for photons, neutrons, and light ions.

    Science.gov (United States)

    Stewart, Robert D; Streitmatter, Seth W; Argento, David C; Kirkby, Charles; Goorley, John T; Moffitt, Greg; Jevremovic, Tatjana; Sandison, George A

    2015-11-07

    To account for particle interactions in the extracellular (physical) environment, information from the cell-level Monte Carlo damage simulation (MCDS) for DNA double strand break (DSB) induction has been integrated into the general purpose Monte Carlo N-particle (MCNP) radiation transport code system. The effort to integrate these models is motivated by the need for a computationally efficient model to accurately predict particle relative biological effectiveness (RBE) in cell cultures and in vivo. To illustrate the approach and highlight the impact of the larger scale physical environment (e.g. establishing charged particle equilibrium), we examined the RBE for DSB induction (RBEDSB) of x-rays, (137)Cs γ-rays, neutrons and light ions relative to γ-rays from (60)Co in monolayer cell cultures at various depths in water. Under normoxic conditions, we found that (137)Cs γ-rays are about 1.7% more effective at creating DSB than γ-rays from (60)Co (RBEDSB  =  1.017) whereas 60-250 kV x-rays are 1.1 to 1.25 times more efficient at creating DSB than (60)Co. Under anoxic conditions, kV x-rays may have an RBEDSB up to 1.51 times as large as (60)Co γ-rays. Fission neutrons passing through monolayer cell cultures have an RBEDSB that ranges from 2.6 to 3.0 in normoxic cells, but may be as large as 9.93 for anoxic cells. For proton pencil beams, Monte Carlo simulations suggest an RBEDSB of about 1.2 at the tip of the Bragg peak and up to 1.6 a few mm beyond the Bragg peak. Bragg peak RBEDSB increases with decreasing oxygen concentration, which may create opportunities to apply proton dose painting to help address tumor hypoxia. Modeling of the particle RBE for DSB induction across multiple physical and biological scales has the potential to aid in the interpretation of laboratory experiments and provide useful information to advance the safety and effectiveness of hadron therapy in the treatment of cancer.

  2. Simple replication methods for producing nanoslits in thermoplastics and the transport dynamics of double-stranded DNA through these slits.

    Science.gov (United States)

    Chantiwas, Rattikan; Hupert, Mateusz L; Pullagurla, Swathi R; Balamurugan, Subramanian; Tamarit-López, Jesús; Park, Sunggook; Datta, Proyag; Goettert, Jost; Cho, Yoon-Kyoung; Soper, Steven A

    2010-12-01

    Mixed-scale nano- and microfluidic networks were fabricated in thermoplastics using simple and robust methods that did not require the use of sophisticated equipment to produce the nanostructures. High-precision micromilling (HPMM) and photolithography were used to generate mixed-scale molding tools that were subsequently used for producing fluidic networks into thermoplastics such as poly(methyl methacrylate), PMMA, cyclic olefin copolymer, COC, and polycarbonate, PC. Nanoslit arrays were imprinted into the polymer using a nanoimprinting tool, which was composed of an optical mask with patterns that were 2-7 µm in width and a depth defined by the Cr layer (100 nm), which was deposited onto glass. The device also contained a microchannel network that was hot embossed into the polymer substrate using a metal molding tool prepared via HPMM. The mixed-scale device could also be used as a master to produce a polymer stamp, which was made from polydimethylsiloxane, PDMS, and used to generate the mixed-scale fluidic network in a single step. Thermal fusion bonding of the cover plate to the substrate at a temperature below their respective T(g) was accomplished by oxygen plasma treatment of both the substrate and cover plate, which significantly reduced thermally induced structural deformation during assembly: ∼6% for PMMA and ∼9% for COC nanoslits. The electrokinetic transport properties of double-stranded DNA (dsDNA) through the polymeric nanoslits (PMMA and COC) were carried out. In these polymer devices, the dsDNA demonstrated a field-dependent electrophoretic mobility with intermittent transport dynamics. DNA mobilities were found to be 8.2 ± 0.7 × 10(-4) cm(2) V(-1) s(-1) and 7.6 ± 0.6 × 10(-4) cm(2) V(-1) s(-1) for PMMA and COC, respectively, at a field strength of 25 V cm(-1). The extension factors for λ-DNA were 0.46 in PMMA and 0.53 in COC for the nanoslits (2-6% standard deviation).

  3. Rapid MCNP simulation of DNA double strand break (DSB) relative biological effectiveness (RBE) for photons, neutrons, and light ions

    Science.gov (United States)

    Stewart, Robert D.; Streitmatter, Seth W.; Argento, David C.; Kirkby, Charles; Goorley, John T.; Moffitt, Greg; Jevremovic, Tatjana; Sandison, George A.

    2015-11-01

    To account for particle interactions in the extracellular (physical) environment, information from the cell-level Monte Carlo damage simulation (MCDS) for DNA double strand break (DSB) induction has been integrated into the general purpose Monte Carlo N-particle (MCNP) radiation transport code system. The effort to integrate these models is motivated by the need for a computationally efficient model to accurately predict particle relative biological effectiveness (RBE) in cell cultures and in vivo. To illustrate the approach and highlight the impact of the larger scale physical environment (e.g. establishing charged particle equilibrium), we examined the RBE for DSB induction (RBEDSB) of x-rays, 137Cs γ-rays, neutrons and light ions relative to γ-rays from 60Co in monolayer cell cultures at various depths in water. Under normoxic conditions, we found that 137Cs γ-rays are about 1.7% more effective at creating DSB than γ-rays from 60Co (RBEDSB  =  1.017) whereas 60-250 kV x-rays are 1.1 to 1.25 times more efficient at creating DSB than 60Co. Under anoxic conditions, kV x-rays may have an RBEDSB up to 1.51 times as large as 60Co γ-rays. Fission neutrons passing through monolayer cell cultures have an RBEDSB that ranges from 2.6 to 3.0 in normoxic cells, but may be as large as 9.93 for anoxic cells. For proton pencil beams, Monte Carlo simulations suggest an RBEDSB of about 1.2 at the tip of the Bragg peak and up to 1.6 a few mm beyond the Bragg peak. Bragg peak RBEDSB increases with decreasing oxygen concentration, which may create opportunities to apply proton dose painting to help address tumor hypoxia. Modeling of the particle RBE for DSB induction across multiple physical and biological scales has the potential to aid in the interpretation of laboratory experiments and provide useful information to advance the safety and effectiveness of hadron therapy in the treatment of cancer.

  4. Frequent and efficient use of the sister chromatid for DNA double-strand break repair during budding yeast meiosis.

    Directory of Open Access Journals (Sweden)

    Tamara Goldfarb

    Full Text Available Recombination between homologous chromosomes of different parental origin (homologs is necessary for their accurate segregation during meiosis. It has been suggested that meiotic inter-homolog recombination is promoted by a barrier to inter-sister-chromatid recombination, imposed by meiosis-specific components of the chromosome axis. Consistent with this, measures of Holliday junction-containing recombination intermediates (joint molecules [JMs] show a strong bias towards inter-homolog and against inter-sister JMs. However, recombination between sister chromatids also has an important role in meiosis. The genomes of diploid organisms in natural populations are highly polymorphic for insertions and deletions, and meiotic double-strand breaks (DSBs that form within such polymorphic regions must be repaired by inter-sister recombination. Efforts to study inter-sister recombination during meiosis, in particular to determine recombination frequencies and mechanisms, have been constrained by the inability to monitor the products of inter-sister recombination. We present here molecular-level studies of inter-sister recombination during budding yeast meiosis. We examined events initiated by DSBs in regions that lack corresponding sequences on the homolog, and show that these DSBs are efficiently repaired by inter-sister recombination. This occurs with the same timing as inter-homolog recombination, but with reduced (2- to 3-fold yields of JMs. Loss of the meiotic-chromosome-axis-associated kinase Mek1 accelerates inter-sister DSB repair and markedly increases inter-sister JM frequencies. Furthermore, inter-sister JMs formed in mek1Δ mutants are preferentially lost, while inter-homolog JMs are maintained. These findings indicate that inter-sister recombination occurs frequently during budding yeast meiosis, with the possibility that up to one-third of all recombination events occur between sister chromatids. We suggest that a Mek1-dependent reduction in

  5. DNA double-strand break induction in Ku80-deficient CHO cells following Boron Neutron Capture Reaction

    Directory of Open Access Journals (Sweden)

    Masunaga Shinichiro

    2011-09-01

    Full Text Available Abstract Background Boron neutron capture reaction (BNCR is based on irradiation of tumors after accumulation of boron compound. 10B captures neutrons and produces an alpha (4He particle and a recoiled lithium nucleus (7Li. These particles have the characteristics of high linear energy transfer (LET radiation and have marked biological effects. The purpose of this study is to verify that BNCR will increase cell killing and slow disappearance of repair protein-related foci to a greater extent in DNA repair-deficient cells than in wild-type cells. Methods Chinese hamster ovary (CHO-K1 cells and a DNA double-strand break (DSB repair deficient mutant derivative, xrs-5 (Ku80 deficient CHO mutant cells, were irradiated by thermal neutrons. The quantity of DNA-DSBs following BNCR was evaluated by measuring the phosphorylation of histone protein H2AX (gamma-H2AX and 53BP1 foci using immunofluorescence intensity. Results Two hours after neutron irradiation, the number of gamma-H2AX and 53BP1 foci in the CHO-K1 cells was decreased to 36.5-42.8% of the levels seen 30 min after irradiation. In contrast, two hours after irradiation, foci levels in the xrs-5 cells were 58.4-69.5% of those observed 30 min after irradiation. The number of gamma-H2AX foci in xrs-5 cells at 60-120 min after BNCT correlated with the cell killing effect of BNCR. However, in CHO-K1 cells, the RBE (relative biological effectiveness estimated by the number of foci following BNCR was increased depending on the repair time and was not always correlated with the RBE of cytotoxicity. Conclusion Mutant xrs-5 cells show extreme sensitivity to ionizing radiation, because xrs-5 cells lack functional Ku-protein. Our results suggest that the DNA-DSBs induced by BNCR were not well repaired in the Ku80 deficient cells. The RBE following BNCR of radio-sensitive mutant cells was not increased but was lower than that of radio-resistant cells. These results suggest that gamma-ray resistant cells have

  6. Nucleosome resection at a double-strand break during Non-Homologous Ends Joining in mammalian cells - implications from repressive chromatin organization and the role of ARTEMIS

    Directory of Open Access Journals (Sweden)

    De Benedetti Arrigo

    2011-01-01

    Full Text Available Abstract Background The S. cerevisiae mating type switch model of double-strand break (DSB repair, utilizing the HO endonuclease, is one of the best studied systems for both Homologous Recombination Repair (HRR and direct ends-joining repair (Non-Homologous Ends Joining - NHEJ. We have recently transposed that system to a mammalian cell culture model taking advantage of an adenovirus expressing HO and an integrated genomic target. This made it possible to compare directly the mechanism of repair between yeast and mammalian cells for the same type of induced DSB. Studies of DSB repair have emphasized commonality of features, proteins and machineries between organisms, and differences when conservation is not found. Two proteins that stand out that differ between yeast and mammalian cells are DNA-PK, a protein kinase that is activated by the presence of DSBs, and Artemis, a nuclease whose activity is modulated by DNA-PK and ATM. In this report we describe how these two proteins may be involved in a specific pattern of ends-processing at the DSB, particularly in the context of heterochromatin. Findings We previously published that the repair of the HO-induced DSB was generally accurate and occurred by simple rejoining of the cohesive 3'-overhangs generated by HO. During continuous passage of those cells in the absence of puromycin selection, the locus appears to have become more heterochromatic and silenced by displaying several features. 1 The site had become less accessible to cleavage by the HO endonuclease; 2 the expression of the puro mRNA, which confers resistance to puromycin, had become reduced; 3 occupancy of nucleosomes at the site (ChIP for histone H3 was increased, an indicator for more condensed chromatin. After reselection of these cells by addition of puromycin, many of these features were reversed. However, even the reselected cells were not identical in the pattern of cleavage and repair as the cells when originally created

  7. RNA interference: an exciting new target validation tool of drug action and therapeutic approach on cardiovascular diseases

    Institute of Scientific and Technical Information of China (English)

    HeMing

    2005-01-01

    RNA interference (RNAi) is an evolutionarily conserved mechanism for silencing gene expression by targeted degradation of mRNA. Short double-stranded RNAs, known as small interfering RNAs (siRNA), are incorporated into an RNA-induced silencing complex that directs degradation of RNA containing a homologous sequence.

  8. The long and short of antiviral defense: small RNA-based immunity in insects

    NARCIS (Netherlands)

    Bronkhorst, A.W.; Rij, R.P. van

    2014-01-01

    The host RNA interference (RNAi) pathway of insects senses virus infection and induces an antiviral response to restrict virus replication. Dicer-2 detects viral double-stranded RNA, produced by RNA and DNA viruses, and generates viral small interfering RNAs (vsiRNAs). Recent small RNA profiling stu

  9. Massive parallel analysis of the binding specificity of histone-like protein HU to single- and double-stranded DNA with generic oligodeoxyribonucleotide microchips.

    Energy Technology Data Exchange (ETDEWEB)

    Krylov, A. S.; Zasedateleva, O. A.; Prokopenko, D. V.; Rouviere-Yaniv, J.; Mirzabekov, A. D.; Biochip Technology Center; Engelhardt Inst. of Molecular Biology; Inst. de Biologie Physico-Chimique

    2001-06-15

    A generic hexadeoxyribonucleotide microchip has been applied to test the DNA-binding properties of HU histone-like bacterial protein, which is known to have a low sequence specificity. All 4096 hexamers flanked within 8mers by degenerate bases at both the 3'- and 5'-ends were immobilized within the 100 x 100 x 20 mm polyacrylamide gel pads of the microchip. Single-stranded immobilized oligonucleotides were converted in some experiments to the double-stranded form by hybridization with a specified mixture of 8mers. The DNA interaction with HU was characterized by three type of measurements: (i) binding of FITC-labeled HU to microchip oligonucleotides; (ii) melting curves of complexes of labeled HU with single-stranded microchip oligonucleotides; (iii) the effect of HU binding on melting curves of microchip double-stranded DNA labeled with another fluorescent dye, Texas Red. Large numbers of measurements of these parameters were carried out in parallel for all or many generic microchip elements in real time with a multi-wavelength fluorescence microscope. Statistical analysis of these data suggests some preference for HU binding to G/C-rich single-stranded oligonucleotides. HU complexes with double-stranded microchip 8mers can be divided into two groups in which HU binding either increased the melting temperature (T{sub m}) of duplexes or decreased it. The stabilized duplexes showed some preference for presence of the sequence motifs AAG, AGA and AAGA. In the second type of complex, enriched with A/T base pairs, the destabilization effect was higher for longer stretches of A/T duplexes. Binding of HU to labeled duplexes in the second type of complex caused some decrease in fluorescence. This decrease also correlates with the higher A/T content and lower T{sub m}. The results demonstrate that generic microchips could be an efficient approach in analysis of sequence specificity of proteins.

  10. Influence of different iodinated contrast media on the induction of DNA double-strand breaks after in vitro X-ray irradiation.

    Science.gov (United States)

    Deinzer, Christoph K W; Danova, Daniela; Kleb, Beate; Klose, Klaus J; Heverhagen, Johannes T

    2014-01-01

    The objective of this work was to examine differences in DNA double-strand break induction in peripheral blood lymphocytes after in vitro X-ray irradiation between iodinated contrast agents. Four different iodinated X-ray contrast agents--three of them with two different iodine concentrations--and mannitol (negative control; concentration of 150 mg mannitol per ml blood) were pipetted into blood samples so that there was a concentration of 0, 7.5 or 15 mg of iodine per ml blood in the samples. Negative controls without contrast medium (0 mg of iodine per ml blood) were also processed for every irradiation dose. The tubes were exposed to 0, 20 or 500 mGy in vitro X-ray irradiation. After that, the lymphocytes were separated by using density-gradient centrifugation. Fluorescence microscopy was applied to determine the average number of γH2AX-foci per lymphocyte in the presence or absence of different contrast media or mannitol. Differences in the number of γH2AX-foci were statistically analysed by one-way ANOVA and post-hoc Tukey's honestly significant difference test. Iodinated contrast agents led to a statistically significant increase in DNA double-strand breaks after in vitro irradiation. This effect increased statistically significant with rising radiation dose and appeared independent of the contrast agent used (iopromid, iodixanol, iomeprol, iopamidol). A statistically significant difference in DNA damage between the different tested contrast agents was not found. Therefore, the increase in DNA double-strand breaks depends solely on the amount of iodine applied. For evaluation of clinical consequences, our findings could be tested in further animal studies.

  11. Replication-Dependent and Transcription-Dependent Mechanisms of DNA Double-Strand Break Induction by the Topoisomerase 2-Targeting Drug Etoposide

    OpenAIRE

    Margaret Tammaro; Peri Barr; Brett Ricci; Hong Yan

    2013-01-01

    Etoposide is a DNA topoisomerase 2-targeting drug widely used for the treatment of cancer. The cytoxicity of etoposide correlates with the generation of DNA double-strand breaks (DSBs), but the mechanism of how it induces DSBs in cells is still poorly understood. Catalytically, etoposide inhibits the re-ligation reaction of Top2 after it nicks the two strands of DNA, trapping it in a cleavable complex consisting of two Top2 subunits covalently linked to the 5' ends of DNA (Top2cc). Top2cc is ...

  12. Conditional deletion of Nbs1 in murine cells reveals its role in branching repair pathways of DNA double-strand breaks

    OpenAIRE

    Yang, Yun-Gui; Saidi, Amal; Frappart, Pierre-Olivier; Min, WooKee; Barrucand, Christelle; Dumon-Jones, Valérie; Michelon, Jocelyne; Herceg, Zdenko; Wang, Zhao-Qi

    2006-01-01

    NBS1 forms a complex with MRE11 and RAD50 (MRN) that is proposed to act on the upstream of two repair pathways of DNA double-strand break (DSB), homologous repair (HR) and non-homologous end joining (NHEJ). However, the function of Nbs1 in these processes has not fully been elucidated in mammals due to the lethal phenotype of cells and mice lacking Nbs1. Here, we have constructed mouse Nbs1-null embryonic fibroblasts and embryonic stem cells, through the Cre-loxP and sequential gene targeting...

  13. Budding yeast ATM/ATR control meiotic double-strand break (DSB) levels by down-regulating Rec114, an essential component of the DSB-machinery.

    OpenAIRE

    Carballo, Jesús A.; Panizza, Silvia; Serrentino, Maria Elisabetta; Anthony L Johnson; Geymonat, Marco; Borde, Valérie; Klein, Franz; Cha, Rita S.

    2013-01-01

    An essential feature of meiosis is Spo11 catalysis of programmed DNA double strand breaks (DSBs). Evidence suggests that the number of DSBs generated per meiosis is genetically determined and that this ability to maintain a pre-determined DSB level, or "DSB homeostasis", might be a property of the meiotic program. Here, we present direct evidence that Rec114, an evolutionarily conserved essential component of the meiotic DSB-machinery, interacts with DSB hotspot DNA, and that Tel1 and Mec1, t...

  14. The control in cis of the position and the amount of the ARG4 meiotic double-strand break of Saccharomyces cerevisiae.

    OpenAIRE

    de Massy, B.; Nicolas, A.

    1993-01-01

    During meiosis, a transient DNA double-strand break (DSB) occurs in the promoter region (positions -200/-185) of the Saccharomyces cerevisiae ARG4 gene and is a likely intermediate in the initiation of meiotic gene conversion events in this region. We report here a functional analysis of the ARG4 DSB based on the study of various deletions in this chromosomal region. We have identified several cis-acting elements located within the -465/+3 region of the ARG4 promoter that control the formatio...

  15. TU-CD-303-02: Beyond Radiation Induced Double Strand Breaks - a New Horizon for Radiation Therapy Research

    Energy Technology Data Exchange (ETDEWEB)

    Chang, S. [UNC School of Medicine (United States)

    2015-06-15

    Recent advances in cancer research have shed new light on the complex processes of how therapeutic radiation initiates changes at cellular, tissue, and system levels that may lead to clinical effects. These new advances may transform the way we use radiation to combat certain types of cancers. For the past two decades many technological advancements in radiation therapy have been largely based on the hypothesis that direct radiation-induced DNA double strand breaks cause cell death and thus tumor control and normal tissue damage. However, new insights have elucidated that in addition to causing cellular DNA damage, localized therapeutic radiation also initiates cascades of complex downstream biological responses in tissue that extend far beyond where therapeutic radiation dose is directly deposited. For instance, studies show that irradiated dying tumor cells release tumor antigens that can lead the immune system to a systemic anti-cancer attack throughout the body of cancer patient; targeted irradiation to solid tumor also increases the migration of tumor cells already in bloodstream, the seeds of potential metastasis. Some of the new insights may explain the long ago discovered but still unexplained non-localized radiation effects (bystander effect and abscopal effect) and the efficacy of spatially fractionated radiation therapy (microbeam radiation therapy and GRID therapy) where many “hot” and “cold” spots are intentionally created throughout the treatment volume. Better understanding of the mechanisms behind the non-localized radiation effects creates tremendous opportunities to develop new and integrated cancer treatment strategies that are based on radiotherapy, immunology, and chemotherapy. However, in the multidisciplinary effort to advance new radiobiology, there are also tremendous challenges including a lack of multidisciplinary researchers and imaging technologies for the microscopic radiation-induced responses. A better grasp of the essence of

  16. Creating Directed Double-strand Breaks with the Ref Protein: A Novel Rec A-Dependent Nuclease from Bacteriophage P1

    Energy Technology Data Exchange (ETDEWEB)

    Gruenig, Marielle C.; Lu, Duo; Won, Sang Joon; Dulberger, Charles L.; Manlick, Angela J.; Keck, James L.; Cox, Michael M. (UW)

    2012-03-16

    The bacteriophage P1-encoded Ref protein enhances RecA-dependent recombination in vivo by an unknown mechanism. We demonstrate that Ref is a new type of enzyme; that is, a RecA-dependent nuclease. Ref binds to ss- and dsDNA but does not cleave any DNA substrate until RecA protein and ATP are added to form RecA nucleoprotein filaments. Ref cleaves only where RecA protein is bound. RecA functions as a co-nuclease in the Ref/RecA system. Ref nuclease activity can be limited to the targeted strands of short RecA-containing D-loops. The result is a uniquely programmable endonuclease activity, producing targeted double-strand breaks at any chosen DNA sequence in an oligonucleotide-directed fashion. We present evidence indicating that cleavage occurs in the RecA filament groove. The structure of the Ref protein has been determined to 1.4 {angstrom} resolution. The core structure, consisting of residues 77-186, consists of a central 2-stranded {beta}-hairpin that is sandwiched between several {alpha}-helical and extended loop elements. The N-terminal 76 amino acid residues are disordered; this flexible region is required for optimal activity. The overall structure of Ref, including several putative active site histidine residues, defines a new subclass of HNH-family nucleases. We propose that enhancement of recombination by Ref reflects the introduction of directed, recombinogenic double-strand breaks.

  17. De novo-engineered transcription activator-like effector (TALE) hybrid nuclease with novel DNA binding specificity creates double-strand breaks

    KAUST Repository

    Mahfouz, Magdy M.

    2011-01-24

    Site-specific and rare cutting nucleases are valuable tools for genome engineering. The generation of double-strand DNA breaks (DSBs) promotes homologous recombination in eukaryotes and can facilitate gene targeting, additions, deletions, and inactivation. Zinc finger nucleases have been used to generate DSBs and subsequently, for genome editing but with low efficiency and reproducibility. The transcription activator-like family of type III effectors (TALEs) contains a central domain of tandem repeats that could be engineered to bind specific DNA targets. Here, we report the generation of a Hax3-based hybrid TALE nuclease with a user-selected DNA binding specificity. We show that the engineered TALE nuclease can bind to its target sequence in vitro and that the homodimeric TALE nuclease can cleave double-stranded DNA in vitro if the DNA binding sites have the proper spacing and orientation. Transient expression assays in tobacco leaves suggest that the hybrid nuclease creates DSB in its target sequence, which is subsequently repaired by nonhomologous end-joining repair. Taken together, our data show the feasibility of engineering TALE-based hybrid nucleases capable of generating site-specific DSBs and the great potential for site-specific genome modification in plants and eukaryotes in general.

  18. Mechanism of adsorption of single and double stranded DNA on gold and silver nanoparticles: Investigating some important parameters in bio-sensing applications.

    Science.gov (United States)

    Farkhari, Nahid; Abbasian, Sara; Moshaii, Ahmad; Nikkhah, Maryam

    2016-12-01

    The mechanism of adsorption of single and double stranded DNAs on colloidal gold and silver nanoparticles has been studied by measuring the resistance of the nanoparticles, surrounded by various oligonucleotides, against salt induced aggregation. It is shown that both single and double stranded DNAs can be adsorbed on the metal nanoparticles and the adsorption strength is determined by the interaction between various bases of DNA and the nanoparticles. By changing the salt concentration, the difference between adsorption of various DNA strands on the nanoparticles can be specified. The results indicate that a key parameter in success of a sensing assay of DNA hybridization is the salt concentration which should be greater than a minimum threshold depending on the nanoparticles characteristics. We have also investigated the interaction mechanism between various DNA bases with the metal nanoparticles. For both gold and silver nanoparticles, adenine has the highest and thymine has the lowest attachment to the nanoparticles. From surface enhanced Raman spectroscopy (SERS) data of various bases in the presence of gold nanoparticles, the probable interaction points in the bases with the nanoparticles have been determined, which are mainly the nitrogen sites of these oligonucleotides.

  19. Creating directed double-strand breaks with the Ref protein: a novel RecA-dependent nuclease from bacteriophage P1.

    Science.gov (United States)

    Gruenig, Marielle C; Lu, Duo; Won, Sang Joon; Dulberger, Charles L; Manlick, Angela J; Keck, James L; Cox, Michael M

    2011-03-11

    The bacteriophage P1-encoded Ref protein enhances RecA-dependent recombination in vivo by an unknown mechanism. We demonstrate that Ref is a new type of enzyme; that is, a RecA-dependent nuclease. Ref binds to ss- and dsDNA but does not cleave any DNA substrate until RecA protein and ATP are added to form RecA nucleoprotein filaments. Ref cleaves only where RecA protein is bound. RecA functions as a co-nuclease in the Ref/RecA system. Ref nuclease activity can be limited to the targeted strands of short RecA-containing D-loops. The result is a uniquely programmable endonuclease activity, producing targeted double-strand breaks at any chosen DNA sequence in an oligonucleotide-directed fashion. We present evidence indicating that cleavage occurs in the RecA filament groove. The structure of the Ref protein has been determined to 1.4 Å resolution. The core structure, consisting of residues 77-186, consists of a central 2-stranded β-hairpin that is sandwiched between several α-helical and extended loop elements. The N-terminal 76 amino acid residues are disordered; this flexible region is required for optimal activity. The overall structure of Ref, including several putative active site histidine residues, defines a new subclass of HNH-family nucleases. We propose that enhancement of recombination by Ref reflects the introduction of directed, recombinogenic double-strand breaks.

  20. Oncogenic ras-driven cancer cell vesiculation leads to emission of double-stranded DNA capable of interacting with target cells

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Tae Hoon; Chennakrishnaiah, Shilpa [Montreal Children’s Hospital, Research Institute of McGill University Health Centre, McGill University, Montreal, Quebec (Canada); Audemard, Eric [McGill University and Genome Quebec Innovation Centre, Montreal, Quebec (Canada); Montermini, Laura; Meehan, Brian [Montreal Children’s Hospital, Research Institute of McGill University Health Centre, McGill University, Montreal, Quebec (Canada); Rak, Janusz, E-mail: janusz.rak@mcgill.ca [Montreal Children’s Hospital, Research Institute of McGill University Health Centre, McGill University, Montreal, Quebec (Canada)

    2014-08-22

    Highlights: • Oncogenic H-ras stimulates emission of extracellular vesicles containing double-stranded DNA. • Vesicle-associated extracellular DNA contains mutant N-ras sequences. • Vesicles mediate intercellular transfer of mutant H-ras DNA to normal fibroblasts where it remains for several weeks. • Fibroblasts exposed to vesicles containing H-ras DNA exhibit increased proliferation. - Abstract: Cell free DNA is often regarded as a source of genetic cancer biomarkers, but the related mechanisms of DNA release, composition and biological activity remain unclear. Here we show that rat epithelial cell transformation by the human H-ras oncogene leads to an increase in production of small, exosomal-like extracellular vesicles by viable cancer cells. These EVs contain chromatin-associated double-stranded DNA fragments covering the entire host genome, including full-length H-ras. Oncogenic N-ras and SV40LT sequences were also found in EVs emitted from spontaneous mouse brain tumor cells. Disruption of acidic sphingomyelinase and the p53/Rb pathway did not block emission of EV-related oncogenic DNA. Exposure of non-transformed RAT-1 cells to EVs containing mutant H-ras DNA led to the uptake and retention of this material for an extended (30 days) but transient period of time, and stimulated cell proliferation. Thus, our study suggests that H-ras-mediated transformation stimulates vesicular emission of this histone-bound oncogene, which may interact with non-transformed cells.

  1. Members of the RAD52 Epistasis Group Contribute to Mitochondrial Homologous Recombination and Double-Strand Break Repair in Saccharomyces cerevisiae.

    Science.gov (United States)

    Stein, Alexis; Kalifa, Lidza; Sia, Elaine A

    2015-11-01

    Mitochondria contain an independently maintained genome that encodes several proteins required for cellular respiration. Deletions in the mitochondrial genome have been identified that cause several maternally inherited diseases and are associated with certain cancers and neurological disorders. The majority of these deletions in human cells are flanked by short, repetitive sequences, suggesting that these deletions may result from recombination events. Our current understanding of the maintenance and repair of mtDNA is quite limited compared to our understanding of similar events in the nucleus. Many nuclear DNA repair proteins are now known to also localize to mitochondria, but their function and the mechanism of their action remain largely unknown. This study investigated the contribution of the nuclear double-strand break repair (DSBR) proteins Rad51p, Rad52p and Rad59p in mtDNA repair. We have determined that both Rad51p and Rad59p are localized to the matrix of the mitochondria and that Rad51p binds directly to mitochondrial DNA. In addition, a mitochondrially-targeted restriction endonuclease (mtLS-KpnI) was used to produce a unique double-strand break (DSB) in the mitochondrial genome, which allowed direct analysis of DSB repair in vivo in Saccharomyces cerevisiae. We find that loss of these three proteins significantly decreases the rate of spontaneous deletion events and the loss of Rad51p and Rad59p impairs the repair of induced mtDNA DSBs.

  2. Radiation-induced DNA Double Strand Breaks and Their Modulations by Treatments with Moringa oleifera Lam. Leaf Extracts: A Cancer Cell Culture Model

    Directory of Open Access Journals (Sweden)

    K. Boonsirichai

    2014-04-01

    Full Text Available Gamma radiation brings deleterious effects upon human cells by inducing oxidative stress and DNA damages. Antioxidants have been shown to confer protective effects on irradiated normal cells. Moringa oleifera Lam. is a widely used nutritional supplement with antioxidant activities. This report showed that antioxidant-containing supplements, in addition to protecting normal cells, could protect cancer cells against genotoxic effects of gamma radiation. -H2AX immunofluorescent foci were utilized as an indicator of radiation-induced DNA double strand breaks. MCF-7 human breast adenocarcinoma cells were irradiated with 2-8 Gy gamma radiation. A linear relationship between the formation of -H2AX foci and radiation dose was observed with an average of 10 foci per cell per Gy. A 30-minute pretreatment of the cells with either the aqueous or the ethanolic extract of M. oleifera leaves could partially protect the cells from radiation-induced DNA double strand breaks. A pretreatment with 500 µg/mL aqueous extract reduced the number of foci formed by 15% when assayed at 30 minutes post-irradiation. The ethanolic extract was more effective; 500 µg/mL of its concentration reduced the number of foci among irradiated cells by 30%. The results indicated that irradiated cancer cells responded similarly to nutritional supplements containing antioxidants as irradiated normal cells. These natural antioxidants could confer protective effects upon cancer cells against gamma radiation

  3. Dynamics and Cell-Type Specificity of the DNA Double-Strand Break Repair Protein RecN in the Developmental Cyanobacterium Anabaena sp. Strain PCC 7120.

    Directory of Open Access Journals (Sweden)

    Sheng Hu

    Full Text Available DNA replication and repair are two fundamental processes required in life proliferation and cellular defense and some common proteins are involved in both processes. The filamentous cyanobacterium Anabaena sp. strain PCC 7120 is capable of forming heterocysts for N2 fixation in the absence of a combined-nitrogen source. This developmental process is intimately linked to cell cycle control. In this study, we investigated the localization of the DNA double-strand break repair protein RecN during key cellular events, such as chromosome damaging, cell division, and heterocyst differentiation. Treatment by a drug causing DNA double-strand breaks (DSBs induced reorganization of the RecN focus preferentially towards the mid-cell position. RecN-GFP was absent in most mature heterocysts. Furthermore, our results showed that HetR, a central player in heterocyst development, was involved in the proper positioning and distribution of RecN-GFP. These results showed the dynamics of RecN in DSB repair and suggested a differential regulation of DNA DSB repair in vegetative cell and heterocysts. The absence of RecN in mature heterocysts is compatible with the terminal nature of these cells.

  4. Dynamics and Cell-Type Specificity of the DNA Double-Strand Break Repair Protein RecN in the Developmental Cyanobacterium Anabaena sp. Strain PCC 7120.

    Science.gov (United States)

    Hu, Sheng; Wang, Jinglan; Wang, Li; Zhang, Cheng-Cai; Chen, Wen-Li

    2015-01-01

    DNA replication and repair are two fundamental processes required in life proliferation and cellular defense and some common proteins are involved in both processes. The filamentous cyanobacterium Anabaena sp. strain PCC 7120 is capable of forming heterocysts for N2 fixation in the absence of a combined-nitrogen source. This developmental process is intimately linked to cell cycle control. In this study, we investigated the localization of the DNA double-strand break repair protein RecN during key cellular events, such as chromosome damaging, cell division, and heterocyst differentiation. Treatment by a drug causing DNA double-strand breaks (DSBs) induced reorganization of the RecN focus preferentially towards the mid-cell position. RecN-GFP was absent in most mature heterocysts. Furthermore, our results showed that HetR, a central player in heterocyst development, was involved in the proper positioning and distribution of RecN-GFP. These results showed the dynamics of RecN in DSB repair and suggested a differential regulation of DNA DSB repair in vegetative cell and heterocysts. The absence of RecN in mature heterocysts is compatible with the terminal nature of these cells.

  5. De novo-engineered transcription activator-like effector (TALE) hybrid nuclease with novel DNA binding specificity creates double-strand breaks.

    Science.gov (United States)

    Mahfouz, Magdy M; Li, Lixin; Shamimuzzaman, Md; Wibowo, Anjar; Fang, Xiaoyun; Zhu, Jian-Kang

    2011-02-08

    Site-specific and rare cutting nucleases are valuable tools for genome engineering. The generation of double-strand DNA breaks (DSBs) promotes homologous recombination in eukaryotes and can facilitate gene targeting, additions, deletions, and inactivation. Zinc finger nucleases have been used to generate DSBs and subsequently, for genome editing but with low efficiency and reproducibility. The transcription activator-like family of type III effectors (TALEs) contains a central domain of tandem repeats that could be engineered to bind specific DNA targets. Here, we report the generation of a Hax3-based hybrid TALE nuclease with a user-selected DNA binding specificity. We show that the engineered TALE nuclease can bind to its target sequence in vitro and that the homodimeric TALE nuclease can cleave double-stranded DNA in vitro if the DNA binding sites have the proper spacing and orientation. Transient expression assays in tobacco leaves suggest that the hybrid nuclease creates DSB in its target sequence, which is subsequently repaired by nonhomologous end-joining repair. Taken together, our data show the feasibility of engineering TALE-based hybrid nucleases capable of generating site-specific DSBs and the great potential for site-specific genome modification in plants and eukaryotes in general.

  6. ZIP4H (TEX11 deficiency in the mouse impairs meiotic double strand break repair and the regulation of crossing over.

    Directory of Open Access Journals (Sweden)

    Carrie A Adelman

    2008-03-01

    Full Text Available We have recently shown that hypomorphic Mre11 complex mouse mutants exhibit defects in the repair of meiotic double strand breaks (DSBs. This is associated with perturbation of synaptonemal complex morphogenesis, repair and regulation of crossover formation. To further assess the Mre11 complex's role in meiotic progression, we identified testis-specific NBS1-interacting proteins via two-hybrid screening in yeast. In this screen, Zip4h (Tex11, a male germ cell specific X-linked gene was isolated. Based on sequence and predicted structural similarity to the S. cerevisiae and A. thaliana Zip4 orthologs, ZIP4H appears to be the mammalian ortholog. In S. cerevisiae and A. thaliana, Zip4 is a meiosis-specific protein that regulates the level of meiotic crossovers, thus influencing homologous chromosome segregation in these organisms. As is true for hypomorphic Nbs1 (Nbs1(DeltaB/DeltaB mice, Zip4h(-/Y mutant mice were fertile. Analysis of spermatocytes revealed a delay in meiotic double strand break repair and decreased crossover formation as inferred from DMC1 and MLH1 staining patterns, respectively. Achiasmate chromosomes at the first meiotic division were also observed in Zip4h(-/Y mutants, consistent with the observed reduction in MLH1 focus formation. These results indicate that meiotic functions of Zip4 family members are conserved and support the view that the Mre11 complex and ZIP4H interact functionally during the execution of the meiotic program in mammals.

  7. Coordinateendonucleolytic 5' and 3' trimming of terminally blocked blunt DNA double-strand break ends by Artemis nuclease and DNA-dependent protein kinase

    Energy Technology Data Exchange (ETDEWEB)

    Povirk, Lawrence; Yannone, Steven M.; Khan, Imran S.; Zhou, Rui-Zhe; Zhou, Tong; Valerie, Kristoffer; F., Lawrence

    2008-02-18

    Previous work showed that, in the presence of DNA-PK, Artemis slowly trims 3'-phosphoglycolate-terminated blunt ends. To examine the trimming reaction in more detail, long internally labeled DNA substrates were treated with Artemis. In the absence of DNA-PK, Artemis catalyzed extensive 5' {yields} 3' exonucleolytic resection of double-stranded DNA. This resection required a 5'-phosphate but did not require ATP, and was accompanied by endonucleolytic cleavage of the resulting 3' overhang. In the presence of DNA-PK, Artemis-mediated trimming was more limited, was ATP-dependent, and did not require a 5'-phosphate. For a blunt end with either a 3'-phosphoglycolate or 3'-hydroxyl terminus, endonucleolytic trimming of 2-4 nucleotides from the 3'-terminal strand was accompanied by trimming of 6 nucleotides from the 5'-terminal strand. The results suggest that autophosphorylated DNA-PK suppresses the exonuclease activity of Artemis toward blunt-ended DNA, and promotes slow and limited endonucleolytic trimming of the 5'-terminal strand, resulting in short 3' overhangs that are trimmed endonucleolytically. Thus, Artemis and DNA-PK can convert terminally blocked DNA ends of diverse geometry and chemical structure to a form suitable for polymerase mediated patching and ligation, with minimal loss of terminal sequence. Such processing could account for the very small deletions often found at DNA double-strand break repair sites.

  8. In vivo expression of a single viral DNA-binding protein generates systemic lupus erythematosus-related autoimmunity to double-stranded DNA and histones.

    Science.gov (United States)

    Moens, U; Seternes, O M; Hey, A W; Silsand, Y; Traavik, T; Johansen, B; Rekvig, O P

    1995-01-01

    Although the origin of autoimmune antibodies to double-stranded DNA is not known, the variable-region structures of such antibodies indicate that they are produced in response to antigen-selective stimulation. In accordance with this, results from experiments using artificial complexes of DNA and DNA-binding polypeptides for immunizations have indicated that DNA may induce these antibodies. Hence, the immunogenicity of DNA in vivo may depend upon other structures or processes that may render DNA immunogenic. We report that in vivo expression of a single DNA-binding protein, the polyoma virus T antigen, is sufficient to initiate production of anti-double-stranded DNA and anti-histone antibodies but not a panel of other autoantigens. Expression of a mutant, non-DNA-binding T antigen did result in strong production of antibodies to the T antigen, but only borderline levels of antibodies to DNA and no detectable antibodies to histones. Nonexpressing plasmid DNA containing the complete cDNA sequence for T antigen did not evoke such immune responses, indicating that DNA by itself is not immunogenic in vivo. The results represent a conceptual advance in understanding a potential molecular basis for initiation of autoimmunity in systemic lupus erythematosus. PMID:8618908

  9. Screening of Modified RNA duplexes

    DEFF Research Database (Denmark)

    Schyth, Brian Dall; Bramsen, Jesper Bertram; Kjems, Jørgen

    Because of sequence specific gene targeting activity siRNAs are regarded as promising active compounds in gene medicine. But one serious problem with delivering siRNAs as treatment is the now well-established non-specific activities of some RNA duplexes. Cellular reactions towards double stranded...... RNAs include the 2´-5´ oligoadenylate synthetase system, the protein kinase R, RIG-I and Toll-like receptor activated pathways all resulting in antiviral defence mechanism. We have previously shown that antiviral innate immune reactions against double stranded RNAs could be detected in vivo as partial...... protection against a fish pathogenic virus. This protection corresponded with an interferon response in the fish. Here we use this fish model to screen siRNAs containing various chemical modifications of the RNA backbone for their antiviral activity, the overall aim being identification of an siRNA form...

  10. Epstein-Barr virus (EBV)-encoded small RNA is released from EBV-infected cells and activates signaling from toll-like receptor 3

    OpenAIRE

    2009-01-01

    Epstein-Barr virus–encoded small RNA (EBER) is nonpolyadenylated, noncoding RNA that forms stem-loop structure by intermolecular base-pairing, giving rise to double-stranded RNA (dsRNA)–like molecules, and exists abundantly in EBV-infected cells. Here, we report that EBER induces signaling from the Toll-like receptor 3 (TLR3), which is a sensor of viral double-stranded RNA (dsRNA) and induces type I IFN and proinflammatory cytokines. A substantial amount of EBER, which was sufficient to induc...

  11. HIC1 (hypermethylated in cancer 1) SUMOylation is dispensable for DNA repair but is essential for the apoptotic DNA damage response (DDR) to irreparable DNA double-strand breaks (DSBs).

    Science.gov (United States)

    Paget, Sonia; Dubuissez, Marion; Dehennaut, Vanessa; Nassour, Joe; Harmon, Brennan T; Spruyt, Nathalie; Loison, Ingrid; Abbadie, Corinne; Rood, Brian R; Leprince, Dominique

    2017-01-10

    The tumor suppressor gene HIC1 (Hypermethylated In Cancer 1) encodes a transcriptional repressor mediating the p53-dependent apoptotic response to irreparable DNA double-strand breaks (DSBs) through direct transcriptional repression of SIRT1. HIC1 is also essential for DSB repair as silencing of endogenous HIC1 in BJ-hTERT fibroblasts significantly delays DNA repair in functional Comet assays. HIC1 SUMOylation favours its interaction with MTA1, a component of NuRD complexes. In contrast with irreparable DSBs induced by 16-hours of etoposide treatment, we show that repairable DSBs induced by 1 h etoposide treatment do not increase HIC1 SUMOylation or its interaction with MTA1. Furthermore, HIC1 SUMOylation is dispensable for DNA repair since the non-SUMOylatable E316A mutant is as efficient as wt HIC1 in Comet assays. Upon induction of irreparable DSBs, the ATM-mediated increase of HIC1 SUMOylation is independent of its effector kinase Chk2. Moreover, irreparable DSBs strongly increase both the interaction of HIC1 with MTA1 and MTA3 and their binding to the SIRT1 promoter. To characterize the molecular mechanisms sustained by this increased repression potential, we established global expression profiles of BJ-hTERT fibroblasts transfected with HIC1-siRNA or control siRNA and treated or not with etoposide. We identified 475 genes potentially repressed by HIC1 with cell death and cell cycle as the main cellular functions identified by pathway analysis. Among them, CXCL12, EPHA4, TGFβR3 and TRIB2, also known as MTA1 target-genes, were validated by qRT-PCR analyses. Thus, our data demonstrate that HIC1 SUMOylation is important for the transcriptional response to non-repairable DSBs but dispensable for DNA repair.

  12. Development of Studies on RNA Interference

    Institute of Scientific and Technical Information of China (English)

    Li ZHANG; Yaqiong ZHANG; Lina SHE; Wenting XU; Yangying JIA; Shiqing XIE; WenliSUN; Quan LIANG

    2012-01-01

    RNA interference (RNAi), caused by endogenous or exogenous double- stranded RNA (dsRNA) homologous with target genes, refers to gene silencing widely existing in animals and plants. It was first found in plants, and now it has developed into a kind of biotechnology as well as an important approach in post- genome era. This paper is to summarize the achievements of studies on RNAi tech- nology in basic biology, medicine, pharmacy, botany and other fields.

  13. The Regularities of the Induction and Reparation of DNA Double Strand Breaks in Human Lymphocytes after Irradiation by Carbon Ions with High Energy

    CERN Document Server

    Boreyko, A V

    2005-01-01

    The regularities of the induction of DNA double strand breaks (DSB) in human lymphocytes after irradiation by different doses of accelerated carbon ions (480 MeV/nucleon, LET = 10.6 keV/$\\mu $m) and $\\gamma $-rays $^{60}$?? by using of comet assay were investigated. It was shown that dependence of DSB formation increases linearly with growing of the dose of carbon ions and $\\gamma $-rays. The biological effectiveness of carbon ions with high energy was similar to $\\gamma $-rays. The kinetics of DSB reparation in human lymphocytes after irradiation by both carbon ions and $\\gamma $-rays was studied. It is revealed that the reparation proceeds effectively with heavy ion and $\\gamma $-ray irradiation.

  14. A role for the malignant brain tumour (MBT domain protein LIN-61 in DNA double-strand break repair by homologous recombination.

    Directory of Open Access Journals (Sweden)

    Nicholas M Johnson

    Full Text Available Malignant brain tumour (MBT domain proteins are transcriptional repressors that function within Polycomb complexes. Some MBT genes are tumour suppressors, but how they prevent tumourigenesis is unknown. The Caenorhabditis elegans MBT protein LIN-61 is a member of the synMuvB chromatin-remodelling proteins that control vulval development. Here we report a new role for LIN-61: it protects the genome by promoting homologous recombination (HR for the repair of DNA double-strand breaks (DSBs. lin-61 mutants manifest numerous problems associated with defective HR in germ and somatic cells but remain proficient in meiotic recombination. They are hypersensitive to ionizing radiation and interstrand crosslinks but not UV light. Using a novel reporter system that monitors repair of a defined DSB in C. elegans somatic cells, we show that LIN-61 contributes to HR. The involvement of this MBT protein in HR raises the possibility that MBT-deficient tumours may also have defective DSB repair.

  15. Rad10 exhibits lesion-dependent genetic requirements for recruitment to DNA double-strand breaks in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Moore, Destaye M; Karlin, Justin; González-Barrera, Sergio;

    2009-01-01

    . Here we show that yeast strains expressing fluorescently labeled Rad10 protein (Rad10-YFP) form foci in response to double-strand breaks (DSBs) induced by a site-specific restriction enzyme, I-SceI or by ionizing radiation (IR). Additionally, for endonuclease-induced DSBs, Rad10-YFP localization to DSB...... sites depends on both RAD51 and RAD52, but not MRE11 while IR-induced breaks do not require RAD51. Finally, Rad10-YFP colocalizes with Rad51-CFP and with Rad52-CFP at DSB sites, indicating a temporal overlap of Rad52, Rad51 and Rad10 functions at DSBs. These observations are consistent with a putative...... role of Rad10 protein in excising overhanging DNA ends after homology searching and refine the potential role(s) of the Rad1-Rad10 complex in DSB repair in yeast....

  16. The deubiquitylating enzyme USP44 counteracts the DNA double-strand break response mediated by the RNF8 and RNF168 ubiquitin ligases

    DEFF Research Database (Denmark)

    Mosbech, Anna; Lukas, Claudia; Bekker-Jensen, Simon;

    2013-01-01

    Protein recruitment to DNA double-strand breaks (DSBs) relies on ubiquitylation of the surrounding chromatin by the RING finger ubiquitin ligases RNF8 and RNF168. Flux through this pathway is opposed by several deubiquitylating enzymes (DUBs), including OTUB1 and USP3. By analyzing the effect...... of individually overexpressing the majority of human DUBs on RNF8/RNF168-mediated 53BP1 retention at DSB sites, we found that USP44 and USP29 powerfully inhibited this response at the level of RNF168 accrual. Both USP44 and USP29 promoted efficient deubiquitylation of histone H2A, but unlike USP44, USP29...... displayed non-specific reactivity towards ubiquitylated substrates. Moreover, USP44 but not other H2A DUBs was recruited to RNF168-generated ubiquitylation products at DSB sites. Individual depletion of these DUBs only mildly enhanced accumulation of ubiquitin conjugates and 53BP1 at DSBs, suggesting...

  17. Removal of nonhomologous DNA ends in double-strand break recombination: The role of the yeast ultraviolet repair gene RAD1

    Energy Technology Data Exchange (ETDEWEB)

    Fishman-Lobell, J.; Habert, J.E. (Brandeis Univ., Waltham, MA (United States))

    1992-10-15

    Double-strand breaks (DSBs) in Saccharomyces cerevisiae can be repaired by gene conversions or by deletions resulting from single-strand annealing between direct repeats of homologous sequences. Although rad1 mutants are resistant to x-rays and can complete DSB-mediated mating-type switching, they could not complete recombination when the ends of the break contained approximately 60 base pairs of nonhomology. Recombination was restored when the ends of the break were made homologous to donor sequences. Additionally, the absence of RAD1 led to the frequent appearance of a previously unobserved type of recombination product. These data suggest RAD1 is required to remove nonhomologous DNA from the 3{prime} ends of recombining DNA, a process analogous to the excision of photodimers during repair of ultraviolet-damaged DNA.

  18. Mito