WorldWideScience

Sample records for by-product double-stranded rna

  1. Double-Stranded RNA Resists Condensation

    Science.gov (United States)

    Li, Li; Pabit, Suzette A.; Meisburger, Steve P.; Pollack, Lois

    2011-03-01

    Much attention has been focused on DNA condensation because of its fundamental biological importance. The recent discovery of new roles for RNA duplexes demands efficient packaging of double-stranded RNA for therapeutics. Here we report measurements of short DNA and RNA duplexes in the presence of trivalent ions. Under conditions where UV spectroscopy indicates condensation of DNA duplexes into (insoluble) precipitates, RNA duplexes remain soluble. Small angle x-ray scattering results suggest that the differing surface topologies of RNA and DNA may be crucial in generating the attractive forces that result in precipitation.

  2. Double-stranded RNA resists condensation

    OpenAIRE

    Li, Li; Pabit, Suzette A.; Meisburger, Steve P.; Pollack, Lois

    2011-01-01

    Much attention has focused on DNA condensation because of its fundamental biological importance. The recent discovery of new roles for RNA duplexes demands efficient packaging of dsRNA for therapeutics. Here we report measurements of short DNA and RNA duplexes in the presence of trivalent ions. Under conditions where UV spectroscopy indicates condensation of DNA duplexes into (insoluble) precipitates, RNA duplexes remain soluble. SAXS results suggest that the differing surface topologies of R...

  3. RNA-directed repair of DNA double-strand breaks.

    Science.gov (United States)

    Yang, Yun-Gui; Qi, Yijun

    2015-08-01

    DNA double-strand breaks (DSBs) are among the most deleterious DNA lesions, which if unrepaired or repaired incorrectly can cause cell death or genome instability that may lead to cancer. To counteract these adverse consequences, eukaryotes have evolved a highly orchestrated mechanism to repair DSBs, namely DNA-damage-response (DDR). DDR, as defined specifically in relation to DSBs, consists of multi-layered regulatory modes including DNA damage sensors, transducers and effectors, through which DSBs are sensed and then repaired via DNAprotein interactions. Unexpectedly, recent studies have revealed a direct role of RNA in the repair of DSBs, including DSB-induced small RNA (diRNA)-directed and RNA-templated DNA repair. Here, we summarize the recent discoveries of RNA-mediated regulation of DSB repair and discuss the potential impact of these novel RNA components of the DSB repair pathway on genomic stability and plasticity. PMID:25960340

  4. Double-stranded RNA under force and torque: Similarities to and striking differences from double-stranded DNA

    OpenAIRE

    Lipfert, Jan; Skinner, Gary M.; Keegstra, Johannes M.; Hensgens, Toivo; Jager, Tessa; Dulin, David; Köber, Mariana; Yu, Zhongbo; Donkers, Serge P.; Chou, Fang-Chieh; Das, Rhiju; Dekker, Nynke H.

    2014-01-01

    RNA, like DNA, can form double helices held together by the pairing of complementary bases, and such helices are ubiquitous in functional RNAs. Here we apply external forces and torques to individual double-stranded RNA molecules to determine the mechanical properties and conformational transitions of these fundamental biological building blocks. For small forces and torques, RNA helices behave like elastic rods, and we have determined their bending, stretching, and twisting stiffness. Surpri...

  5. 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

  6. A closely related group of RNA-dependent RNA polymerases from double-stranded RNA viruses.

    OpenAIRE

    Bruenn, J A

    1993-01-01

    Probably one of the first proteinaceous enzymes was an RNA-dependent RNA polymerase (RDRP). Although there are several conserved motifs present in the RDRPs of most positive and double-stranded RNA (dsRNA) viruses, the RDRPs of the dsRNA viruses show no detectable sequence similarity outside the conserved motifs. There is now, however, a group of dsRNA viruses of lower eucaryotes whose RDRPs are detectably similar. The origin of this sequence similarity appears to be common descent from one o...

  7. 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...l) Show TLR3: interferon induction by double-stranded RNA including poly(I:C). PubmedID 18262679 Title TLR3:... interferon induction by double-stranded RNA including poly(I:C). Authors Matsumo

  8. Viral Proteins That Bind Double-Stranded RNA: Countermeasures Against Host Antiviral Responses

    OpenAIRE

    Krug, Robert M.

    2014-01-01

    Several animal viruses encode proteins that bind double-stranded RNA (dsRNA) to counteract host dsRNA-dependent antiviral responses. This article discusses the structure and function of the dsRNA-binding proteins of influenza A virus and Ebola viruses (EBOVs).

  9. A New Protocol for Extraction of Double-Stranded RNA from Plants

    Science.gov (United States)

    There are several unidentified virus-like diseases of blueberry that have been recalcitrant to standard methods of virus characterization based on double-stranded RNA (dsRNA). Relatives of these viruses yield large amounts of dsRNA in hosts other than blueberry. Modifications in dsRNA extraction p...

  10. Identification of an RNA Silencing Suppressor from a Plant Double-Stranded RNA Virus

    OpenAIRE

    Cao, Xuesong; Zhou, Peng; Zhang, Xiaoming; Zhu, Shifeng; Zhong, Xuehua; Xiao, Qi; Ding, Biao; Li, Yi

    2005-01-01

    RNA silencing is a mechanism which higher plants and animals have evolved to defend against viral infection in addition to regulation of gene expression for growth and development. As a counterdefense, many plant and some animal viruses studied to date encode RNA silencing suppressors (RSS) that interfere with various steps of the silencing pathway. In this study, we report the first identification of an RSS from a plant double-stranded RNA (dsRNA) virus. Pns10, encoded by S10 of Rice dwarf p...

  11. Differential recognition of double-stranded RNA by RIG-I–like receptors in antiviral immunity

    OpenAIRE

    Saito, Takeshi; Gale, Michael

    2008-01-01

    Retinoic acid–inducible gene (RIG)-I–like receptors (RLRs) are cytosolic RNA helicases that sense viral RNA and trigger signaling pathways that induce the production of type I interferons (IFNs) and proinflammatory cytokines. RLRs recognize distinct and overlapping sets of viruses, but the mechanisms that dictate this specificity were unknown. A new study now provides evidence for size-based discrimination of double-stranded RNA (dsRNA) by RLRs and suggests how host cells recognize a variety ...

  12. Template Role of Double-Stranded RNA in Tombusvirus Replication

    OpenAIRE

    Kovalev, Nikolay; Pogany, Judit; Nagy, Peter D.

    2014-01-01

    Replication of plus-strand RNA [(+)RNA] viruses of plants is a relatively simple process that involves complementary minus-strand RNA [(−)RNA] synthesis and subsequent (+)RNA synthesis. However, the actual replicative form of the (−)RNA template in the case of plant (+)RNA viruses is not yet established unambiguously. In this paper, using a cell-free replication assay supporting a full cycle of viral replication, we show that replication of Tomato bushy stunt virus (TBSV) leads to the formati...

  13. Genome Sequence of Saccharomyces cerevisiae Double-Stranded RNA Virus L-A-28.

    Science.gov (United States)

    Konovalovas, Aleksandras; Serviené, Elena; Serva, Saulius

    2016-01-01

    We cloned and sequenced the complete genome of the L-A-28 virus from the Saccharomyces cerevisiae K28 killer strain. This sequence completes the set of currently identified L-A helper viruses required for expression of double-stranded RNA-originated killer phenotypes in baking yeast. PMID:27313294

  14. 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.

  15. 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.

  16. Double-Stranded RNA Binding May Be a General Plant RNA Viral Strategy To Suppress RNA Silencing

    OpenAIRE

    Mérai, Zsuzsanna; Kerényi, Zoltán; Kertész, Sándor; Magna, Melinda; Lakatos, Lóránt; Silhavy, Dániel

    2006-01-01

    In plants, RNA silencing (RNA interference) is an efficient antiviral system, and therefore successful virus infection requires suppression of silencing. Although many viral silencing suppressors have been identified, the molecular basis of silencing suppression is poorly understood. It is proposed that various suppressors inhibit RNA silencing by targeting different steps. However, as double-stranded RNAs (dsRNAs) play key roles in silencing, it was speculated that dsRNA binding might be a g...

  17. Relationships among the positive strand and double-strand RNA viruses as viewed through their RNA-dependent RNA polymerases.

    OpenAIRE

    Bruenn, J A

    1991-01-01

    The sequences of 50 RNA-dependent RNA polymerases (RDRPs) from 43 positive strand and 7 double strand RNA (dsRNA) viruses have been compared. The alignment permitted calculation of distances among the 50 viruses and a resultant dendrogram based on every amino acid, rather than just those amino acids in the conserved motifs. Remarkably, a large subgroup of these viruses, including vertebrate, plant, and insect viruses, forms a single cluster whose only common characteristic is exploitation of ...

  18. Response of airway epithelial cells to double-stranded RNA in an allergic environment

    OpenAIRE

    Herbert, Cristan; Zeng, Qing-Xiang; Shanmugasundaram, Ramesh; Garthwaite, Linda; Oliver, Brian G.; Kumar, Rakesh K.

    2014-01-01

    Background Respiratory viral infections are the most common trigger of acute exacerbations in patients with allergic asthma. The anti-viral response of airway epithelial cells (AEC) may be impaired in asthmatics, while cytokines produced by AEC may drive the inflammatory response. We investigated whether AEC cultured in the presence of Th2 cytokines associated with an allergic environment exhibited altered responses to double-stranded RNA, a virus-like stimulus. Methods We undertook prelimina...

  19. Double-stranded RNA viral infection in Cuban Trichomonas vaginalis isolates

    Directory of Open Access Journals (Sweden)

    Jorge Fraga

    2005-12-01

    Full Text Available Trichomonas vaginalis can be infected with double-stranded RNA (dsRNA viruses designated T. vaginalis virus (TVV, which may have important implications for trichomonal virulence and disease pathogenesis. We tested for TVV in 40 fresh T. vaginalis isolates from Cuban patients by total extraction of nucleic acids (DNA and RNA. TVV was detected in 22 (55% of the 40 T. vaginalis isolates. This gives an estimate of the infection rate of Cuban T. vaginalis isolates by the dsRNA virus. Future research should focus on the association between trichomonosis symptoms and the presence of TVV.

  20. Double-stranded RNA viral infection in Cuban Trichomonas vaginalis isolates

    OpenAIRE

    Jorge Fraga; Lázara Rojas; Idalia Sariego; Aymé Fernández-Calienes

    2005-01-01

    Trichomonas vaginalis can be infected with double-stranded RNA (dsRNA) viruses designated T. vaginalis virus (TVV), which may have important implications for trichomonal virulence and disease pathogenesis. We tested for TVV in 40 fresh T. vaginalis isolates from Cuban patients by total extraction of nucleic acids (DNA and RNA). TVV was detected in 22 (55%) of the 40 T. vaginalis isolates. This gives an estimate of the infection rate of Cuban T. vaginalis isolates by the dsRNA virus. Future re...

  1. Sequence Selective Recognition of Double-Stranded RNA at Physiologically Relevant Conditions Using PNA-Peptide Conjugates

    OpenAIRE

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

    2013-01-01

    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-...

  2. Visualizing repetitive diffusion activity of double-strand RNA binding proteins by single molecule fluorescence assays.

    Science.gov (United States)

    Koh, Hye Ran; Wang, Xinlei; Myong, Sua

    2016-08-01

    TRBP, one of double strand RNA binding proteins (dsRBPs), is an essential cofactor of Dicer in the RNA interference pathway. Previously we reported that TRBP exhibits repetitive diffusion activity on double strand (ds)RNA in an ATP independent manner. In the TRBP-Dicer complex, the diffusion mobility of TRBP facilitates Dicer-mediated RNA cleavage. Such repetitive diffusion of dsRBPs on a nucleic acid at the nanometer scale can be appropriately captured by several single molecule detection techniques. Here, we provide a step-by-step guide to four different single molecule fluorescence assays by which the diffusion activity of dsRBPs on dsRNA can be detected. One color assay, termed protein induced fluorescence enhancement enables detection of unlabeled protein binding and diffusion on a singly labeled RNA. Two-color Fluorescence Resonance Energy Transfer (FRET) in which labeled dsRBPs is applied to labeled RNA, allows for probing the motion of protein along the RNA axis. Three color FRET reports on the diffusion movement of dsRBPs from one to the other end of RNA. The single molecule pull down assay provides an opportunity to collect dsRBPs from mammalian cells and examine the protein-RNA interaction at single molecule platform. PMID:27012177

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

    OpenAIRE

    Villanueva, Francisco; Sabanadzovic, Sead; Valverde, Rodrigo A.; Navas-Castillo, Jesús

    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).

  4. 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. PMID:27332119

  5. PKR, the double stranded RNA-dependent protein kinase as a critical target in Alzheimer's disease

    OpenAIRE

    Morel, Milena; Couturier, Julien; Lafay-Chebassier, Claire; Paccalin, Marc; Page, Guylène

    2009-01-01

    Amyloid β-peptide (Aβ) deposits and neurofibrillary tangles are key hallmarks in Alzheimer's disease (AD). Aβ stimulates many signal transducers involved in the neuronal death. However, many mechanisms remain to be elucidated because no definitive therapy of AD exists. Some studies have focused on the control of translation which involves eIF2 and eIF4E, main eukaryotic factors of initiation. The availability of these factors depends on the activation of the double-stranded RNA-dependent prot...

  6. [Characteristics of virus double-stranded RNA, isolated from microscopic fungi parasitizing on sugar beet].

    Science.gov (United States)

    Mel'nychuk, M D; Spyrydonov, V H; Oleksiienko, I P

    2005-01-01

    We have carried out comparative studies of double-stranded RNA (dsRNA) of viral nature isolated from sugar beet leaves and from mycelium of microscopic fungi using different methods such as PAAG electrophoresis and by polymerase chain reaction (PCR). It was shown that the fragments of dsRNA from sugar beet leaves and from mycelium microscopic fungi had the identical electrophoretic pattern and the same size (1.8 and 2.0 kbp). Using PCR technique it was shown, that isolated dsRNA have a common template for amplification. Electron microscopy of PCR-positive mycelium allows us to detect the virus particles of the spherical form with diameter 30-40 nm. The obtained data confirm our previous suppositions, concerning the belonging of isolated dsRNAs (size 1.8 and 2.0 kbp) to new mycovirus targeted a microscopic fungus, instead of beet cryptic viruses. PMID:16250236

  7. 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.

  8. Double-stranded RNA viral infection of Trichomonas vaginalis and correlation with genetic polymorphism of isolates.

    Science.gov (United States)

    Fraga, Jorge; Rojas, Lazara; Sariego, Idalia; Fernández-Calienes, Ayme

    2011-02-01

    Trichomonas vaginalis can be infected with double-stranded RNA (dsRNA) viruses known as T. vaginalis virus (TVV). This viral infection may have important implications for trichomonal virulence and disease pathogenesis. The objective of this study was to determine the possible correlation between the T. vaginalis genetic polymorphism and the isolate infection with TVV. The Random Amplified Polymorphic DNA (RAPD) technique was used to determine genetic differences among 37 isolates of T. vaginalis using a panel of 30 random primers and these genetic data were correlated with the infection of isolates with TVV. The trees drawn based on RAPD data showed significantly association with the presence of TVV (P = 0.028) demonstrating the existence of concordance between the genetic relatedness and the presence of TVV in T. vaginalis isolates. This result could point to a predisposition of T. vaginalis for the viral enters and/or survival. PMID:20875411

  9. 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.

  10. 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

  11. 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.

  12. Functional expression of double-stranded RNA-dependent protein kinase in rat intestinal epithelial cells.

    Science.gov (United States)

    Sato, Nagahiro; Morimoto, Hiroyuki; Baba, Ryoko; Nakamata, Junichi; Doi, Yoshiaki; Yamaguchi, Koji

    2010-05-01

    Intestinal epithelial cells (IECs) are exposed to external environment, microbial and viral products, and serve as essential barriers to antigens. Recent studies have shown that IECs express Toll-like receptors (TLRs) and respond to microbial components. The antimicrobial and antiviral barriers consist of many molecules including TLRs. To investigate the further component of this barrier in intestine, we examined the expression of double-stranded RNA-dependent protein kinase (PKR). PKR is a player in the cellular antiviral response and phosphorylates alpha-subunit of the eukaryotic translation initiation factor 2 (eIF-2alpha) to block protein synthesis and induces apoptosis. In this study, we showed that the expression of PKR was restricted to the cytoplasm of absorptive epithelial cells in the intestine of adult rat. We also demonstrated that PKR was expressed in the cultured rat intestinal epithelial cells (IEC-6). The level of PKR protein expression and the activity of alkaline phosphatase (ALP) increased in the cultured IEC-6 cells in a time-dependent manner. Inhibition of PKR by the 2-aminopurine treatment decreased ALP activity in the IEC-6 cells. Treatment of IEC-6 cells with synthetic double-stranded RNA (dsRNA) induced cell death in a dose-dependent manner. The addition of hydrocortisone also provoked suppression of PKR expression and ALP activity. This modulation might be mediated by signal transducers and activators of transcription-1 (STAT-1) protein. We concluded that PKR is expressed in IECs as potent barriers to antigens and is a possible modulator of the differentiation of rat IECs. PMID:20213745

  13. Activation of 2'-5' oligoadenylate synthetase by single-stranded and double-stranded RNA aptamers

    DEFF Research Database (Denmark)

    Hartmann, R; Norby, P L; Martensen, P M;

    1998-01-01

    . Competition experiments indicate that the binding site for the small RNAs on the 2'-5' oligoadenylate synthetase molecule at least partially overlaps that for the synthetic double-stranded RNA, poly(I).poly(C). Several of the RNAs function as potent activators of 2'-5' oligoadenylate synthetase in vitro......, although there is no correlation between binding affinity and ability to activate. The RNA aptamers having the strongest activation potential appear to have few base-paired regions. This suggests that 2'-5' oligoadenylate synthetase, which has previously been believed to be activated only by double-stranded...

  14. A single transgene locus triggers both transcriptional and post-transcriptional silencing through double-stranded RNA production

    OpenAIRE

    Mourrain, Philippe; Blokland, van, R.; Kooter, Jan; Vaucheret, Hervé

    2007-01-01

    Silencing of a target locus by an unlinked silencing locus can result from transcription inhibition (transcriptional gene silencing; TGS) or mRNA degradation (post-transcriptional gene silencing; PTGS), owing to the production of double-stranded RNA (dsRNA) corresponding to promoter or transcribed sequences, respectively. The involvement of distinct cellular components in each process suggests that dsRNA-induced TGS and PTGS likely result from the diversification of an ancient common mechanis...

  15. Aureusvirus P14 Is an Efficient RNA Silencing Suppressor That Binds Double-Stranded RNAs without Size Specificity‡

    OpenAIRE

    Mérai, Zsuzsanna; Kerényi, Zoltán; Molnár, Attila; Barta, Endre; Válóczi, Anna; Bisztray, György; Havelda, Zoltán; Burgyán, József; Silhavy, Dániel

    2005-01-01

    RNA silencing is a conserved eukaryotic gene regulatory system in which sequence specificity is determined by small RNAs. Plant RNA silencing also acts as an antiviral mechanism; therefore, viral infection requires expression of a silencing suppressor. The mechanism and the evolution of silencing suppression are still poorly understood. Tombusvirus open reading frame (ORF) 5-encoded P19 is a size-selective double-stranded RNA (dsRNA) binding protein that suppresses silencing by sequestering d...

  16. Editing of HIV-1 RNA by the double-stranded RNA deaminase ADAR1 stimulates viral infection

    OpenAIRE

    Doria, Margherita; Neri, Francesca; Gallo, Angela; Farace, Maria Giulia; Michienzi, Alessandro

    2009-01-01

    Adenosine deaminases that act on dsRNA (ADARs) are enzymes that target double-stranded regions of RNA converting adenosines into inosines (A-to-I editing) thus contributing to genome complexity and fine regulation of gene expression. It has been described that a member of the ADAR family, ADAR1, can target viruses and affect their replication process. Here we report evidence showing that ADAR1 stimulates human immuno deficiency virus type 1 (HIV-1) replication by using both editing-dependent ...

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

    International Nuclear Information System (INIS)

    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

  18. 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.

  19. 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

  20. Disruption of Specific RNA-RNA Interactions in a Double-Stranded RNA Virus Inhibits Genome Packaging and Virus Infectivity.

    Science.gov (United States)

    Fajardo, Teodoro; Sung, Po-Yu; Roy, Polly

    2015-12-01

    Bluetongue virus (BTV) causes hemorrhagic disease in economically important livestock. The BTV genome is organized into ten discrete double-stranded RNA molecules (S1-S10) which have been suggested to follow a sequential packaging pathway from smallest to largest segment during virus capsid assembly. To substantiate and extend these studies, we have investigated the RNA sorting and packaging mechanisms with a new experimental approach using inhibitory oligonucleotides. Putative packaging signals present in the 3'untranslated regions of BTV segments were targeted by a number of nuclease resistant oligoribonucleotides (ORNs) and their effects on virus replication in cell culture were assessed. ORNs complementary to the 3' UTR of BTV RNAs significantly inhibited virus replication without affecting protein synthesis. Same ORNs were found to inhibit complex formation when added to a novel RNA-RNA interaction assay which measured the formation of supramolecular complexes between and among different RNA segments. ORNs targeting the 3'UTR of BTV segment 10, the smallest RNA segment, were shown to be the most potent and deletions or substitution mutations of the targeted sequences diminished the RNA complexes and abolished the recovery of viable viruses using reverse genetics. Cell-free capsid assembly/RNA packaging assay also confirmed that the inhibitory ORNs could interfere with RNA packaging and further substitution mutations within the putative RNA packaging sequence have identified the recognition sequence concerned. Exchange of 3'UTR between segments have further demonstrated that RNA recognition was segment specific, most likely acting as part of the secondary structure of the entire genomic segment. Our data confirm that genome packaging in this segmented dsRNA virus occurs via the formation of supramolecular complexes formed by the interaction of specific sequences located in the 3' UTRs. Additionally, the inhibition of packaging in-trans with inhibitory ORNs

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

    OpenAIRE

    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 grov...

  2. Crystallization of the avian reovirus double-stranded RNA-binding and core protein σA

    Energy Technology Data Exchange (ETDEWEB)

    Hermo-Parrado, X. Lois; Guardado-Calvo, Pablo [Departamento de Bioquímica y Biología Molecular, Facultad de Farmacia, Universidad de Santiago de Compostela, Campus Sur, E-15782 Santiago de Compostela (Spain); Llamas-Saiz, Antonio L. [Unidad de Difracción de Rayos X, Laboratorio Integral de Dinámica y Estructura de Biomoléculas José R. Carracido, Edificio CACTUS, Universidad de Santiago de Compostela, Campus Sur, E-15782 Santiago de Compostela (Spain); Fox, Gavin C. [Spanish CRG Beamline BM16, European Synchrotron Radiation Facility (ESRF), 6 Rue Jules Horowitz, BP 220, F-38043 Grenoble (France); Vazquez-Iglesias, Lorena; Martínez-Costas, José; Benavente, Javier [Departamento de Bioquímica y Biología Molecular, Facultad de Farmacia, Universidad de Santiago de Compostela, Campus Sur, E-15782 Santiago de Compostela (Spain); Raaij, Mark J. van, E-mail: vanraaij@usc.es [Departamento de Bioquímica y Biología Molecular, Facultad de Farmacia, Universidad de Santiago de Compostela, Campus Sur, E-15782 Santiago de Compostela (Spain); Unidad de Difracción de Rayos X, Laboratorio Integral de Dinámica y Estructura de Biomoléculas José R. Carracido, Edificio CACTUS, Universidad de Santiago de Compostela, Campus Sur, E-15782 Santiago de Compostela (Spain)

    2007-05-01

    The avian reovirus double-stranded RNA-binding and core protein σA has been crystallized in space group P1, with unit-cell parameters a = 103.2, b = 129.9, c = 144.0 Å, α = 93.8, β = 105.1, γ = 98.2°. A complete data set has been collected to 2.3 Å resolution and analyzed. The avian reovirus protein σA plays a dual role: it is a structural protein forming part of the transcriptionally active core, but it has also been implicated in the resistance of the virus to interferon by strongly binding double-stranded RNA and thus inhibiting the double-stranded RNA-dependent protein kinase. The σA protein has been crystallized from solutions containing ammonium sulfate at pH values around 6. Crystals belonging to space group P1, with unit-cell parameters a = 103.2, b = 129.9, c = 144.0 Å, α = 93.8, β = 105.1, γ = 98.2° were grown and a complete data set has been collected to 2.3 Å resolution. The self-rotation function suggests that σA may form symmetric arrangements in the crystals.

  3. Crystallization of the avian reovirus double-stranded RNA-binding and core protein σA

    International Nuclear Information System (INIS)

    The avian reovirus double-stranded RNA-binding and core protein σA has been crystallized in space group P1, with unit-cell parameters a = 103.2, b = 129.9, c = 144.0 Å, α = 93.8, β = 105.1, γ = 98.2°. A complete data set has been collected to 2.3 Å resolution and analyzed. The avian reovirus protein σA plays a dual role: it is a structural protein forming part of the transcriptionally active core, but it has also been implicated in the resistance of the virus to interferon by strongly binding double-stranded RNA and thus inhibiting the double-stranded RNA-dependent protein kinase. The σA protein has been crystallized from solutions containing ammonium sulfate at pH values around 6. Crystals belonging to space group P1, with unit-cell parameters a = 103.2, b = 129.9, c = 144.0 Å, α = 93.8, β = 105.1, γ = 98.2° were grown and a complete data set has been collected to 2.3 Å resolution. The self-rotation function suggests that σA may form symmetric arrangements in the crystals

  4. MDA-5 activation by cytoplasmic double-stranded RNA impairs endothelial function and aggravates atherosclerosis.

    Science.gov (United States)

    Asdonk, Tobias; Steinmetz, Martin; Krogmann, Alexander; Ströcker, Christine; Lahrmann, Catharina; Motz, Inga; Paul-Krahe, Kathrin; Flender, Anna; Schmitz, Theresa; Barchet, Winfried; Hartmann, Gunther; Nickenig, Georg; Zimmer, Sebastian

    2016-09-01

    Recent studies have highlighted the relevance of viral nucleic acid immunorecognition by pattern recognition receptors in atherogenesis. Melanoma differentiation associated gene 5 (MDA-5) belongs to the intracellular retinoic acid inducible gene-I like receptors and its activation promotes pro-inflammatory mechanisms. Here, we studied the effect of MDA-5 stimulation in vascular biology. To gain insights into MDA-5 dependent effects on endothelial function, cultured human coronary artery endothelial cells (HCAEC) were transfected with the synthetic MDA-5 agonist polyIC (long double-stranded RNA). Human coronary endothelial cell expressed MDA-5 and reacted with receptor up-regulation upon stimulation. Reactive oxygen species formation, apoptosis and the release of pro-inflammatory cytokines was enhanced, whereas migration was significantly reduced in response to MDA-5 stimulation. To test these effects in vivo, wild-type mice were transfected with 32.5 μg polyIC/JetPEI or polyA/JetPEI as control every other day for 7 days. In polyIC-treated wild-type mice, endothelium-dependent vasodilation and re-endothelialization was significantly impaired, vascular oxidative stress significantly increased and circulating endothelial microparticles and circulating endothelial progenitor cells significantly elevated compared to controls. Importantly, these effects could be abrogated by MDA-5 deficiency in vivo. Finally, chronic MDA-5 stimulation in Apolipoprotein E/toll-like receptor 3 (TLR3) double(-) deficient (ApoE(-/-) /TLR3(-/-) ) mice-enhanced atherosclerotic plaque formation. This study demonstrates that MDA-5 stimulation leads to endothelial dysfunction, and has the potential to aggravate atherosclerotic plaque burden in murine atherosclerosis. Thus, the spectrum of relevant innate immune receptors in vascular diseases and atherogenesis might not be restricted to TLRs but also encompasses the group of RLRs including MDA-5. PMID:27130701

  5. Zygotic Expression of the Double-Stranded RNA Binding Motif Protein Drb2p Is Required for DNA Elimination in the Ciliate Tetrahymena thermophila ▿

    OpenAIRE

    Motl, Jason A.; Chalker, Douglas L.

    2011-01-01

    Double-stranded RNA binding motif (DSRM)-containing proteins play many roles in the regulation of gene transcription and translation, including some with tandem DSRMs that act in small RNA biogenesis. We report the characterization of the genes for double-stranded RNA binding proteins 1 and 2 (DRB1 and DRB2), two genes encoding nuclear proteins with tandem DSRMs in the ciliate Tetrahymena thermophila. Both proteins are expressed throughout growth and development but exhibit distinct peaks of ...

  6. Enhanced Whitefly Resistance in Transgenic Tobacco Plants Expressing Double Stranded RNA of v-ATPase A Gene

    OpenAIRE

    Thakur, Nidhi; Upadhyay, Santosh Kumar; Verma, Praveen C.; Chandrashekar, Krishnappa; Tuli, Rakesh; Singh, Pradhyumna K.

    2014-01-01

    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 cr...

  7. Characterization of a novel double-stranded RNA mycovirus conferring hypovirulence from the phytopathogenic fungus Botryosphaeria dothidea.

    Science.gov (United States)

    Zhai, Lifeng; Xiang, Jun; Zhang, Meixin; Fu, Min; Yang, Zuokun; Hong, Ni; Wang, Guoping

    2016-06-01

    A novel double-stranded RNA (dsRNA) virus, designated as Botryosphaeria dothidea RNA virus 1 (BdRV1), isolated from a hypovirulent strain YZN115 of Botryosphaeria dothidea was biologically and molecularly characterized. The genome of BdRV1 comprises of five dsRNAs. Each dsRNA contains a single open reading frame. The proteins encoded by dsRNA1-4 shared significant amino acid identities of 55%, 47%, 43% and 53% with the corresponding proteins of Aspergillus fumigatus tetramycovirus-1. DsRNA1, 3, and 4 of BdRV1 encoded an RNA-dependent RNA polymerase, a viral methyltransferase, and a P-A-S-rich protein, respectively. Function of proteins encoded by the dsRNA2 and dsRNA5 were unknown. BdRV1 conferred hypovirulence for its host and could be transmitted through conidia and hyphae contact. PMID:27015523

  8. TRIP: a novel double stranded RNA binding protein which interacts with the leucine rich repeat of flightless I.

    Science.gov (United States)

    Wilson, S A; Brown, E C; Kingsman, A J; Kingsman, S M

    1998-08-01

    A northwestern screen of a CHO-K1 cell line cDNA library with radiolabelled HIV-1 TAR RNA identified a novel TAR RNA interacting protein, TRIP. The human trip cDNA was also cloned and its expression is induced by phorbol esters. The N-terminus of TRIP shows high homology to the coiled coil domain of FLAP, a protein which binds the leucine-rich repeat (LRR) of Flightless I (FLI) and the interaction of TRIP with the FLI LRR has been confirmed in vitro . TRIP does not bind single stranded DNA or RNA significantly and binds double stranded DNA weakly. In contrast, TRIP binds double stranded RNA with high affinity and two molecules of TRIP bind the TAR stem. The RNA binding domain has been identified and encompasses a lysine-rich motif. A TRIP-GFP fusion is localised in the cytoplasm and excluded from the nucleus. FLI has a C-terminal gelsolin-like domain which binds actin and therefore the association of TRIP with the FLI LRR may provide a link between the actin cytoskeleton and RNA in mammalian cells. PMID:9671805

  9. Double-Stranded RNA Is Produced by Positive-Strand RNA Viruses and DNA Viruses but Not in Detectable Amounts by Negative-Strand RNA Viruses

    OpenAIRE

    Weber, Friedemann; Wagner, Valentina; Rasmussen, Simon B; Hartmann, Rune; Paludan, Søren R.

    2006-01-01

    Double-stranded RNA (dsRNA) longer than 30 bp is a key activator of the innate immune response against viral infections. It is widely assumed that the generation of dsRNA during genome replication is a trait shared by all viruses. However, to our knowledge, no study exists in which the production of dsRNA by different viruses is systematically investigated. Here, we investigated the presence and localization of dsRNA in cells infected with a range of viruses, employing a dsRNA-specific antibo...

  10. Editing of HIV-1 RNA by the double-stranded RNA deaminase ADAR1 stimulates viral infection

    Science.gov (United States)

    Doria, Margherita; Neri, Francesca; Gallo, Angela; Farace, Maria Giulia; Michienzi, Alessandro

    2009-01-01

    Adenosine deaminases that act on dsRNA (ADARs) are enzymes that target double-stranded regions of RNA converting adenosines into inosines (A-to-I editing) thus contributing to genome complexity and fine regulation of gene expression. It has been described that a member of the ADAR family, ADAR1, can target viruses and affect their replication process. Here we report evidence showing that ADAR1 stimulates human immuno deficiency virus type 1 (HIV-1) replication by using both editing-dependent and editing-independent mechanisms. We show that over-expression of ADAR1 in HIV-1 producer cells increases viral protein accumulation in an editing-independent manner. Moreover, HIV-1 virions generated in the presence of over-expressed ADAR1 but not an editing-inactive ADAR1 mutant are released more efficiently and display enhanced infectivity, as demonstrated by challenge assays performed with T cell lines and primary CD4+ T lymphocytes. Finally, we report that ADAR1 associates with HIV-1 RNAs and edits adenosines in the 5′ untranslated region (UTR) and the Rev and Tat coding sequence. Overall these results suggest that HIV-1 has evolved mechanisms to take advantage of specific RNA editing activity of the host cell and disclose a stimulatory function of ADAR1 in the spread of HIV-1. PMID:19651874

  11. Analysis of gene repair tracts from Cas9/gRNA double-stranded breaks in the human CFTR gene.

    Science.gov (United States)

    Hollywood, Jennifer A; Lee, Ciaran M; Scallan, Martina F; Harrison, Patrick T

    2016-01-01

    To maximise the efficiency of template-dependent gene editing, most studies describe programmable and/or RNA-guided endonucleases that make a double-stranded break at, or close to, the target sequence to be modified. The rationale for this design strategy is that most gene repair tracts will be very short. Here, we describe a CRISPR Cas9/gRNA selection-free strategy which uses deep sequencing to characterise repair tracts from a donor plasmid containing seven nucleotide differences across a 216 bp target region in the human CFTR gene. We found that 90% of the template-dependent repair tracts were >100 bp in length with equal numbers of uni-directional and bi-directional repair tracts. The occurrence of long repair tracts suggests that a single gRNA could be used with variants of the same template to create or correct specific mutations within a 200 bp range, the size of ~80% of human exons. The selection-free strategy used here also allowed detection of non-homologous end joining events in many of the homology-directed repair tracts. This indicates a need to modify the donor, possibly by silent changes in the PAM sequence, to prevent creation of a second double-stranded break in an allele that has already been correctly edited by homology-directed repair. PMID:27557525

  12. Double-stranded RNA confers both preventive and therapeutic effects against Penaeus stylirostris densovirus (PstDNV) in Litopenaeus vannamei.

    Science.gov (United States)

    Ho, Teerapong; Yasri, Pratchayapong; Panyim, Sakol; Udomkit, Apinunt

    2011-01-01

    Penaeus stylirostris densovirus (PstDNV) infection is found widespread in peneaid shrimp, especially in economically important species such as black tiger shrimp Penaeus monodon and Pacific white shrimp Litopenaeus vannamei. Although effective prevention method for viral diseases is not well established in shrimp, the treatment with viral specific double-stranded RNA (dsRNA) or siRNA has given promising results. In present study, dsRNAs corresponding to non-structural (ORF1 and ORF2 overlapping sequence) and structural (ORF3) genes of PstDNV were investigated for their potency to inhibit PstDNV replication in the shrimp. Periodically injection of either ORF1-2 dsRNA or ORF3 dsRNA at three days interval into L. vannamei resulted in substantial inhibition of PstDNV infection. In addition, a possibility for a therapeutic application of dsRNA in PstDNV-infected shrimp was demonstrated by the efficient suppression of PstDNV replication in L. vannamei when the ORF1-2 dsRNA was delivered into the shrimp within 24h post-PstDNV injection. Hence, our results established both the preventive and therapeutic potency of dsRNA to inhibit PstDNV in L. vannamei that could be applied as a potential treatment of PstDNV infection in shrimp. PMID:20869997

  13. Inhibition of Plasmodium falciparum proliferation in vitro by double-stranded RNA directed against malaria histone deacetylase

    International Nuclear Information System (INIS)

    Acetylation and deacetylation of histones play important roles in transcription regulation, cell cycle progression and development events. The steady state status of histone acetylation is controlled by a dynamic equilibrium between competing histone acetylase and deacetylase (HDAC). We have used long PfHDAC-1 double-stranded (ds)RNA to interfere with its cognate mRNA expression and determined the effect on malaria parasite growth and development. Chloroquine- and pyrimethamine-resistant Plasmodium falciparum K1 strain was exposed to 1-25 μg of dsRNA/ml of culture for 48 h and growth was determined by [3H]-hypoxanthine incorporation and microscopic examination. Parasite culture treated with 10 μg/ml pfHDAC-1 dsRNA exhibited 47% growth inhibition when compared with either untreated control or culture treated with an unrelated dsRNA. PfHDAC-1 dsRNA specifically blocked maturation of trophozoite to schizont stages and decreased PfHDAC-1 transcript 44% in treated trophozoites. These results indicate the potential of HDAC-1 as a target for development of novel antimalarials.

  14. The Cellular TAR RNA Binding Protein, TRBP, Promotes HIV-1 Replication Primarily by Inhibiting the Activation of Double-Stranded RNA-Dependent Kinase PKR▿

    OpenAIRE

    Sanghvi, Viraj R.; Steel, Laura F

    2011-01-01

    The TAR RNA binding protein, TRBP, is a cellular double-stranded RNA (dsRNA) binding protein that can promote the replication of HIV-1 through interactions with the viral TAR element as well as with cellular proteins that affect the efficiency of translation of viral transcripts. The structured TAR element, present on all viral transcripts, can impede efficient translation either by sterically blocking access of translation initiation factors to the 5′-cap or by activating the dsRNA-dependent...

  15. Complete sequence of a double-stranded RNA from the phytopathogenic fungus Erysiphe cichoracearum that might represent a novel endornavirus.

    Science.gov (United States)

    Du, Zhenguo; Lin, Wenzhong; Qiu, Ping; Liu, Xiaojuan; Guo, Lingfang; Wu, Kangcheng; Zhang, Songbai; Wu, Zujian

    2016-08-01

    A double-stranded RNA (dsRNA) HBJZ1506 recovered from the phytopathogenic fungus Erysiphe cichoracearum infecting Calendula officinalis in Jingzhou, Hubei Province, China, was sequenced. HBJZ1506 comprises 11,908 nucleotides (nt) and contains a 11,859-nt-long open reading frame (ORF) coding for a polypeptide that is 61 % identical to that of a putative endornavirus named grapevine endophyte endornavirus (GeEV). The putative polyprotein has an RNA-dependent RNA polymerase (RdRp) domain and an RNA helicase domain, which show homology to and have an arrangement that is similar to that of their counterparts in approved or putative endornaviruses. In a phylogenetic tree constructed using amino acid sequences of the RdRp region of HBJZ1506 and selected endornaviruses, HBJZ1506 clustered with endornaviruses and formed a well-supported monophyletic branch with GeEV. These results suggest that HBJZ1506 might represent a novel endornavirus, for which the name Erysiphe cichoracearum endornavirus (EcEV) is proposed. PMID:27255746

  16. 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.

  17. 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.

  18. Synergy of CpG oligodeoxynucleotide and double-stranded RNA (poly I:C) on nitric oxide induction in chicken peripheral blood monocytes

    Science.gov (United States)

    Toll-like receptors (TLRs) recognize microbial components and initiate the innate immune responses that control microbial infections. We have investigated the innate immune response of chicken monocytes to ligands of TLR3 and TLR9, poly I:C, the analog of viral double-stranded RNA, and CpG-ODN, bac...

  19. A new virus discovered by immunocapture of double-stranded RNA, a rapid method for virus enrichment in metagenomic studies.

    Science.gov (United States)

    Blouin, Arnaud G; Ross, Howard A; Hobson-Peters, Jody; O'Brien, Caitlin A; Warren, Ben; MacDiarmid, Robin

    2016-09-01

    Next-generation sequencing technologies enable the rapid identification of viral infection of diseased organisms. However, despite a consistent decrease in sequencing costs, it is difficult to justify their use in large-scale surveys without a virus sequence enrichment technique. As the majority of plant viruses have an RNA genome, a common approach is to extract the double-stranded RNA (dsRNA) replicative form, to enrich the replicating virus genetic material over the host background. The traditional dsRNA extraction is time-consuming and labour-intensive. We present an alternative method to enrich dsRNA from plant extracts using anti-dsRNA monoclonal antibodies in a pull-down assay. The extracted dsRNA can be amplified by reverse transcriptase-polymerase chain reaction and sequenced by next-generation sequencing. In our study, we have selected three distinct plant hosts: Māori potato (Solanum tuberosum), rengarenga (Arthropodium cirratum) and broadleaved dock (Rumex obtusifolius) representing a cultivated crop, a New Zealand-native ornamental plant and a weed, respectively. Of the sequence data obtained, 31-74% of the reads were of viral origin, and we identified five viruses including Potato virus Y and Potato virus S in potato; Turnip mosaic virus in rengarenga (a new host record); and in the dock sample Cherry leaf roll virus and a novel virus belonging to the genus Macluravirus. We believe that this new assay represents a significant opportunity to upscale virus ecology studies from environmental, primary industry and/or medical samples. PMID:26990372

  20. Recognition of Double Stranded RNA by Guanidine-Modified Peptide Nucleic Acids (GPNA)

    OpenAIRE

    Gupta, Pankaj; Muse, Oluwatoyosi; Rozners, Eriks

    2011-01-01

    Double helical RNA has become an attractive target for molecular recognition because many non-coding RNAs play important roles in control of gene expression. Recently, we discovered that short peptide nucleic acids (PNA) bind strongly and sequence selectively to a homopurine tract of double helical RNA via triple helix formation. Herein we tested if the molecular recognition of RNA can be enhanced by α-guanidine modification of PNA. Our study was motivated by the discovery of Ly and co-worker...

  1. 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. PMID:16341944

  2. Systemic gene silencing in plants triggered by fluorescent nanoparticle-delivered double-stranded RNA

    Science.gov (United States)

    Jiang, Li; Ding, Lian; He, Bicheng; Shen, Jie; Xu, Zejun; Yin, Meizhen; Zhang, Xiaolan

    2014-08-01

    A cationic fluorescence nanoparticle efficiently enters plants with high transfection efficacy. Applying a mixture of G2/dsRNA to the model plant, Arabidopsis root, leads to significant reduction in the expression of important developmental genes and results in apparent phenotypes. This study reports a non-viral gene nanocarrier which triggers gene silencing in plants and leads to systemic phenotypes.A cationic fluorescence nanoparticle efficiently enters plants with high transfection efficacy. Applying a mixture of G2/dsRNA to the model plant, Arabidopsis root, leads to significant reduction in the expression of important developmental genes and results in apparent phenotypes. This study reports a non-viral gene nanocarrier which triggers gene silencing in plants and leads to systemic phenotypes. Electronic supplementary information (ESI) available. See DOI: 10.1039/c4nr03481c

  3. 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.

  4. 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.

  5. Novel 5'/3'RACE Method for Amplification and Determination of Single-Stranded RNAs Through Double-Stranded RNA (dsRNA) Intermediates.

    Science.gov (United States)

    Pankovics, Péter; Boros, Ákos; Reuter, Gábor

    2015-12-01

    To acquire the full-length sequences and to determine the 5'/3'ends of the RNA genomes and mRNA transcripts using the rapid amplification of cDNA ends (RACE) protocols-via cDNA or mRNA templates-are a great challenge. This 4-steps RNA-based RACE method uses different ways to determine the RNA ends through a double-stranded (ds) RNA intermediate (dsRNA-RACE). In the first step a complementary RNA strand is synthesised by Phi6 RNA replicase enzyme next to the template ssRNA forming a dsRNA intermediate. The following steps include adapter ligation, nucleic acid purification and two classical methods with minor modifications reverse transcription and polymerase chain reaction. The dsRNA-RACE protocol could be used in wide variety of ssRNA (cellular, viral, bacterial, etc.) templates in the field of microbiology and cellular biology and suitable for the amplification of full-length RNAs including the 5'/3'ends. This is a novel, expansively utilizable molecular tool with fewer disadvantages than the existing 5'/3'RACE approaches. PMID:26315976

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

    International Nuclear Information System (INIS)

    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

  7. 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.

  8. Rapid isolation of both double-stranded RNA and PCR-suitable DNA from the obligate biotrophic phytopathogenic fungus Uncinula necator using a commercially available reagent.

    Science.gov (United States)

    Délye, C; Corio-Costet, M F

    1998-10-01

    A method for rapid extraction of both double-stranded RNA (dsRNA) and DNA from an obligate biotrophic phytopathogenic fungus is described. Lyophilised fungal material is incubated in a commercial guanidium thiocyanate reagent. Proteins and cell debris are centrifuged by chloroform precipitation. After precipitation in isopropanol and washing in 75% ethanol, nucleic acids are resuspended in water (10 microl/mg fungal dry weight). DsRNA is directly visualised by agarose gel electrophoresis. DNA contained in 10-fold dilutions of the samples proved to be suitable for PCR-based experiments. PMID:9779614

  9. A wild-type Botrytis cinerea strain co-infected by double-stranded RNA mycoviruses presents hypovirulence-associated traits

    OpenAIRE

    Potgieter, Christiaan A.; Castillo, Antonio; Castro, Miguel; Cottet, Luis; Morales, Angélica

    2013-01-01

    Background Botrytis cinerea CCg378 is a wild-type strain infected with two types of double-stranded RNA (dsRNA) mycoviruses and which presents hypovirulence-associated traits. The objectives of the present study were to characterize the mycoviruses and investigate their relationship with the low virulence degree of the fungal host. Results B. cinerea CCg378 contains five dsRNA molecules that are associated with two different types of isometric viral particles of 32 and 23 nm in diameter, form...

  10. Double-stranded RNA-mediated interference of dumpy genes in Bursaphelenchus xylophilus by feeding on filamentous fungal transformants.

    Science.gov (United States)

    Wang, Meng; Wang, Diandong; Zhang, Xi; Wang, Xu; Liu, Wencui; Hou, Xiaomeng; Huang, Xiaoyin; Xie, Bingyan; Cheng, Xinyue

    2016-05-01

    RNA interference (RNAi) is a valuable tool for studying gene function in vivo and provides a functional genomics platform in a wide variety of organisms. The pinewood nematode, Bursaphelenchus xylophilus, is a prominent invasive plant-parasitic nematode and has become a serious worldwide threat to forest ecosystems. Presently, the complete genome sequence of B. xylophilus has been published, and research involving genome-wide functional analyses is likely to increase. In this study, we describe the construction of an effective silencing vector, pDH-RH, which contains a transcriptional unit for a hairpin loop structure. Utilising this vector, double-stranded (ds)RNAs with sequences homologous to the target genes can be expressed in a transformed filamentous fungus via Agrobacterium tumefaciens-mediated transformation technology, and can subsequently induce the knockdown of target gene mRNA expression in B. xylophilus by allowing the nematode to feed on the fungal transformants. Four dumpy genes (Bx-dpy-2, 4, 10 and 11) were used as targets to detect RNAi efficiency. By allowing the nematode to feed on target gene-transformed Fusarium oxysporum strains, target transcripts were knocked down 34-87% compared with those feeding on the wild-type strain as determined by real-time quantitative PCR (RT-qPCR). Morphological RNAi phenotypes were observed, displaying obviously reduced body length; weak dumpy or small (short and thin) body size; or general abnormalities. Moreover, compensatory regulation and non-specific silencing of dpy genes were found in B. xylophilus. Our results indicate that RNAi delivery by feeding in B. xylophilus is a successful technique. This platform may provide a new opportunity for undertaking RNAi-based, genome-wide gene functional studies in vitro in B. xylophilus. Moreover, as B. xylophilus feeds on endophytic fungi when a host has died, RNAi feeding technology will offer the prospect for developing a novel control strategy for the nematode

  11. Prosthetic labelling of double-stranded small interfering RNA (s.i.RNA) using the bromo-acetamide [18F]fpybra reagent

    International Nuclear Information System (INIS)

    Introduction. - Small interfering RNAs (s.i.RNA) are double-stranded oligonucleotides able to induce a specific post-transcriptional inhibition of genes. S.i.RNA is one of the most powerful tools to achieve gene silencing in cells, but is limited in vivo because of the poor pharmacological properties of RNAs. In order to evaluate the in vivo pharmacokinetics of some selected s.i.RNA with Positron Emission Tomography (PET), the corresponding oligonucleotidic sequences have been modified in order to increase their resistance toward nucleases and then labelled with fluorine-18. Methods. - S.i.RNA (21 mer, non-modified or the corresponding 20-Fluoro- and 2-OMe-modified sequences) have been labelled with fluorine using the following two-step process: (A) prosthetic conjugation of one strand with [18F]F.Py.Br.A. [1] which was carried out in a mixture of methanol and phosphate buffer saline (pH 7.4) at 80 degrees C for 15 min. The corresponding [18F]conjugated oligonucleotide was then HPLC-purified, desalted, and its concentration measured at 260 nm. (B) The [18F]conjugated oligonucleotide was then hybridized with its complementary strand in stoichiometric condition (in 100 m M NaCl at 90 degrees C for 1 min and 25 degrees C for 10 min) to give the desired [18F]conjugated s.i.RNA. Quality controls of single- and double stranded [18F]conjugated oligonucleotides were performed by non-denaturing polyacrylamide gel electrophoresis (P.A.G.E.) with the corresponding [19F]conjugated oligonucleotides as references, enzymatically labelled with [32P]phosphate at their 5 end. Results. - Typically, up to 0.55 GBq of pure [18F]conjugated s.i.RNA can be produced in 160 min with a specific radioactivity up to 212 GBq/micro-mol at the end of synthesis, starting from 20.3 to 24.0 GBq of a [18F]F batch. They were found to be at least 95% radiochemically pure. Complete hybridization of the two strands was confirmed by non-denaturing P.A.G.E. analysis. All the [18F]conjugated s.i.RNA have

  12. Inhibition of L-Deleted Foot-and-Mouth Disease Virus Replication by Alpha/Beta Interferon Involves Double-Stranded RNA-Dependent Protein Kinase

    OpenAIRE

    Chinsangaram, Jarasvech; Koster, Marla; Grubman, Marvin J.

    2001-01-01

    We previously demonstrated that the ability of foot-and-mouth disease virus (FMDV) to form plaques in cell culture is associated with the suppression of alpha/beta interferon (IFN-α/β). In the present study, we used Escherichia coli-expressed porcine and bovine IFN-α or -β individually to demonstrate that each was equally effective in inhibiting FMDV replication. The block in FMDV replication appeared to be at the level of protein translation, suggesting a role for double-stranded RNA-depende...

  13. An adenovirus-based vaccine with a double-stranded RNA adjuvant protects mice and ferrets against H5N1 avian influenza in oral delivery models.

    Science.gov (United States)

    Scallan, Ciaran D; Tingley, Debora W; Lindbloom, Jonathan D; Toomey, James S; Tucker, Sean N

    2013-01-01

    An oral gene-based avian influenza vaccine would allow rapid development and simplified distribution, but efficacy has previously been difficult to achieve by the oral route. This study assessed protection against avian influenza virus challenge using a chimeric adenovirus vector expressing hemagglutinin and a double-stranded RNA adjuvant. Immunized ferrets and mice were protected upon lethal challenge. Further, ferrets immunized by the peroral route induced cross-clade neutralizing antibodies, and the antibodies were selective against hemagglutinin, not the vector. Similarly, experiments in mice demonstrated selective immune responses against HA with peroral delivery and the ability to circumvent preexisting vector immunity. PMID:23155123

  14. RNA Activation of the Vascular Endothelial Growth Factor Gene (VEGF) Promoter by Double-Stranded RNA and Hypoxia: Role of Noncoding VEGF Promoter Transcripts.

    Science.gov (United States)

    Lopez, Pascal; Wagner, Kay-Dietrich; Hofman, Paul; Van Obberghen, Emmanuel

    2016-05-15

    RNA activation (RNAa) is a gene regulation process in which promoter-targeted short double-stranded RNAs (dsRNAs) or microRNAs (miRs) induce target gene expression at the transcriptional level. Here, we investigate the presence of cryptic promoter transcripts within the VEGF promoter. Single-strand sense and antisense noncoding vascular endothelial growth factor (NcVEGF) promoter transcripts are identified, and their respective expression is studied in cells transfected with a VEGF promoter targeted dsRNA, namely, dsVEGF706, in hypoxic cells and in human malignant lung tissues. Interestingly, in dsVEGF706-transfected, as well as in hypoxic cells, NcVEGF expression levels increase coordinately with coding VEGF expression. Ago2 interaction with both sense and antisense NcVEGFs is increased in hypoxic cells, whereas in dsVEGF706-transfected cells, Ago2 and the antisense strand of the dsRNA interact specifically with the sense NcVEGF transcript. Furthermore, both dsVEGF706 and ectopic NcVEGF transcripts are able to activate the VEGF promoter endogenously present or in a reporter construct. Finally, using small interfering RNA targeting Ago2, we show that RNAa plays a role in the maintenance of increased VEGF and NcVEGF expression after hypoxia. Given the central role of VEGF in major human diseases, including cancer, this novel molecular mechanism is poised to reveal promising possibilities for therapeutic interventions. PMID:26976645

  15. Zygotic Expression of the Double-Stranded RNA Binding Motif Protein Drb2p Is Required for DNA Elimination in the Ciliate Tetrahymena thermophila ▿

    Science.gov (United States)

    Motl, Jason A.; Chalker, Douglas L.

    2011-01-01

    Double-stranded RNA binding motif (DSRM)-containing proteins play many roles in the regulation of gene transcription and translation, including some with tandem DSRMs that act in small RNA biogenesis. We report the characterization of the genes for double-stranded RNA binding proteins 1 and 2 (DRB1 and DRB2), two genes encoding nuclear proteins with tandem DSRMs in the ciliate Tetrahymena thermophila. Both proteins are expressed throughout growth and development but exhibit distinct peaks of expression, suggesting different biological roles. In support of this, we show that expression of DRB2 is essential for vegetative growth while DRB1 expression is not. During conjugation, Drb1p and Drb2p localize to distinct nuclear foci. Cells lacking all DRB1 copies are able to produce viable progeny, although at a reduced rate relative to wild-type cells. In contrast, cells lacking germ line DRB2 copies, which thus cannot express Drb2p zygotically, fail to produce progeny, arresting late into conjugation. This arrest phenotype is accompanied by a failure to organize the essential DNA rearrangement protein Pdd1p into DNA elimination bodies and execute DNA elimination and chromosome breakage. These results implicate zygotically expressed Drb2p in the maturation of these nuclear structures, which are necessary for reorganization of the somatic genome. PMID:22021239

  16. H2AX phosphorylation in response to DNA double-strand break formation during bystander signalling: Effect of micro-RNA knockdown

    International Nuclear Information System (INIS)

    Upon DNA double-strand break (DSB) formation, hundreds of H2AX molecules in the chromatin flanking the break site are phosphorylated on serine residue 139, termed gamma-H2AX, so that virtually every DSB site in a nucleus can be visualised within 10 min of its formation using an antibody to gamma-H2AX. One application of this sensitive assay is to examine the induction of DNA double-strand damage in subtle non-targeted cellular effects such as the bystander effect. Here whether micro-RNA (miRNA) serve as a primary signalling mechanism for bystander effect propagation by comparing matched human colon carcinoma cell lines with wild-type or depleted levels of mature miRNAs was investigated. No major differences were found in the levels of induced gamma-H2AX foci in the tested cell lines, indicating that though miRNAs play a role in bystander effect manifestation, they appear not to be the primary bystander signalling molecules in the formation of bystander effect-induced DSBs. (authors)

  17. Solution structure of the SL1 RNA of the M1 double-stranded RNA virus of Saccharomyces cerevisiae.

    OpenAIRE

    Yoo, J. S.; Cheong, H. K.; Lee, B.J.; Kim, Y. B.; Cheong, C

    2001-01-01

    The 20-nucleotide SL1 VBS RNA, 5'-GGAGACGC[GAUUC]GCGCUCC (bulged A underlined and loop bases in brackets), plays a crucial role in viral particle binding to the plus strand and packaging of the RNA. Its structure was determined by NMR spectroscopy. Structure calculations gave a precisely defined structure, with an average pairwise root mean square deviation (RMSD) of 1.28 A for the entire molecule, 0.57 A for the loop region (C8-G14), and 0.46 A for the bulge region (G4-G7, C15-C17). Base sta...

  18. The cellular TAR RNA binding protein, TRBP, promotes HIV-1 replication primarily by inhibiting the activation of double-stranded RNA-dependent kinase PKR.

    Science.gov (United States)

    Sanghvi, Viraj R; Steel, Laura F

    2011-12-01

    The TAR RNA binding protein, TRBP, is a cellular double-stranded RNA (dsRNA) binding protein that can promote the replication of HIV-1 through interactions with the viral TAR element as well as with cellular proteins that affect the efficiency of translation of viral transcripts. The structured TAR element, present on all viral transcripts, can impede efficient translation either by sterically blocking access of translation initiation factors to the 5'-cap or by activating the dsRNA-dependent kinase, PKR. Several mechanisms by which TRBP can facilitate translation of viral transcripts have been proposed, including the binding and unwinding of TAR and the suppression of PKR activation. Further, TRBP has been identified as a cofactor of Dicer in the processing of microRNAs (miRNAs), and sequestration of TRBP by TAR in infected cells has been proposed as a viral countermeasure to potential host cell RNA interference-based antiviral activities. Here, we have addressed the relative importance of these various roles for TRBP in HIV-1 replication. Using Jurkat T cells, primary human CD4(+) T cells, and additional cultured cell lines, we show that depletion of TRBP has no effect on viral replication when PKR activation is otherwise blocked. Moreover, the presence of TAR-containing mRNAs does not affect the efficacy of cellular miRNA silencing pathways. These results establish that TRBP, when expressed at physiological levels, promotes HIV-1 replication mainly by suppressing the PKR-mediated antiviral response, while its contribution to HIV-1 replication through PKR-independent pathways is minimal. PMID:21937648

  19. 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). PMID:27387642

  20. 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.

  1. The involvement of clathrin-mediated endocytosis and two Sid-1-like transmembrane proteins in double-stranded RNA uptake in the Colorado potato beetle midgut.

    Science.gov (United States)

    Cappelle, K; de Oliveira, C F R; Van Eynde, B; Christiaens, O; Smagghe, G

    2016-06-01

    RNA interference (RNAi) is a powerful tool in entomology and shows promise as a crop protection strategy, but variability in its efficiency across different insect species limits its applicability. For oral uptake of the double-stranded RNA (dsRNA), the RNAi trigger, two different mechanisms are known: systemic RNA interference deficient-1 (Sid-1) transmembrane channel-mediated uptake and clathrin-mediated endocytosis. So far, a wide range of experiments has been conducted, confirming the involvement of one of the pathways in dsRNA uptake, but never both pathways in the same species. We investigated the role of both pathways in dsRNA uptake in the Colorado potato beetle, Leptinotarsa decemlineata, known to have an efficient RNAi response. Through RNAi-of-RNAi experiments, we demonstrated the contribution of two different sid-1-like (sil) genes, silA and silC, and clathrin heavy chain and the 16kDa subunit of the vacuolar H(+) ATPase (vha16), elements of the endocytic pathway, to the RNAi response. Furthermore, the sid-1-like genes were examined through phylogenetic and hydrophobicity analysis. This article reports for the first time on the involvement of two pathways in dsRNA uptake in an insect species and stresses the importance of evaluating both pathways through a well-devised reporter system in any future experiments on cellular dsRNA uptake. PMID:26959524

  2. A Transformed Bacterium Expressing Double-Stranded RNA Specific to Integrin β1 Enhances Bt Toxin Efficacy against a Polyphagous Insect Pest, Spodoptera exigua.

    Directory of Open Access Journals (Sweden)

    Eunseong Kim

    Full Text Available Oral toxicity of double-stranded RNA (dsRNA specific to integrin β1 subunit (SeINT was known in a polyphagous insect pest, Spodoptera exigua. For an application of the dsRNA to control the insect pest, this study prepared a transformed Escherichia coli expressing dsRNA specific to SeINT.The dsRNA expression was driven by T7 RNA polymerase overexpressed by an inducer in the transformed E. coli. The produced dsRNA amount was proportional to the number of the cultured bacteria. The transformed bacteria gave a significant oral toxicity to S. exigua larvae with a significant reduction of the SeINT expression. The resulting insect mortality increased with the fed number of the bacteria. Pretreatment with an ultra-sonication to disrupt bacterial cell wall/membrane significantly increased the insecticidal activity of the transformed bacteria. The larvae treated with the transformed bacteria suffered tissue damage in the midgut epithelium, which exhibited a marked loss of cell-cell contacts and underwent a remarkable cell death. Moreover, these treated larvae became significantly susceptible to a Cry toxin derived from Bacillus thuringiensis (Bt.This study provides a novel and highly efficient application technique to use dsRNA specific to an integrin gene by mixing with a biopesticide, Bt.

  3. Triplex-Forming Peptide Nucleic Acid Probe Having Thiazole Orange as a Base Surrogate for Fluorescence Sensing of Double-stranded RNA.

    Science.gov (United States)

    Sato, Takaya; Sato, Yusuke; Nishizawa, Seiichi

    2016-08-01

    We have developed a new fluorescent sensing probe for double-stranded RNA (dsRNA) by integrating thiazole orange (TO) as a base surrogate into triplex-forming PNA. Our probe forms the thermally stable triplex with the target dsRNA at acidic pH; and the triplex formation is accompanied by the remarkable light-up response of the TO unit. The binding of our probe to the target dsRNA proceeds very rapidly, allowing real-time monitoring of the triplex formation. Importantly, we found the TO base surrogate in our probe functions as a universal base for the base pair opposite the TO unit in the triplex formation. Furthermore, the TO unit is significantly more responsive for the fully matched dsRNA sequence compared to the mismatch-containing sequences, which enables the analysis of the target dsRNA sequence at the single-base pair resolution. The binding and sensing functions of our probe are described for the development of fluorescent probes applicable to sensing biologically relevant dsRNA. PMID:27442229

  4. 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.

  5. Characterization of virus/double-stranded RNA-dependent induction of antimicrobial peptide hepcidin in trout macrophages

    Science.gov (United States)

    Hepcidin is an antimicrobial peptide responsive to bacterial infection. We report the characterization of a virus/doublestranded RNA (dsRNA) induction of hepcidin in rainbow trout (Oncorhynchus mykiss). Increased level of hepcidin mRNA was observed in trout macrophage RTS11 cells treated with poly...

  6. Identification of NH…N hydrogen bonds by magic angle spinning solid state NMR in a double-stranded RNA associated with myotonic dystrophy

    Science.gov (United States)

    Leppert, Jörg; Urbinati, Carl R.; Häfner, Sabine; Ohlenschläger, Oliver; Swanson, Maurice S.; Görlach, Matthias; Ramachandran, Ramadurai

    2004-01-01

    RNA plays a central role in biological processes and exhibits a variety of secondary and tertiary structural features that are often stabilized via hydrogen bonds. The distance between the donor and acceptor nitrogen nuclei involved in NH…N hydrogen bonds in nucleic acid base pairs is typically in the range of 2.6–2.9 Å. Here, we show for the first time that such spatial proximity between 15N nitrogen nuclei can be conveniently monitored via magic angle spinning solid state NMR on a uniformly 15N-labelled RNA. The presence of NH…N hydrogen bonds is reflected as cross-peaks between the donor and acceptor nitrogen nuclei in 2D 15N dipolar chemical shift correlation spectra. The RNA selected for this experimental study was a CUG repeat expansion implicated in the neuromuscular disease myotonic dystrophy. The results presented provide direct evidence that the CUG repeat expansion adopts a double-stranded conformation. PMID:14973225

  7. 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.

  8. TRIP: a novel double stranded RNA binding protein which interacts with the leucine rich repeat of flightless I.

    OpenAIRE

    Wilson, S A; Brown, E C; Kingsman, A J; Kingsman, S M

    1998-01-01

    A northwestern screen of a CHO-K1 cell line cDNA library with radiolabelled HIV-1 TAR RNA identified a novel TAR RNA interacting protein, TRIP. The human trip cDNA was also cloned and its expression is induced by phorbol esters. The N-terminus of TRIP shows high homology to the coiled coil domain of FLAP, a protein which binds the leucine-rich repeat (LRR) of Flightless I (FLI) and the interaction of TRIP with the FLI LRR has been confirmed in vitro . TRIP does not bind single stranded DNA or...

  9. Stability and extractability of double-stranded RNA of pangola stunt and sugarcane Fiji disease viruses in dried plant tissues.

    Science.gov (United States)

    Karan, M; Hicks, S; Harding, R M; Teakle, D S

    1991-06-01

    When leaves infected with pangola stunt virus (PaSV) were dried at 23, 37, 50, 70 or 105 degrees C, the dsRNA was stable and could be extracted after aerobic storage at room temperature for 1 month, although at 105 degrees C the amount obtained was reduced. The dsRNA was also recovered after leaves were freeze dried and stored in vacuo at room temperature for 6 months, or were dried and stored aerobically at room temperature for 10.5 months. dsRNA of sugarcane Fiji disease virus (FDV) was also stable when infected leaves were dried at 23, 37, 50 or 105 degrees C and stored aerobically for 3 months or for at least 6 months when infected leaves were dried at 70 degrees C. The unexpected high stability and extractability of both PaSV and FDV dsRNA when dried in leaves at low or high temperatures and stored at room temperature indicate that these, and probably other plant-infecting reoviruses, can be transported readily in desiccated host tissue between different countries for later extraction and comparison of their dsRNAs. PMID:1939508

  10. Conserved Surface Features Form the Double-stranded RNA Binding Site of Non-structural Protein 1 (NS1) from Influenza A and B Viruses

    Energy Technology Data Exchange (ETDEWEB)

    Yin,C.; Khan, J.; Swapna, G.; Ertekin, A.; Krug, R.; Tong, L.; Montelione, G.

    2007-01-01

    Influenza A viruses cause a highly contagious respiratory disease in humans and are responsible for periodic widespread epidemics with high mortality rates. The influenza A virus NS1 protein (NS1A) plays a key role in countering host antiviral defense and in virulence. The 73-residue N-terminal domain of NS1A (NS1A-(1-73)) forms a symmetric homodimer with a unique six-helical chain fold. It binds canonical A-form double-stranded RNA (dsRNA). Mutational inactivation of this dsRNA binding activity of NS1A highly attenuates virus replication. Here, we have characterized the unique structural features of the dsRNA binding surface of NS1A-(1-73) using NMR methods and describe the 2.1-{angstrom} x-ray crystal structure of the corresponding dsRNA binding domain from human influenza B virus NS1B-(15-93). These results identify conserved dsRNA binding surfaces on both NS1A-(1-73) and NS1B-(15-93) that are very different from those indicated in earlier 'working models' of the complex between dsRNA and NS1A-(1-73). The combined NMR and crystallographic data reveal highly conserved surface tracks of basic and hydrophilic residues that interact with dsRNA. These tracks are structurally complementary to the polyphosphate backbone conformation of A-form dsRNA and run at an {approx}45{sup o} angle relative to the axes of helices {alpha}2/{alpha}2'. At the center of this dsRNA binding epitope, and common to NS1 proteins from influenza A and B viruses, is a deep pocket that includes both hydrophilic and hydrophobic amino acids. This pocket provides a target on the surface of the NS1 protein that is potentially suitable for the development of antiviral drugs targeting both influenza A and B viruses.

  11. 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.

  12. Double-stranded RNA-dependent protein kinase is required for bone calcification in MC3T3-E1 cells in vitro.

    Science.gov (United States)

    Yoshida, Kaya; Okamura, Hirohiko; Amorim, Bruna Rabelo; Ozaki, Akiko; Tanaka, Hiroaki; Morimoto, Hiroyuki; Haneji, Tatsuji

    2005-11-15

    In this study, we demonstrated that double-stranded RNA-dependent protein kinase (PKR) is required for the calcification of osteoblasts via the signal transducers and activators of transcription 1alpha (STAT1alpha) signaling in vitro. A dominant-negative mutant PKR cDNA, in which the amino acid lysine at 296 was replaced with arginine and which does not have catalytic activity, was transfected into mouse osteoblastic MC3T3-E1 cells; thereby, we established cells that stably expressed the PKR mutant gene (PKR-K/R). Phosphorylation of PKR was not stimulated by polyinosic-polycytidylic acid in the mutant cells. The PKR-K/R mutant cells exhibited up-regulated cell growth and had low alkaline phosphatase (ALP) activity. The PKR-K/R mutant cells were not able to form bone nodules in vitro. In the PKR-K/R mutant cells, runt-related gene 2 (Runx2)-mediated transcription decreased compared with the levels in the control cells. The expression of STAT1alpha protein increased and the protein was translocated to the nucleus in the PKR-K/R mutant cells. When the expression of STAT1alpha protein in PKR mutant cells was suppressed using RNAi, the activity of Runx2-mediated transcription recovered to the control level. Our results indicate that PKR is a stimulator of Runx2 transcription and is a negative modulator of STAT1alpha expression. Our findings also suggest that PKR plays important roles in the differentiation and calcification of osteoblasts by modulating STAT1alpha and/or Runx2 expression. PMID:16216244

  13. Plant-feeding insects harbor double-stranded RNA viruses encoding a novel proline-alanine rich protein and a polymerase distantly related to that of fungal viruses

    Science.gov (United States)

    Novel double-stranded RNAs (~8 kbp) were isolated from three cornered alfalfa hopper (Spissistilus festinus) and beet leafhopper (Circulifer tenellus), two plant-feeding hemipteran insect pests. Genomes of the two new viruses, designated as Spissistilus festinus virus 1 (SpFV1) and Circulifer tenell...

  14. Novel double-stranded RNA viruses of plant-feeding insects encode a serine-alanine-proline rich protein and a polymerase distantly related to fungal viruses

    Science.gov (United States)

    Novel double stranded RNAs (~8 kbp) were isolated from the three cornered alfalfa hopper (Spissistilus festinus) and beet leafhopper (Circulifer tenellus), two plant-feeding hemipteran insect pests. Genome organization of the two new viruses, designated as Spissistilus festinus virus 1 (SpFV1) and ...

  15. 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

  16. Recognition of double-stranded DNA using energetically activated duplexes with interstrand zippers of 1-, 2-or 4-pyrenyl-functionalized O2 '-alkylated RNA monomers

    DEFF Research Database (Denmark)

    Karmakar, Saswata; Madsen, Andreas Stahl; Guenther, Dale C.;

    2014-01-01

    '-alkylated uridine monomers X-Z by means of thermal denaturation experiments, optical spectroscopy, force-field simulations and recognition experiments using DNA hairpins as model targets. We demonstrate that Invaders with +1 interstrand zippers of X or Y monomers efficiently recognize mixed-sequence DNA...... hairpins with single nucleotide fidelity. Intercalator-mediated unwinding and activation of the double-stranded probe, coupled with extraordinary stabilization of probe target duplexes (Delta T-m/modification up to +14.0 degrees C), provides the driving force for dsDNA recognition. In contrast, Z...

  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 to...... displacement of one strand of the DNA locally by the PNA hybridization....

  18. Double-stranded RNA interferes in a sequence-specific manner with the infection of representative members of the two viroid families

    International Nuclear Information System (INIS)

    Infection by viroids, non-protein-coding circular RNAs, occurs with the accumulation of 21-24 nt viroid-derived small RNAs (vd-sRNAs) with characteristic properties of small interfering RNAs (siRNAs) associated to RNA silencing. The vd-sRNAs most likely derive from dicer-like (DCL) enzymes acting on viroid-specific dsRNA, the key elicitor of RNA silencing, or on the highly structured genomic RNA. Previously, viral dsRNAs delivered mechanically or agroinoculated have been shown to interfere with virus infection in a sequence-specific manner. Here, we report similar results with members of the two families of nuclear- and chloroplast-replicating viroids. Moreover, homologous vd-sRNAs co-delivered mechanically also interfered with one of the viroids examined. The interference was sequence-specific, temperature-dependent and, in some cases, also dependent on the dose of the co-inoculated dsRNA or vd-sRNAs. The sequence-specific nature of these effects suggests the involvement of the RNA induced silencing complex (RISC), which provides sequence specificity to RNA silencing machinery. Therefore, viroid titer in natural infections might be regulated by the concerted action of DCL and RISC. Viroids could have evolved their secondary structure as a compromise between resistance to DCL and RISC, which act preferentially against RNAs with compact and relaxed secondary structures, respectively. In addition, compartmentation, association with proteins or active replication might also help viroids to elude their host RNA silencing machinery

  19. Identification and characterization of a double-stranded RNA- reovirus temperature-sensitive mutant defective in minor core protein mu2.

    OpenAIRE

    Coombs, K M

    1996-01-01

    A newly identified temperature-sensitive mutant whose defect was mapped to the reovirus M1 gene (minor core protein mu2) was studied to better understand the functions of this virion protein. Sequence determination of the Ml gene of this mutant (tsH11.2) revealed a predicted methionine-to-threonine alteration at amino acid 399 and a change from proline to histidine at amino acid 414. The mutant made normal amounts of single-stranded RNA, both in in vitro transcriptase assays and in infected c...

  20. 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.

  1. 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.......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....

  2. Synergistic effect of combined double-stranded RNA and imiquimod stimulation%双链RNA和咪喹莫特联合刺激的协同作用研究

    Institute of Scientific and Technical Information of China (English)

    李萃; 王慧琪; 王文静; 狄静芳; 曾山; 林羿

    2009-01-01

    目的 研究双链RNA(double-stranded RNA,dsRNA)和咪喹莫特联合刺激是否具有协同效应.方法 在Toll样受体3(TLR3)的特异性刺激剂dsRNA、TLR7的特异性刺激剂咪唪莫特(R837)单独刺激或二者联合刺激条件下,检测BALB/c×C57BL/6和NH细胞缺陷的非肥胖型糖尿病(NOD)×C57BL/6小鼠胚胎吸收率.采用小鼠体内注射上述刺激剂的方法 ,流式细胞术检测子宫CD45+细胞内细胞因子表达水平.为进一步鉴定CD45+细胞身份,在体外培养系统中采用dsRNA和咪喹莫特刺激胎盘和底蜕膜来源的子官CD3+T细胞和CD49b+NK细胞,并检测细胞内细胞因子表达水平.采用丝裂原激活的蛋白激酶(mitogen-activated protein kinase,MAPK)抑制剂SP600125和PD98059阻断细胞因子表达水平的增加.结果 此 dsRNA和咪喹莫特联合刺激对胚胎吸收率的增高具有协同作用,同时对CD45+细胞内TNF-α和IFN-γ的表达增强具有协同刺激作用.进一步细胞鉴定研究显示,虽然在BALB/c小鼠CD3+细胞和CD49b+NK细胞中均可发现这种协同效应,但是在NOD小鼠,这种细胞因子水平的增高应主要归因于CD3+T细胞,因为在CD49b+NK细胞不显示这种细胞因子增高趋势.上述刺激剂合用的协同效应町部分地被JNK(Jun N-terminal kinase)MAPK抑制剂SP600125阻断,而几乎被ERK(extracellular signal.regulated kinase)MAPK抑制剂PD98059所完全阻断.结论 增强的TLR3和TLR7联合信号可能是通过NOD小鼠Th1型T细胞,而不是NK细胞所传导.ERK MAPK途径可能在TLR3和TLR7参与的细胞信息传递过程中发挥关键性作用.%Objective To investigate the effect of combined double-stranded RNA (dsRNA) and imiquimod stimulation on uterine immune cells. Methods In BALB/c × C57BL/6 mice and non-obese dia-betic (NOD) × C57BI/6 mice, embryo resorption rate was detected in the presence or absence of Toll-like receptor 3 (TLR3) agonist dsRNA [poly( 1: C)], TLR7 agonist imiquimod ( R837), or their

  3. Fragmentation in DNA double-strand breaks

    International Nuclear Information System (INIS)

    DNA double strand breaks are important lesions induced by irradiations. Random breakage model or quantification supported by this concept is suitable to analyze DNA double strand break data induced by low LET radiation, but deviation from random breakage model is more evident in high LET radiation data analysis. In this work we develop a new method, statistical fragmentation model, to analyze the fragmentation process of DNA double strand breaks. After charged particles enter the biological cell, they produce ionizations along their tracks, and transfer their energies to the cells and break the cellular DNA strands into fragments. The probable distribution of the fragments is obtained under the condition in which the entropy is maximum. Under the approximation E≅E0 + E1l + E2l2, the distribution functions are obtained as exp(αl + βl2). There are two components, the one proportional to exp(βl2), mainly contributes to the low mass fragment yields, the other component, proportional to exp(αl), decreases slowly as the mass of the fragments increases. Numerical solution of the constraint equations provides parameters α and β. Experimental data, especially when the energy deposition is higher, support the statistical fragmentation model. (authors)

  4. Primer-dependent and primer-independent initiation of double stranded RNA synthesis by purified arabidopsis RNA-dependent RNA polymerases RDR2 and RDR6

    DEFF Research Database (Denmark)

    Devert, Anthony; Fabre, Nicolas; Floris, Maina Huguette Joséphine;

    2015-01-01

    RNA) 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...

  5. DNA double strand break induction in yeast

    International Nuclear Information System (INIS)

    The induction of DNA double strand breaks (DSBs) by accelerated heavy ions was systematically measured in diploid yeast cells. Particles were provided by the accelerators at GSI, Darmstadt, and HMI, Berlin. DNA was separated using pulsed field gel electrophoresis and the intensity of the largest bands used to determine the loss of molecular weight. Since the DNA content of each chromosome is exactly known absolute values for DSB induction can be measured without calibration procedures. Ions used range from protons to uranium with LET values between 2 and about 15,000 keV.μm-1. Induction cross sections increase in the lower LET region approaching a plateau around 200 keV.μm-1. With higher LET values the dependence can no longer be described by a common curve with each ion showing a specific behaviour. With very heavy particles the influence of the penumbra becomes obvious: cross sections decrease with LET because of the reduced penumbra extensions. Classical target theory would predict cross sections to follow a simple saturation function which is not substantiated by the data. Track structure analysis as introduced by Butts and Katz in Radiat. Res. 30 855-71 1967 is also not able to predict the experimental results. A semi-empirical fit indicates a linear-quadratic dependence of induction cross sections on LET up to about 1000 keV.μm-1. RBE for DSB induction rises above unity reaching a maximum of about 2.5 around 200 keV.μm-1. This is different from many experiments in mammalian cells and is presumably due to differences in chromatin structure since yeast cells seem to lack a functional H1 histone. (author)

  6. 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.

  7. 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

  8. DNA double-strand break signaling and human disorders

    Directory of Open Access Journals (Sweden)

    Bohgaki Toshiyuki

    2010-11-01

    Full Text Available Abstract DNA double-strand breaks are among the most serious types of DNA damage and their signaling and repair is critical for all cells and organisms. The repair of both induced and programmed DNA breaks is fundamental as demonstrated by the many human syndromes, neurodegenerative diseases, immunodeficiency and cancer associated with defective repair of these DNA lesions. Homologous recombination and non-homologous end-joining pathways are the two major DNA repair pathways responsible for mediating the repair of DNA double-strand breaks. The signaling of DNA double-strand breaks is critical for cells to orchestrate the repair pathways and maintain genomic integrity. This signaling network is highly regulated and involves a growing number of proteins and elaborated posttranslational modifications including phosphorylation and ubiquitylation. Here, we highlight the recent progress in the signaling of DNA double-strand breaks, the major proteins and posttranslational modifications involved and the diseases and syndromes associated with impaired signaling of these breaks.

  9. Repair of double-strand breaks in Micrococcus radiodurans

    International Nuclear Information System (INIS)

    Micrococcus radiodurans has been shown to sustain double-strand breaks in its DNA after exposure to x-radiation. Following sublethal doses of x-rays (200 krad in oxygen or less), the cells were able to repair these breaks, and an intermediate fast-sedimenting DNA component seemed to be involved in the repair process

  10. Dysfunctional mammalian telomeres join to double-strand breaks

    International Nuclear Information System (INIS)

    Telomeres are highly specialized nucleoprotein structures that maintain genomic stability by protecting the ends of linear chromosomes, an essential function inferred cytogenetically from the end-to-end fusions that result when telomeric end-capping fails. In striking contrast to natural chromosomal termini, broken chromosome ends produced by DNA double strand breaks (DSB) are highly recombinogenic, and represent a major threat to the integrity of the cell's genome due to their potential for causing chromosomal rearrangements that contribute to genomic instability and tumorigenesis. Previously, we demonstrated that effective end-capping of mammalian telomeres has a seemingly paradoxical requirement for proteins more commonly associated with DNA DSB repair. Ku70, Ku80, and DNA-PKcs all participate in DSB repair through non-homologous end-joining. Somewhat surprisingly, mutations in any of these genes cause spontaneous chromosomal end-to-end fusions that maintain large blocks of telomeric sequence at the points of fusion and are not a consequence of telomere shortening. We have also shown that nascent telomeres produced via leading-strand DNA synthesis are especially susceptible to these end-to-end fusions. Here we report that impaired end-capping in DNA-PKcs-deficient backgrounds not only allows these dysfunctional telomeres to join to one another, but also to broken chromosome ends created by radiation-induced DSB. Mouse cell lines were exposed to graded doses of gamma-rays and examined utilizing the strand-specific fluorescence in situ hybridization technique of CO-FISH, in order to distinguish true telomere-to-DSB events from telomere-to-telomere fusions. Telomere-to-DSB fusions were observed in a dose-dependent fashion in each mutant cell line analyzed, including BALB/c, which is both radiosensitive and susceptible to radiogenic mammary cancer. Telomere-DSB fusion represents a new and sensitive method of detecting telomere dysfunction as measured by failure of

  11. Electrodeless dielectrophoresis of single- and double-stranded DNA.

    OpenAIRE

    Chou, Chia-Fu; Tegenfeldt, Jonas O.; Bakajin, Olgica; Chan, Shirley S; Cox, Edward C; Darnton, Nicholas; Duke, Thomas; Austin, Robert H.

    2002-01-01

    Dielectrophoretic trapping of molecules is typically carried out using metal electrodes to provide high field gradients. In this paper we demonstrate dielectrophoretic trapping using insulating constrictions at far lower frequencies than are feasible with metallic trapping structures because of water electrolysis. We demonstrate that electrodeless dielectrophoresis (EDEP) can be used for concentration and patterning of both single-strand and double-strand DNA. A possible mechanism for DNA pol...

  12. 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.

  13. Double-Stranded Water on Stepped Platinum Surfaces

    Science.gov (United States)

    Kolb, Manuel J.; Farber, Rachael G.; Derouin, Jonathan; Badan, Cansin; Calle-Vallejo, Federico; Juurlink, Ludo B. F.; Killelea, Daniel R.; Koper, Marc T. M.

    2016-04-01

    The interaction of platinum with water plays a key role in (electro)catalysis. Herein, we describe a combined theoretical and experimental study that resolves the preferred adsorption structure of water wetting the Pt(111)-step type with adjacent (111) terraces. Double stranded lines wet the step edge forming water tetragons with dissimilar hydrogen bonds within and between the lines. Our results qualitatively explain experimental observations of water desorption and impact our thinking of solvation at the Pt electrochemical interface.

  14. Deficiency of DNA double-strand break repair and enhanced radiosensitivity in Tip60 silenced cells

    International Nuclear Information System (INIS)

    Objective: To investigate the effect of Tip60 on the cellular radiosensitivity,and to explore the related mechanism. Methods: siRNA and anacardic acid (AA, an inhibitor of Tip60 acetyltransferase) were used to inhibit Tip60 expression and its acetyltransferase activity, respectively. Radiosensitivity was analyzed by colony-forming ability assay. γ-H2AX foci were detected to analyze the DNA double-strand break (DSB). Immunoprecipitation was used to determine the interaction of proteins. Results: siRNA-mediated silencing of Tip60 led to enhanced sensitivity of U2OS cells at 1, 2 Gy after γ-ray irradiation, but had no significant effect at 4 Gy post-irradiation (t=3.364, 3.979, P<0.05).γ-H2AX foci detection indicated that Tip60 silencing resulted in a decreased capability of DNA double-strand break repair at 1, 4 and 8 h after irradiation (t=3.875, 3.183 and 3.175, respectively, P<0.05). The interaction of Tip60 and DNA-PKcs was prompted by ionizing radiation. Anacardic acid largely abrogated the phosphorylation of DNA-PKcs at T2609 site induced by irradiation. Conclusions: Tip60 plays a role in the cellular response to ionizing radiation-induced DNA damage through, at least in part, interacting with DNA-PKcs and regulating its phosphorylation. (authors)

  15. DNA double strand break repair pathway choice following ionizing radiation

    International Nuclear Information System (INIS)

    A DNA double strand break (DSB) is one of the critical DNA lesions leading to cell death if unrepaired. DSB is repaired by two distinct repair pathways, i.e. non-homologous end-joining (NHEJ) or homologous recombination (HR). NHEJ contributes to DSB repair throughout the cell cycle, while HR is active during S/G2 phase following DNA replication. We aim to elucidate the molecular mechanisms underlying DSB repair pathway choice at two ended DSBs in G2 phase following ionizing radiation (IR). Here, we discuss recent work that provides new insights into DSB repair pathways choice including our study. (author)

  16. Activation-induced cytidine deaminase acts on double-strand breaks in vitro.

    Science.gov (United States)

    Shen, Hong Ming

    2007-02-01

    Activation-induced cytidine deaminase (AID) is likely responsible for DNA cytidine deamination, although it may also act as an RNA deaminase. It functions on single-stranded DNA, the non-template strand in double-stranded DNA during transcription, or both strands in supercoiled DNA. To ask whether AID is able to deaminate cytidine at DNA breaks, plasmids, containing a SnaBI site (TAC downward arrowGTA) that forms blunt ends after digestion with SnaBI, were generated. If AID deaminates cytidine at the upstream blunt end, the ATG start codon in either of two drug resistance genes will be regenerated after ligation and replication in UDG-null E. coli cells. This study shows that AID targets cytidine at the break. The extent of deamination activity beyond the break is correlated with the base composition in the break region. If the break region is A, T-rich, C > T transitions are extensive. However, when the break region is not A, T-rich, mutations are mainly restricted to the break, similar to findings in vivo. The results indicate that AID has activity on double strand breaks (DSBs). Based on previous and current findings, a somatic hypermutation (SHM) model is proposed, in which collision between the transcription apparatus and the replication fork generates DSBs. After AID acts on break ends, the error-prone DNA repair machinery fixes and creates mutations. PMID:16697045

  17. Mitochondrial DNA repairs double-strand breaks in yeast chromosomes.

    Science.gov (United States)

    Ricchetti, M; Fairhead, C; Dujon, B

    1999-11-01

    The endosymbiotic theory for the origin of eukaryotic cells proposes that genetic information can be transferred from mitochondria to the nucleus of a cell, and genes that are probably of mitochondrial origin have been found in nuclear chromosomes. Occasionally, short or rearranged sequences homologous to mitochondrial DNA are seen in the chromosomes of different organisms including yeast, plants and humans. Here we report a mechanism by which fragments of mitochondrial DNA, in single or tandem array, are transferred to yeast chromosomes under natural conditions during the repair of double-strand breaks in haploid mitotic cells. These repair insertions originate from noncontiguous regions of the mitochondrial genome. Our analysis of the Saccharomyces cerevisiae mitochondrial genome indicates that the yeast nuclear genome does indeed contain several short sequences of mitochondrial origin which are similar in size and composition to those that repair double-strand breaks. These sequences are located predominantly in non-coding regions of the chromosomes, frequently in the vicinity of retrotransposon long terminal repeats, and appear as recent integration events. Thus, colonization of the yeast genome by mitochondrial DNA is an ongoing process. PMID:10573425

  18. 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...... substantial efforts are currently devoted to the development of alternative strategies that overcome the limitations observed with the classic approaches. In 2005, we introduced Invader locked nucleic acids (LNAs), i.e., double-stranded probes that are activated for mixed-sequence recognition of dsDNA through...... monomers. We compare the thermal denaturation characteristics of double-stranded probes featuring different interstrand zippers of pyrene-functionalized monomers based on 2'-amino-α-l-LNA, 2'-N-methyl-2'-amino-DNA, and RNA scaffolds. Insights from fluorescence spectroscopy, molecular modeling, and NMR...

  19. X-radiation induced double-strand DNA breaks in rat bone marrow cells

    International Nuclear Information System (INIS)

    The method of sedimentation in a neutral sucrose gradient was used to study y doublestranded dna in a total population of rat bone marrow cells. As a resul of cell lysis in neutral conditions the fragments of double-stranded dna were fo ormed having the molecular mass of (3+-0.3)x109D. A study was made of the dynamics of accumulation of dna double-strand breaks after irradiation of a cell l suspension. It was shown that the yield of double-strand breaks and ratio between single- and double-strand breaks in bone marrow cells were similar to th hose of cultured L5178Y cells

  20. Do DNA Double-Strand Breaks Drive Aging?

    Science.gov (United States)

    White, Ryan R; Vijg, Jan

    2016-09-01

    DNA double-strand breaks (DSBs) are rare, but highly toxic, lesions requiring orchestrated and conserved machinery to prevent adverse consequences, such as cell death and cancer-causing genome structural mutations. DSBs trigger the DNA damage response (DDR) that directs a cell to repair the break, undergo apoptosis, or become senescent. There is increasing evidence that the various endpoints of DSB processing by different cells and tissues are part of the aging phenotype, with each stage of the DDR associated with specific aging pathologies. In this Perspective, we discuss the possibility that DSBs are major drivers of intrinsic aging, highlighting the dynamics of spontaneous DSBs in relation to aging, the distinct age-related pathologies induced by DSBs, and the segmental progeroid phenotypes in humans and mice with genetic defects in DSB repair. A model is presented as to how DSBs could drive some of the basic mechanisms underlying age-related functional decline and death. PMID:27588601

  1. Relationships between DNA double-strand breaks and chromosomal aberrations

    International Nuclear Information System (INIS)

    Evidence suggests that double strand breaks are induced linearly with radiation dose at frequencies of 30-40 DSB/cell/Gy. It seems possible that there is a fast component not normally related to the induction of chromosomal aberrations, and a second slower component underlying the observed joining of chromosome and chromatid breaks. Radiation induces a mixture of blunt and cohesive-ended DSB probably with a preponderance of the latter which are much less effective at inducing aberrations. Visible chromatid breaks are also induced linearly with dose at much lower frequency than DSB and rejoin with a half-time reminiscent of slowly repairing DSB. It is possible that this slow rejoining reflects underlying repair of biologically important DSB. Rejoining of chromatid breaks and misjoining giving rise to exchanges are thought to be determined by different mechanisms. (UK)

  2. Radiation induced DNA double-strand breaks in radiology

    International Nuclear Information System (INIS)

    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. Evidence for fully conjugated double-stranded cycles.

    Science.gov (United States)

    Standera, Malte; Häfliger, Rolf; Gershoni-Poranne, Renana; Stanger, Amnon; Jeschke, Gunnar; van Beek, Jacco D; Bertschi, Louis; Schlüter, A Dieter

    2011-10-17

    A chain of circumstantial evidence for the existence of the first fully conjugated, double-stranded cycles is presented. The products have the structure of the belt-region of fullerene C(84)(D(2)) and carry either four hexyl chains or four phenyl groups. The unsubstituted parent cycle is also presented. The chain of evidence is mainly based on mass spectrometric analysis and trapping reactions, the latter being supported by quantum mechanical calculations. It is also of importance that the phenyl-substituted and unsubstituted products cannot undergo a [1,5] hydrogen shift, the only reasonable side-reaction that recently could not be excluded for the alkyl-substituted analogue. It is concluded that the fully aromatic targets truly exist in the gas phase. Whether they can be generated in solution under the applied conditions cannot yet be firmly decided; theoretical evidence speaks against. PMID:21915923

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

    Science.gov (United States)

    Morales, Maria E.; Derbes, Rebecca S.; Ade, Catherine M.; Ortego, Jonathan C.; Stark, Jeremy; Deininger, Prescott L.; Roy-Engel, Astrid M.

    2016-01-01

    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. PMID:26966913

  5. Altered radiation recovery by DNA double-strand break inducers

    International Nuclear Information System (INIS)

    Identical biphasic time-dependent profiles of cell survival were obtained in V79 fibroblasts exposed to a split-dose protocol consisting of a fixed dose of γ-rays followed, at a variable time interval, either by a second exposure to radiation, or by contact with an equi-toxic amount of antitumor drugs acting to produce DNA double-strand breaks. The drugs used in this context were the neocarcinostatin antibiotic (NCS), which preferentially cleaves DNA in the linker region of nucleosomes, and etoposide (VP), whose major target is topoisomerase IIα, a nuclear matrix fraction-linked enzyme acting to relieve topological constraints in replicating DNA and mitotic chromosomes. Radiation-induced DNA strand break rejoining was not inhibited by either drug. The initial number of DNA strand breaks was consistently found o depend only on the radiation dose and/or on the drug concentration. However, the cytotoxicity they induced in combined treatment was determined in essence by the time elapsed after the first radiation exposure. While resistance to NCS and VP in non-irradiated, synchronized cells peaks in G2 phase of the cell cycle, enhanced drug susceptibility was observed within the radiation-induced G2 block. Concomitant exposure to drug and radiation also resulted in supra-additive cytotoxic interaction. Our data suggest that impaired split-dose radiation recovery dose not proceed from inhibition of DNA damage repair, but rather from additional double-strand breaks produced by drug or radiation during the time cells are in the dynamic process of DNA repair; a time range characterized by a dynamic DNA fragility. (authors)

  6. 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...

  7. Split-dose recovery is due to the repair of DNA double-strand breaks

    International Nuclear Information System (INIS)

    DNA double-strand breaks are the molecular lesions the repair of which leads to the reappearance of the shoulder observed in split-dose experiments. This conclusion is based on results obtained with the help of a diploid yeast mutant rad54-3 which is temperature-conditional for the repair of DNA double-strand breaks. Two repair steps must be met to yield the reappearance of the shoulder on a split-dose survival curve: the repair of double-strand breaks during the interval between two doses and on the nutrient agar plate after the second dose. In yeast lethality may be attributable to either an unrepaired double-strand break (i.e. a double-strand break is a potentially lethal lesion) or to the interaction of two double-strand breaks (misrepair of double-strand breaks). Evidence is presented that the two cellular phenomena of liquid holding recovery (repair of potentially lethal damage) and of split-dose recovery (repair of sublethal damage) are based on the repair of the same molecular lesion, the DNA double-strand break. (author)

  8. Up-regulation of Tumor Suppressor Gene p21WAF1/CIP1 in Human Cells by Small Double Strand RNA%小分子双链RNA对人类细胞中抑癌基因p21表达的上调作用

    Institute of Scientific and Technical Information of China (English)

    胡嘏; 陈忠; 吴嘉; 张勇; 徐华; 杨为民; 叶章群

    2012-01-01

    Objective To screen more human cell lines susceptible to dsP21 322 mediated tumor suppressor gene p21WAF1/CIP1 activation and investigate whether this process is dependent on p53 expression. Methods A small double strand RNA,dsP21 322,targeting the p21 promoter at position 322 relative to the transcription start site was synthesized. A dsCon trol lacking significant homology to all known human sequences was also synthesized and used as a negative control. Four kinds of human cell lines which expressed different types of p53 protein were chosen,including human osteosarcoma cell line U2 OS (p53 wild type) ,human embryonic kidney cell line 293T(p53 wild type) ,human cervical cancer cell line HeLa(p53 mutant type) and human lung cancer cell line NCI H1299(p53 null). All above cell lines were cultured in vitro and transfected with dsControl or dsP21 322 by Entranster R. RT qPCR and Western blot were applied to detect the expression levels of p21 and p53 mRNA and protein, respectively. Results Seventy two h after transfections, dsP21 322 did not affect the expression of p53 in above four kinds of cell lines,but caused a significant induction in p21 mRNA expression in p53 wild type or p53 mutant cell lines. Compared with dsControl transfections,induction of p21mRNA was 3. 97 ,4. 94 ,and 4. 64 fold in U2 OS,HeLa and 293T cell lines, respectively. Western blot revealed that the elevated levels of p21 protein were strongly correlated to the increase in p21 mRNA expression in these three cell lines. The p21 protein level in dsP21 322 transfections was significantly higher than in ds Control transfections(P<0. 05). However,dsP21 322 was unable to elevate the p21 mRNA and protein levels in p53 null NCI H1299 cell line. Conclusion Activation of p21 expression by dsP21 322 in human cell lines is a pervasive phenomenon. Further more,dsP21 322 failed to elevate the p21 expression in p53 null cell,indicating this process might rely on the expression of p53.%目的 筛选更多的对小分子双链RNA

  9. Looping of anisotropic, short double-stranded DNA

    Science.gov (United States)

    Kim, Harold; Le, Tung

    2013-03-01

    Bending of double-stranded DNA (dsDNA) is associated with fundamental biological processes such as genome packaging and gene regulation, and therefore studying sequence-dependent dsDNA bending is a key to understanding biological impact of DNA sequence beyond the genetic code. Average mechanical behavior of long dsDNA is well described by the wormlike chain model, but sequence-dependent anisotropic bendability and bendedness of dsDNA can in principle lead to abnormally high looping probability at short length scales. Here, we measured the looping probability density (J factor) and kinetics of dsDNA as a function of length and curvature using single-molecule FRET (Förster Resonance Energy Transfer). For theoretical comparison, we calculated the J-factor using a discrete dinucleotide chain model, and also simulated it by Monte Carlo methods. We provide evidences that even when the intrinsic shape of dsDNA is accounted for, the wormlike chain model fails to describe looping dynamics of dsDNA below 200-bp length scale. Georgia Tech FIRE program

  10. 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 (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. PMID:23956222

  11. Ku recruits XLF to DNA double-strand breaks.

    Science.gov (United States)

    Yano, Ken-ichi; Morotomi-Yano, Keiko; Wang, Shih-Ya; Uematsu, Naoya; Lee, Kyung-Jong; Asaithamby, Aroumougame; Weterings, Eric; Chen, David J

    2008-01-01

    XRCC4-like factor (XLF)--also known as Cernunnos--has recently been shown to be involved in non-homologous end-joining (NHEJ), which is the main pathway for the repair of DNA double-strand breaks (DSBs) in mammalian cells. XLF is likely to enhance NHEJ by stimulating XRCC4-ligase IV-mediated joining of DSBs. Here, we report mechanistic details of XLF recruitment to DSBs. Live cell imaging combined with laser micro-irradiation showed that XLF is an early responder to DSBs and that Ku is essential for XLF recruitment to DSBs. Biochemical analysis showed that Ku-XLF interaction occurs on DNA and that Ku stimulates XLF binding to DNA. Unexpectedly, XRCC4 is dispensable for XLF recruitment to DSBs, although photobleaching analysis showed that XRCC4 stabilizes the binding of XLF to DSBs. Our observations showed the direct involvement of XLF in the dynamic assembly of the NHEJ machinery and provide mechanistic insights into DSB recognition. PMID:18064046

  12. 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.

  13. Buried territories: heterochromatic response to DNA double-strand breaks.

    Science.gov (United States)

    Feng, Yi-Li; Xiang, Ji-Feng; Kong, Na; Cai, Xiu-Jun; Xie, An-Yong

    2016-07-01

    Cellular response to DNA double-strand breaks (DSBs), the most deleterious type of DNA damage, is highly influenced by higher-order chromatin structure in eukaryotic cells. Compared with euchromatin, the compacted structure of heterochromatin not only protects heterochromatic DNA from damage, but also adds an extra layer of control over the response to DSBs occurring in heterochromatin. One key step in this response is the decondensation of heterochromatin structure. This decondensation process facilitates the DNA damage signaling and promotes proper heterochromatic DSB repair, thus helping to prevent instability of heterochromatic regions of genomes. This review will focus on the functions of the ataxia telangiectasia mutated (ATM) signaling cascade involving ATM, heterochromatin protein 1 (HP1), Krüppel-associated box (KRAB)-associated protein-1 (KAP-1), tat-interacting protein 60 (Tip60), and many other protein factors in DSB-induced decondensation of heterochromatin and subsequent repair of heterochromatic DSBs. As some subsets of DSBs may be repaired in heterochromatin independently of the ATM signaling, a possible repair model is also proposed for ATM-independent repair of these heterochromatic DSBs. PMID:27151295

  14. Double strand breaks in DNA in vivo and in vitro after 60Co-γ-irradiation

    International Nuclear Information System (INIS)

    The questions of what the correlation is between double strand breaks in DNA in the cell and lethal radiation damage and by means of which possible mechanisms DNA double strand breaks could occur were studied. E. coli served as test system. In addition to this the molecular weight of the DNA from irradiated E. coli as a function of the radiation dose under various conditions was measured. This data was compared on the one hand to the survival of the cell and on the other hand to the formation of DNA double strand breaks in an aqueous buffer system, which in its ionic characteristics was similar to cell fluids. (orig./MG)

  15. Double-Strand Breaks from a Radical Commonly Produced by DNA-Damaging Agents

    OpenAIRE

    Taverna Porro, Marisa L.; Greenberg, Marc M.

    2015-01-01

    Double-strand breaks are widely accepted to be the most toxic form of DNA damage. Molecules that produce double-strand breaks via a single chemical event are typically very cytotoxic and far less common than those that form single-strand breaks. It was recently reported that a commonly formed C4′-radical produces double-strand breaks under aerobic conditions. Experiments described herein indicate that a peroxyl radical initiates strand damage on the complementary strand via C4′-hydrogen atom ...

  16. Hairpin DNA Sequences Bound Strongly by Bleomycin Exhibit Enhanced Double-Strand Cleavage

    OpenAIRE

    Roy, Basab; Hecht, Sidney M.

    2014-01-01

    Clinically used bleomycin A5 has been employed in a study of double-strand cleavage of a library of 10 hairpin DNAs originally selected on the basis of their strong binding to bleomycin. Each of the DNAs underwent double-strand cleavage at more than one site, and all of the cleavage sites were within, or in close proximity to, an eight-base-pair region of the duplex that had been randomized to create the original library. A total of 31 double-strand cleavage sites were identified on the 10 DN...

  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. DNA Double-Strand Break Rejoining in Complex Normal Tissues

    International Nuclear Information System (INIS)

    Purpose: The clinical radiation responses of different organs vary widely and likely depend on the intrinsic radiosensitivities of their different cell populations. Double-strand breaks (DSBs) are the most deleterious form of DNA damage induced by ionizing radiation, and the cells' capacity to rejoin radiation-induced DSBs is known to affect their intrinsic radiosensitivity. To date, only little is known about the induction and processing of radiation-induced DSBs in complex normal tissues. Using an in vivo model with repair-proficient mice, the highly sensitive γH2AX immunofluorescence was established to investigate whether differences in DSB rejoining could account for the substantial differences in clinical radiosensitivity observed among normal tissues. Methods and Materials: After whole body irradiation of C57BL/6 mice (0.1, 0.5, 1.0, and 2.0 Gy), the formation and rejoining of DSBs was analyzed by enumerating γH2AX foci in various organs representative of both early-responding (small intestine) and late-responding (lung, brain, heart, kidney) tissues. Results: The linear dose correlation observed in all analyzed tissues indicated that γH2AX immunofluorescence allows for the accurate quantification of DSBs in complex organs. Strikingly, the various normal tissues exhibited identical kinetics for γH2AX foci loss, despite their clearly different clinical radiation responses. Conclusion: The identical kinetics of DSB rejoining measured in different organs suggest that tissue-specific differences in radiation responses are independent of DSB rejoining. This finding emphasizes the fundamental role of DSB repair in maintaining genomic integrity, thereby contributing to cellular viability and functionality and, thus, tissue homeostasis

  19. 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.

  20. Using Triplex-Forming Oligonucleotide Probes for the Reagentless, Electrochemical Detection of Double-Stranded DNA

    OpenAIRE

    Patterson, Adriana; Caprio, Felice; Vallée-Bélisle, Alexis; Moscone, Danila; Plaxco, Kevin W.; Palleschi, Giuseppe; Ricci, Francesco

    2010-01-01

    We report a reagentless, electrochemical sensor for the detection of double-stranded DNA targets that employs triplex-forming oligonucleotides (TFOs) as its recognition element. These sensors are based on redox-tagged TFO probes strongly chemisorbed onto an interrogating gold electrode. Upon the addition of the relevant double-stranded DNA target, the probe forms a rigid triplex structure via reverse Hoogsteen base pairing in the major groove. The formation of the triplex impedes contact betw...

  1. DNA double-strand break repair: a relentless hunt uncovers new prey.

    Science.gov (United States)

    Sekiguchi, JoAnn M; Ferguson, David O

    2006-01-27

    A major pathway for repair of DNA double-strand breaks is nonhomologous end-joining (NHEJ). In this issue of Cell, and report the discovery of a new NHEJ factor called Cernunnos-XLF. Both groups report that this protein is mutated in a rare inherited human syndrome characterized by severe immunodeficiency, developmental delay, and hypersensitivity to agents that cause DNA double-strand breaks. PMID:16439201

  2. Important DNA repair proteins in DNA double-strand break repair pathways

    International Nuclear Information System (INIS)

    DNA double-strand break repair pathway is one of DNA damage repair pathways. DNA repair genes can repair DNA damage, maintain the integrity of the genetic information and inhibit the formation of tumors. There are two mechanisms-non-homologous end joining and homologous recombination to repair DNA double-strand break. In this review, an overview of important repair proteins of non--homologous end joining and homologous recombination pathways was introduced. (authors)

  3. Highly Efficient Gene Suppression by Chemically Modified 27 Nucleotide Double-Stranded RNAs

    Science.gov (United States)

    Kubo, Takanori; Zhelev, Zhivko; Bakalova, Rumiana; Ohba, Hideki

    2008-02-01

    RNA interference (RNAi) technology, described by Fire and Mello in 1998, is a powerful tool for the suppression of gene expression in mammalian cells. RNAi technology has several advantages over other chemical and genetic drugs. However, several problems in RNAi technology, such as cellular delivery, nuclease stability, and side effects, should be solved before applying it in the clinic. In this study, we focused on the development of novel chemically modified 27 nucleotide (nt) double-stranded RNAs (dsRNAs) with improved biological properties. Our chemically modified 27 nt dsRNAs exhibited an enhanced RNAi activity and a markedly increased stability in cell culture medium (containing 10% serum) in comparison with widely used 21 nt siRNAs and recently reported nonmodified 27 nt dsRNAs. The chemically modified 27 nt dsRNAs also exhibited a strong high long-term gene silencing effect after the 7 d treatment of viable cells. The chemically modified 27 nt dsRNAs in specific positions could be processed to 21 nt siRNAs by a recombinant Dicer enzyme. We suggested that the chemically modified 27 nt dsRNAs could be used for therapeutic applications (as genetic drugs) and bioanalyses.

  4. 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.

  5. Pest control. Full crop protection from an insect pest by expression of long double-stranded RNAs in plastids.

    Science.gov (United States)

    Zhang, Jiang; Khan, Sher Afzal; Hasse, Claudia; Ruf, Stephanie; Heckel, David G; Bock, Ralph

    2015-02-27

    Double-stranded RNAs (dsRNAs) targeted against essential genes can trigger a lethal RNA interference (RNAi) response in insect pests. The application of this concept in plant protection is hampered by the presence of an endogenous plant RNAi pathway that processes dsRNAs into short interfering RNAs. We found that long dsRNAs can be stably produced in chloroplasts, a cellular compartment that appears to lack an RNAi machinery. When expressed from the chloroplast genome, dsRNAs accumulated to as much as 0.4% of the total cellular RNA. Transplastomic potato plants producing dsRNAs targeted against the β-actin gene of the Colorado potato beetle, a notorious agricultural pest, were protected from herbivory and were lethal to its larvae. Thus, chloroplast expression of long dsRNAs can provide crop protection without chemical pesticides. PMID:25722411

  6. Using triplex-forming oligonucleotide probes for the reagentless, electrochemical detection of double-stranded DNA.

    Science.gov (United States)

    Patterson, Adriana; Caprio, Felice; Vallée-Bélisle, Alexis; Moscone, Danila; Plaxco, Kevin W; Palleschi, Giuseppe; Ricci, Francesco

    2010-11-01

    We report a reagentless, electrochemical sensor for the detection of double-stranded DNA targets that employs triplex-forming oligonucleotides (TFOs) as its recognition element. These sensors are based on redox-tagged TFO probes strongly chemisorbed onto an interrogating gold electrode. Upon the addition of the relevant double-stranded DNA target, the probe forms a rigid triplex structure via reverse Hoogsteen base pairing in the major groove. The formation of the triplex impedes contact between the probe's redox moiety and the interrogating electrode, thus signaling the presence of the target. We first demonstrated the proof of principle of this approach by using a well-characterized 22-base polypurine TFO sequence that readily detects a synthetic, double-stranded DNA target. We then confirmed the generalizability of our platform with a second probe, a 19-base polypyrimidine TFO sequence that targets a polypurine tract (PPT) sequence conserved in all HIV-1 strains. Both sensors rapidly and specifically detect their double-stranded DNA targets at concentrations as low as ~10 nM and are selective enough to be employed directly in complex sample matrices such as blood serum. Moreover, to demonstrate real-world applicability of this new sensor platform, we have successfully detected unpurified, double-stranded PCR amplicons containing the relevant conserved HIV-1 sequence. PMID:20936782

  7. Conservation of the rad21 Schizosaccharomyces pombe DNA double-strand break repair gene in mammals

    International Nuclear Information System (INIS)

    Purpose/Objective: Genetic factors are likely to be major determinants of human cellular ionizing radiation sensitivity. DNA double strand breaks (dsbs) are significant ionizing radiation-induced lesions; cellular DNA dsb processing is also important in a number of other contexts. To further the understanding of DNA dsb processing in mammalian cells, we cloned and sequenced mammalian homologs of the rad21 Schizosaccharomyces pombe DNA dsb repair gene. Materials and Methods: The genes were cloned by evolutionary walking, exploiting sequence homology between the yeast and mammalian genes. Results: No major motifs indicative of a particular function were present in the predicted amino acid sequences of the mammalian genes. Alignment of the Rad21 amino acid sequence with its putative homologs showed that similarity was distributed across the length of the proteins, with more highly conserved regions at both termini. The mHR21sp (mouse homolog ofR ad21, S. pombe) and hHR21sp (humanh omolog of Rad21, S. pombe) predicted proteins were 96% identical, whereas the human and S. pombe proteins were 25% identical and 47% similar. RNA blot analysis showed that mHR21sp mRNA was abundant in all adult mouse tissues examined, with highest expression in testis and thymus. In addition to a 3.1kb mRNA transcript in all tissues, an additional 2.2kb transcript was present at a high level in post-meiotic spermatids, white expression of the 3.1kb mRNA in testis was confined to the meiotic compartment. hHR21sp mRNA was cell cycle regulated in human cells, increasing in late S phase to a peak in G2 phase. The level of hHR21sp transcripts was not altered by exposure of normal diploid fibroblasts to 10 Gy ionizing radiation. In situ hybridization showed mHR21sp resided on chromosome 15D3, whereashHR21sp localized to the syntenic 8q24 region. Conclusion: Cloning these novel mammalian genes and characterization of their protein products should contribute to the understanding of cellular DNA dsb

  8. Repair and gamma radiation-induced single- and double-strand breaks in DNA of Escherichia coli

    International Nuclear Information System (INIS)

    Studies in the kinetics of repair of γ-radiation-induced single- and double-strand breaks in DNA of E. coli cells showed that double-strand DNA breaks are rejoined by the following two ways. The first way is conditioned by repair of single-strand breaks and represents the repair of ''oblique'' double-strand breaks in DNA, whereas the second way is conditioned by functioning of the recombination mechanisms and, to all appearance, represents the repair of ''direct'' double-strand breaks in DNA

  9. DNA gyrase action involves the introduction of transient double-strand breaks into DNA.

    OpenAIRE

    Mizuuchi, K; Fisher, L M; O'Dea, M H; Gellert, M

    1980-01-01

    DNA gyrase from Escherichia coli, in the presence of ATP, can both separate catenated DNA circles and unknot knotted DNA. Both these reactions require passage of a DNA segment through a transient double-strand break in DNA. Evidence that transient double-strand breaks are also involved in the supercoiling and relaxing activities of DNA gyrase is derived from experiments showing that the linking number of circular DNA is changed in steps of two. A mechanism is proposed for the action of the en...

  10. 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

  11. RecBCD Enzyme and the Repair of Double-Stranded DNA Breaks

    OpenAIRE

    Dillingham, Mark S.; Kowalczykowski, Stephen C.

    2008-01-01

    Summary: The RecBCD enzyme of Escherichia coli is a helicase-nuclease that initiates the repair of double-stranded DNA breaks by homologous recombination. It also degrades linear double-stranded DNA, protecting the bacteria from phages and extraneous chromosomal DNA. The RecBCD enzyme is, however, regulated by a cis-acting DNA sequence known as Chi (crossover hotspot instigator) that activates its recombination-promoting functions. Interaction with Chi causes an attenuation of the RecBCD enzy...

  12. 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

  13. On the linearity of the dose-effect relationship of DNA double strand breaks

    International Nuclear Information System (INIS)

    Most radiation biologists believe that DNA double-strand breaks are induced linearly with radiation dose for all types of radiation. Since 1985, with the advent of elution and gel electrophoresis techniques which permit the measurement of DNA double-strand breaks induced in mammalian cells at doses having radiobiological relevance, the true nature of the dose-effect relationship has been brought into some doubt. Many investigators measured curvilinear dose-effect relationships and a few found good correlations between the induction of the DNA double-strand breaks and cell survival. We approach the problem pragmatically by assuming that the induction of DNA double-strand breaks by 125I Auger electron emitters incorporated into the DNA of the cells is a linear function of the number of 125I decays, and by comparing the dose-effect relationship for sparsely ionizing radiation against this standard. The conclusion drawn that the curvilinear dose-effect relationships and the correlations with survival are real. (Author)

  14. 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

    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...

  15. 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...

  16. 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

    , 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...

  17. CRISPR-Mediated Base Editing without DNA Double-Strand Breaks.

    Science.gov (United States)

    Plosky, Brian S

    2016-05-19

    Targeting point mutations using CRISPR/Cas9 so far has required efficient homologous recombination (HR) and donor oligonucleotides. In a recent Nature paper, Komor and colleagues (2016) describe a way to make specific base changes that does not depend on HR or donor DNA and does not involve making double-strand breaks. PMID:27203175

  18. Pathway choice in DNA double strand break repair: Observations of a balancing act

    NARCIS (Netherlands)

    I. Brandsma (Inger); D.C. van Gent (Dik)

    2012-01-01

    textabstractProper repair of DNA double strand breaks (DSBs) is vital for the preservation of genomic integrity. There are two main pathways that repair DSBs, Homologous recombination (HR) and Non-homologous end-joining (NHEJ). HR is restricted to the S and G2 phases of the cell cycle due to the req

  19. 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

  20. 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 ...

  1. Entry of double-stranded deoxyribonucleic acid during transformation of Neisseria gonorrhoeae.

    OpenAIRE

    Biswas, G D; Sparling, P F

    1981-01-01

    The state of donor deoxyribonucleic acid after entry into competent cells was examined by assaying the transformed cell lysates for donor-marker transforming activity and density of donor deoxyribonucleic acid in CsCl gradients. The experiments showed that deoxyribonucleic acid entered in native, double-stranded form.

  2. Effects of the environment on the electric conductivity of double-stranded DNA molecules

    NARCIS (Netherlands)

    Malyshev, A. V.; Diaz, E.; Dominguez-Adame, F.; Malyshev, V. A.

    2009-01-01

    We present a theoretical analysis of the effects of the environment on charge transport in double-stranded synthetic poly(G)-poly(C) DNA molecules attached to two ideal leads. Coupling of the DNA to the environment results in two effects: (i) localization of carrier functions due to static disorder

  3. Normal formation and repair of γ-radiation-induced single and double strand DNA breaks in Down syndrome fibroblasts

    International Nuclear Information System (INIS)

    Fibroblasts from patients with Down syndrome (Trisomy 21) were examined for repair capability of γ-radiation-induced single strand and double strand DNA breaks. Formation and repair of DNA breaks were determined by DNA alkaline and non-denaturing elution techniques. Down syndrome fibroblasts were found to repair single strand and double strand breaks as well as fibroblasts from normal controls. (orig.)

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

    International Nuclear Information System (INIS)

    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(230)> rad51-1(300)> rad54-3(360). At 360, rad54-3 cells cannot repair double-strand breaks, while 230, they can. Strains with the rad57-1 mutation can rejoin broken chromosomes at both temperatures. However, the low survival at 360 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

  5. Single-cell microarray enables high-throughput evaluation of DNA double-strand breaks and DNA repair inhibitors.

    Science.gov (United States)

    Weingeist, David M; Ge, Jing; Wood, David K; Mutamba, James T; Huang, Qiuying; Rowland, Elizabeth A; Yaffe, Michael B; Floyd, Scott; Engelward, Bevin P

    2013-03-15

    A key modality of non-surgical cancer management is DNA damaging therapy that causes DNA double-strand breaks that are preferentially toxic to rapidly dividing cancer cells. Double-strand break repair capacity is recognized as an important mechanism in drug resistance and is therefore a potential target for adjuvant chemotherapy. Additionally, spontaneous and environmentally induced DSBs are known to promote cancer, making DSB evaluation important as a tool in epidemiology, clinical evaluation and in the development of novel pharmaceuticals. Currently available assays to detect double-strand breaks are limited in throughput and specificity and offer minimal information concerning the kinetics of repair. Here, we present the CometChip, a 96-well platform that enables assessment of double-strand break levels and repair capacity of multiple cell types and conditions in parallel and integrates with standard high-throughput screening and analysis technologies. We demonstrate the ability to detect multiple genetic deficiencies in double-strand break repair and evaluate a set of clinically relevant chemical inhibitors of one of the major double-strand break repair pathways, non-homologous end-joining. While other high-throughput repair assays measure residual damage or indirect markers of damage, the CometChip detects physical double-strand breaks, providing direct measurement of damage induction and repair capacity, which may be useful in developing and implementing treatment strategies with reduced side effects. PMID:23422001

  6. Feedback inhibition of L1 and alu retrotransposition through altered double strand break repair kinetics

    Directory of Open Access Journals (Sweden)

    Wallace Nicholas A

    2010-10-01

    Full Text Available Abstract Background Cells adapt to various chronic toxic exposures in a multitude of ways to minimize further damage and maximize their growth potential. Expression of L1 elements in the human genome can be greatly deleterious to cells, generating numerous double strand breaks (DSBs. Cells have been reported to respond to chronic DSBs by altering the repair of these breaks, including increasing the rate of homology independent DSB repair. Retrotransposition is strongly affected by proteins involved in DSB repair. Therefore, L1 expression has the potential to be a source of chronic DSBs and thus bring about the changes in cellular environment that could ultimately restrict its own retrotransposition. Results We demonstrate that constitutive L1 expression leads to quicker DSB repair and decreases in the retrotransposition potential of L1 and other retrotransposons dependent on L1 expression for their mobility. This cellular adaptation results in reduced sensitivity to L1 induced toxicity. These effects can be induced by constitutive expression of the functional L1 ORF2 alone, but not by the constitutive expression of an L1 open reading frame 2 with mutations to its endonuclease and reverse transcriptase domains. This adaptation correlates with the relative activity of the L1 introduced into the cells. Conclusions The increased number of DSBs resulting from constitutive expression of L1 results in a more rapid rate of repair. The cellular response to this L1 expression also results in attenuation of retrotransposition and reduced sensitivity of the cells to negative consequences of L1 ORF2 expression. The influence does not appear to be through RNA interference. We believe that the increased rate of DSB repair is the most likely cause of the attenuation of retrotransposition. These alterations act as a fail safe mechanism that allows cells to escape the toxicity associated with the unchecked L1 expression. This gives cells that overexpress L1, such

  7. A model treating the DNA double-strand break repair inhibition by damage clustering

    International Nuclear Information System (INIS)

    A microdosimetric model for the interpretation of radiation induced irreparable DNA double-strand breaks was applied to the biological endpoint of chromosomal aberrations. The model explains irreparable DNA double-strand breaks in terms of break clustering in DNA subunits. The model predicts quite good chromosomal aberrations in gamma- and X-ray irradiated V79 cells and human lymphocytes. In the case of α-particle irradiation the presumption had to be made, that only the cells with indirect events in the nucleus (due to delta-electrons) reach the metaphase and are analysed. With the help of this model we are able to explain the peculiar effectiveness of ultrasoft C-X-rays in human lymphocytes. In addition, an interpretation of experiments with accelerated and spatially correlated particles is given. (author)

  8. Electric field induced charge transfer through single and double-stranded DNA polymer molecules

    OpenAIRE

    Ramos, Marta M. D.; Correia, Helena M. G.

    2011-01-01

    The charge transfer through single-stranded and double-stranded DNA polymer molecules has been the subject of numerous experimental and theoretical studies concerning their applications in molecular electronics. However, the underlying mechanisms responsible for their different electrical conductivity observed in the experiments are poorly understood. Here we use a self-consistent quantum molecular dynamics method to study the effect of an applied electric field along the molecular axis on ch...

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

    OpenAIRE

    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 but also arise as intermediates in several cellular processes (e.g. meiosis). DSBs are among the most genotoxic DNA lesions and their accurate repair is crucial. Genetic instability resulting from ...

  10. Identification of Saccharomyces cerevisiae DNA ligase IV: involvement in DNA double-strand break repair.

    OpenAIRE

    Teo, S H; Jackson, S P

    1997-01-01

    DNA ligases catalyse the joining of single and double-strand DNA breaks, which is an essential final step in DNA replication, recombination and repair. Mammalian cells have four DNA ligases, termed ligases I-IV. In contrast, other than a DNA ligase I homologue (encoded by CDC9), no other DNA ligases have hitherto been identified in Saccharomyces cerevisiae. Here, we report the identification and characterization of a novel gene, LIG4, which encodes a protein with strong homology to mammalian ...

  11. Lack of specificity for antibodies to double stranded DNA found in four commercial kits.

    OpenAIRE

    Kadlubowski, M; Jackson, M.; Yap, P L; Neill, G.

    1991-01-01

    Four commercially available kits (three enzyme linked immunosorbent assays and one modified Farr radioimmunoassay) were compared for their ability to detect specifically autoantibodies to double-stranded DNA (dsDNA) using 66 patient sera. This was assessed by comparing the results of the kits with those from an ELISA specifically measuring antibodies against highly purified dsDNA, single-stranded DNA (ssDNA), native DNA and histones. The RIA and two of the ELISAs seemed equally efficient at d...

  12. The effects of DNA double-strand breaks on mouse oocyte meiotic maturation

    OpenAIRE

    Ma, Jun-Yu; Ou Yang, Ying-Chun; Wang, Zhong-Wei; Wang, Zhen-Bo; Jiang, Zong-Zhe; Luo, Shi-Ming; Hou, Yi; Liu, Zhonghua; Schatten, Heide; Sun, Qing-Yuan

    2013-01-01

    Both endogenous and exogenous factors can induce DNA double-strand breaks (DSBs) in oocytes, which is a potential risk for human-assisted reproductive technology as well as animal nuclear transfer. Here we used bleomycin (BLM) and laser micro-beam dissection (LMD) to induce DNA DSBs in germinal vesicle (GV) stage oocytes and compared the germinal vesicle breakdown (GVBD) rates and first polar body extrusion (PBE) rates between DNA DSB oocytes and untreated oocytes. Employing live cell imaging...

  13. Evaluation of DNA Double Strand Breaks Repair Efficiency in Head and Neck Cancer

    OpenAIRE

    Walczak, Anna; Rusin, Pawel; Dziki, Lukasz; Zielinska-Blizniewska, Hanna; Olszewski, Jurek; Majsterek, Ireneusz

    2012-01-01

    Head and neck cancers (head and neck squamous cell carcinomas [HNSCC]) are a heterogeneous group of neoplasms with varying presenting symptoms, treatment, and expected outcome. There is a need to find an effective way of its treatment at the molecular level. Thus, we should identify the mechanism of cancer cell response to damaging agents' activity, especially at DNA level. Our major goal was to evaluate the efficacy of DNA double strand breaks (DSBs) repair in HTB-43 and SCC-25 cancer cell l...

  14. Mechanisms of chemotherapy-induced human ovarian aging: double strand DNA breaks and microvascular compromise

    OpenAIRE

    Soleimani, Reza; Heytens, Elke; Darzynkiewicz, Zbigniew; Oktay, Kutluk

    2011-01-01

    The mechanism of chemotherapy-induced acceleration of ovarian aging is not fully understood. We used doxorubicin, a widely used cancer chemotherapeutic, in a variety of in vivo xenograft, and in vitro models to investigate the impact of chemotherapy-induced aging on the human ovary. Doxorubicin caused massive double-strand-DNA-breaks in primordial follicles, oocytes, and granulosa cells in a dose dependent fashion as revealed by accumulating γH2AX foci. This damage was associated with apoptot...

  15. G-quadruplex formation in double strand DNA probed by NMM and CV fluorescence

    OpenAIRE

    Kreig, Alex; Calvert, Jacob; Sanoica, Janet; Cullum, Emily; Tipanna, Ramreddy; Myong, Sua

    2015-01-01

    G-quadruplexes (GQs) are alternative DNA secondary structures that can form throughout the human genome and control the replication and transcription of important regulatory genes. Here, we established an ensemble fluorescence assay by employing two GQ-interacting compounds, N-methyl mesoporphyrin IX (NMM) and Crystal Violet (CV). This enables quantitative measurement of the GQ folding propensity and conformation specificity in both single strand (ss) and double strand (ds) DNA. Our GQ mappin...

  16. Crystallization of a flexible polymer in melt in the presence of double-stranded DNA

    International Nuclear Information System (INIS)

    The behavior of a mixture of short fragments of double-stranded DNA and a flexible polymer is studied. The role of the DNA double helix in the formation of the crystalline phase in the flexible polymers melt is considered. It is shown that the presence of DNA rigid fragments in melt results in increase in the melting temperature. A shift of the melting temperature of a perfect crystal as compared to the pure polymer melt is calculated

  17. The dynamics of DNA double-strand breaks and apoptosis in peripheral blood human lymphocytes

    International Nuclear Information System (INIS)

    Two-phase nature of the DNA double-strand breaks (DSBs) formation and apoptosis under the influence of ionizing radiation in normal and chronic lymphocytic leukemia lymphocytes was observed. The highest level of DSBs was in first minutes after irradiation, and then their repair was seen. The dynamics of DSBs and apoptosis were similar in both types of lymphocytes and the correlation of the processes was revealed. (authors)

  18. 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.

  19. Double strand DNA breaks response in Huntington´s disease

    Czech Academy of Sciences Publication Activity Database

    Šolc, Petr; Valášek, Jan; Rausová, Petra; Juhásová, Jana; Juhás, Štefan; Motlík, Jan

    2015-01-01

    Roč. 78, Suppl 2 (2015), s. 15-15. ISSN 1210-7859. [Conference on Animal Models for neurodegenerative Diseases /3./. 08.11.2015-10.11.2015, Liblice] R&D Projects: GA MŠk ED2.1.00/03.0124; GA MŠk(CZ) 7F14308 Institutional support: RVO:67985904 Keywords : Huntington ´s disease * DNA damage * double strand DNA breaks Subject RIV: FH - Neurology

  20. Meiotic double-strand breaks at the interface of chromosome movement, chromosome remodeling, and reductional division

    OpenAIRE

    Storlazzi, Aurora; Tessé, Sophie; Gargano, Silvana; James, Françoise; Kleckner, Nancy; Zickler, Denise

    2003-01-01

    Chromosomal processes related to formation and function of meiotic chiasmata have been analyzed in Sordaria macrospora. Double-strand breaks (DSBs), programmed or γ-rays-induced, are found to promote four major events beyond recombination and accompanying synaptonemal complex formation: (1) juxtaposition of homologs from long-distance interactions to close presynaptic coalignment at midleptotene; (2) structural destabilization of chromosomes at leptotene/zygotene, including sister axis separa...

  1. A New Mechanism of Nonrandom Distribution of DNA Double Strand Breaks

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Since 1996, it has been widely accepted that the distribution of DNA double strand breaks (DSBs) induced by ionizing radiation is nonrandom. The explanation to this phenomenon is focused in two parts. One is the ionizing characteristic of the particles and the other is the high-ordered configuration of chromosome in eukaryote~[1,2]. As reported before~[3], we revealed the nonrandom distribution of DSBs when the deproteinized

  2. Multinuclear non-heme iron complexes for double-strand DNA cleavage

    NARCIS (Netherlands)

    Megens, Rik P.; van den Berg, Tieme A.; de Bruijn, A. Dowine; Feringa, Ben L.; Roelfes, Gerard

    2009-01-01

    The cytotoxicity of the antitumor drug BLM is believed to be related to the ability of the corresponding iron complex (Fe-BLM) to engage in oxidative double-strand DNA cleavage. The iron complex of the ligand N4Py (Fe-N4Py; N4Py - N,N-bis(2-pyridyl)-N-bis(2-pyridyl)methylamine has proven to be a par

  3. The conducting form of gramicidin A is a right-handed double-stranded double helix

    OpenAIRE

    Burkhart, B. M.; Li, N.; Langs, D A; Pangborn, W A; Duax, W L

    1998-01-01

    The linear pentadecapeptide antibiotic, gramicidin D, is a naturally occurring product of Bacillus brevis known to form ion channels in synthetic and natural membranes. The x-ray crystal structures of the right-handed double-stranded double-helical dimers (DSDHℛ) reported here agree with 15N-NMR and CD data on the functional gramicidin D channel in lipid bilayers. These structures demonstrate single-file ion transfer through the channels. The results also indicate that previous crystal struct...

  4. 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. PMID:11539954

  5. Force-Induced Melting and Thermal Melting of a Double-Stranded Biopolymer

    OpenAIRE

    Zhou, Haijun

    2000-01-01

    As a prototype of systems bearing a localization-delocalization transition, the strand-separation (melting) process in a double-stranded biopolymer is studied by a mapping to a quantum-mechanical problem with short-ranged potentials. Both the bounded and the extensive eigenmodes of the corresponding Schrodinger equation are considered and exact expressions for the configurational partition function and free energy are obtained. The force-induced melting is a first order phase transition proce...

  6. Choreographing the double strand break response: Ubiquitin and SUMO control of nuclear architecture

    Directory of Open Access Journals (Sweden)

    Shane M Harding

    2016-06-01

    Full Text Available The cellular response to DNA double strand breaks (DSBs is a multifaceted signaling program that centers on post-translational modifications including phosphorylation, ubiquitylation and SUMOylation. In this review we discuss how ubiquitin and SUMO orchestrate the recognition of DSBs and explore how this influences chromatin organization. We discuss functional outcomes of this response including transcriptional silencing and how pre-existing chromatin states may control the DSB response and the maintenance of genomic stability.

  7. A simple and efficient method for site-directed mutagenesis with double-stranded plasmid DNA.

    OpenAIRE

    Lai, D; Zhu, X.; Pestka, S

    1993-01-01

    A general, simple and efficient method for preparing site-specific mutations in double-stranded plasmid DNA without the need for special plasmids, bacterial strains or reagents is described. Only one synthetic oligonucleotide for each mutation is required, subcloning is unnecessary and a high efficiency of mutation (58-97%) was obtained. If two synthetic oligonucleotide primers are used, two separate mutations can be simultaneously created in a single reaction tube.

  8. Smoking Cessation Reverses DNA Double-Strand Breaks in Human Mononuclear Cells

    OpenAIRE

    Ishida, Mari; Ishida, Takafumi; Tashiro, Satoshi; Uchida, Hitomi; Sakai, Chiemi; Hironobe, Naoya; Miura, Katsuya; Hashimoto, Yu; Arihiro, Koji; Chayama, Kazuaki; Kihara, Yasuki; Yoshizumi, Masao

    2014-01-01

    Objective Cigarette smoking is a major risk factor for atherosclerotic cardiovascular disease, which is responsible for a significant proportion of smoking-related deaths. However, the precise mechanism whereby smoking induces this pathology has not been fully delineated. Based on observation of DNA double-strand breaks (DSBs), the most harmful type of DNA damage, in atherosclerotic lesions, we hypothesized that there is a direct association between smoking and DSBs. The goal of this study wa...

  9. DNA double-strand break repair proteins are required to cap the ends of mammalian chromosomes

    OpenAIRE

    Bailey, Susan M.; Meyne, Julianne; Chen, David J.; Kurimasa, Akihiro; Li, Gloria C.; Lehnert, Bruce E.; Goodwin, Edwin H.

    1999-01-01

    Recent findings intriguingly place DNA double-strand break repair proteins at chromosome ends in yeast, where they help maintain normal telomere length and structure. In the present study, an essential telomere function, the ability to cap and thereby protect chromosomes from end-to-end fusions, was assessed in repair-deficient mouse cell lines. By using fluorescence in situ hybridization with a probe to telomeric DNA, spontaneously occurring chromosome aberrations were examined for telomere ...

  10. A role for Ddc1 in signaling meiotic double-strand breaks at the pachytene checkpoint

    OpenAIRE

    Hong, Eun-Jin Erica; Roeder, G. Shirleen

    2002-01-01

    The pachytene checkpoint prevents meiotic cell cycle progression in response to unrepaired recombination intermediates. We show that Ddc1 is required for the pachytene checkpoint in Saccharomyces cerevisiae. During meiotic prophase, Ddc1 localizes to chromosomes and becomes phosphorylated; these events depend on the formation and processing of double-strand breaks (DSBs). Ddc1 colocalizes with Rad51, a DSB-repair protein, indicating that Ddc1 associates with sites of DSB repair. The Rad24 che...

  11. Enzyme-free detection and quantification of double-stranded nucleic acids.

    Science.gov (United States)

    Feuillie, Cécile; Merheb, Maxime Mohamad; Gillet, Benjamin; Montagnac, Gilles; Hänni, Catherine; Daniel, Isabelle

    2012-08-01

    We have developed a fully enzyme-free SERRS hybridization assay for specific detection of double-stranded DNA sequences. Although all DNA detection methods ranging from PCR to high-throughput sequencing rely on enzymes, this method is unique for being totally non-enzymatic. The efficiency of enzymatic processes is affected by alterations, modifications, and/or quality of DNA. For instance, a limitation of most DNA polymerases is their inability to process DNA damaged by blocking lesions. As a result, enzymatic amplification and sequencing of degraded DNA often fail. In this study we succeeded in detecting and quantifying, within a mixture, relative amounts of closely related double-stranded DNA sequences from Rupicapra rupicapra (chamois) and Capra hircus (goat). The non-enzymatic SERRS assay presented here is the corner stone of a promising approach to overcome the failure of DNA polymerase when DNA is too degraded or when the concentration of polymerase inhibitors is too high. It is the first time double-stranded DNA has been detected with a truly non-enzymatic SERRS-based method. This non-enzymatic, inexpensive, rapid assay is therefore a breakthrough in nucleic acid detection. PMID:22695500

  12. Differential requirement for SUB1 in chromosomal and plasmid double-strand DNA break repair.

    Directory of Open Access Journals (Sweden)

    Lijian Yu

    Full Text Available Non homologous end joining (NHEJ is an important process that repairs double strand DNA breaks (DSBs in eukaryotic cells. Cells defective in NHEJ are unable to join chromosomal breaks. Two different NHEJ assays are typically used to determine the efficiency of NHEJ. One requires NHEJ of linearized plasmid DNA transformed into the test organism; the other requires NHEJ of a single chromosomal break induced either by HO endonuclease or the I-SceI restriction enzyme. These two assays are generally considered equivalent and rely on the same set of NHEJ genes. PC4 is an abundant DNA binding protein that has been suggested to stimulate NHEJ. Here we tested the role of PC4's yeast homolog SUB1 in repair of DNA double strand breaks using different assays. We found SUB1 is required for NHEJ repair of DSBs in plasmid DNA, but not in chromosomal DNA. Our results suggest that these two assays, while similar are not equivalent and that repair of plasmid DNA requires additional factor(s that are not required for NHEJ repair of chromosomal double-strand DNA breaks. Possible roles for Sub1 proteins in NHEJ of plasmid DNA are discussed.

  13. Inhibition of APOBEC3G activity impedes double-stranded DNA repair.

    Science.gov (United States)

    Prabhu, Ponnandy; Shandilya, Shivender M D; Britan-Rosich, Elena; Nagler, Adi; Schiffer, Celia A; Kotler, Moshe

    2016-01-01

    The cellular cytidine deaminase APOBEC3G (A3G) was first described as an anti-HIV-1 restriction factor, acting by directly deaminating reverse transcripts of the viral genome. HIV-1 Vif neutralizes the activity of A3G, primarily by mediating degradation of A3G to establish effective infection in host target cells. Lymphoma cells, which express high amounts of A3G, can restrict Vif-deficient HIV-1. Interestingly, these cells are more stable in the face of treatments that result in double-stranded DNA damage, such as ionizing radiation and chemotherapies. Previously, we showed that the Vif-derived peptide (Vif25-39) efficiently inhibits A3G deamination, and increases the sensitivity of lymphoma cells to ionizing radiation. In the current study, we show that additional peptides derived from Vif, A3G, and APOBEC3F, which contain the LYYF motif, inhibit deamination activity. Each residue in the Vif25-39 sequence moderately contributes to the inhibitory effect, whereas replacing a single residue in the LYYF motif completely abrogates inhibition of deamination. Treatment of A3G-expressing lymphoma cells exposed to ionizing radiation with the new inhibitory peptides reduces double-strand break repair after irradiation. Incubation of cultured irradiated lymphoma cells with peptides that inhibit double-strand break repair halts their propagation. These results suggest that A3G may be a potential therapeutic target that is amenable to peptide and peptidomimetic inhibition. PMID:26460502

  14. 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.

  15. Simulation of the Formation of DNA Double Strand Breaks and Chromosome Aberrations in Irradiated Cells

    Science.gov (United States)

    Plante, Ianik; Ponomarev, Artem L.; Wu, Honglu; Blattnig, Steve; George, Kerry

    2014-01-01

    The formation of DNA double-strand breaks (DSBs) and chromosome aberrations is an important consequence of ionizing radiation. To simulate DNA double-strand breaks and the formation of chromosome aberrations, we have recently merged the codes RITRACKS (Relativistic Ion Tracks) and NASARTI (NASA Radiation Track Image). The program RITRACKS is a stochastic code developed to simulate detailed event-by-event radiation track structure: [1] This code is used to calculate the dose in voxels of 20 nm, in a volume containing simulated chromosomes, [2] The number of tracks in the volume is calculated for each simulation by sampling a Poisson distribution, with the distribution parameter obtained from the irradiation dose, ion type and energy. The program NASARTI generates the chromosomes present in a cell nucleus by random walks of 20 nm, corresponding to the size of the dose voxels, [3] The generated chromosomes are located within domains which may intertwine, and [4] Each segment of the random walks corresponds to approx. 2,000 DNA base pairs. NASARTI uses pre-calculated dose at each voxel to calculate the probability of DNA damage at each random walk segment. Using the location of double-strand breaks, possible rejoining between damaged segments is evaluated. This yields various types of chromosomes aberrations, including deletions, inversions, exchanges, etc. By performing the calculations using various types of radiations, it will be possible to obtain relative biological effectiveness (RBE) values for several types of chromosome aberrations.

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

    International Nuclear Information System (INIS)

    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. 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.

  18. The production and repair of double strand breaks in cells from normal humans and patients with ataxia telangiectasia

    International Nuclear Information System (INIS)

    The production and repair of double strand breaks induced by γ-rays in the DNA of human fibroblasts have been measured by sedimentation in sucrose gradients under non-denaturing conditions. Unirradiated DNA formed a rapidly sedimenting gel. Low doses of radiation released freely sedimenting DNA molecules from this gel. Higher doses reduced the rate of sedimentation of the free DNA due to the introduction of double strand breaks. The breakage efficiency was 1 break/1.3x1010 daltons of DNA/krad. Postirradiation incubation after a high dose of radiation resulted in an increase in molecular weight of the free DNA molecules, and after a low dose the rapidly-sedimenting gel was reformed. These data suggest that double strand breaks are repaired in human fibroblasts. No significant differences were found between fibroblasts from two normal donors and four patients with the radiosensitive disorder, ataxia telangiectasia, in either the production or repair of double strand breaks

  19. Activation of repair and checkpoints by double-strand breaks of DNA. Activational cascade of protein phosphorylation

    International Nuclear Information System (INIS)

    Molecular mechanisms of double-strand breaks repair and checkpoints include phosphorylations of repair and checkpoint-proteins by protein kinases. Chemical modification of proteins has different consequences including activation, changing of affinity to proteins and localization

  20. Single-cell microarray enables high-throughput evaluation of DNA double-strand breaks and DNA repair inhibitors

    OpenAIRE

    Weingeist, David M.; Ge, Jing; Wood, David K.; Mutamba, James T; Huang, Qiuying; Rowland, Elizabeth A.; Yaffe, Michael B.; Floyd, Scott; Engelward, Bevin P.

    2013-01-01

    A key modality of non-surgical cancer management is DNA damaging therapy that causes DNA double-strand breaks that are preferentially toxic to rapidly dividing cancer cells. Double-strand break repair capacity is recognized as an important mechanism in drug resistance and is therefore a potential target for adjuvant chemotherapy. Additionally, spontaneous and environmentally induced DSBs are known to promote cancer, making DSB evaluation important as a tool in epidemiology, clinical evaluatio...

  1. Ku counteracts mobilization of PARP1 and MRN in chromatin damaged with DNA double-strand breaks.

    Science.gov (United States)

    Cheng, Qiao; Barboule, Nadia; Frit, Philippe; Gomez, Dennis; Bombarde, Oriane; Couderc, Bettina; Ren, Guo-Sheng; Salles, Bernard; Calsou, Patrick

    2011-12-01

    In mammalian cells, the main pathway for DNA double-strand breaks (DSBs) repair is classical non-homologous end joining (C-NHEJ). An alternative or back-up NHEJ (B-NHEJ) pathway has emerged which operates preferentially under C-NHEJ defective conditions. Although B-NHEJ appears particularly relevant to genomic instability associated with cancer, its components and regulation are still largely unknown. To get insights into this pathway, we have knocked-down Ku, the main contributor to C-NHEJ. Thus, models of human cell lines have been engineered in which the expression of Ku70/80 heterodimer can be significantly lowered by the conditional induction of a shRNA against Ku70. On Ku reduction in cells, resulting NHEJ competent protein extracts showed a shift from C- to B-NHEJ that could be reversed by addition of purified Ku protein. Using a cellular fractionation protocol after treatment with a strong DSBs inducer followed by western blotting or immunostaining, we established that, among C-NHEJ factors, Ku is the main counteracting factor against mobilization of PARP1 and the MRN complex to damaged chromatin. In addition, Ku limits PAR synthesis and single-stranded DNA production in response to DSBs. These data support the involvement of PARP1 and the MRN proteins in the B-NHEJ route for the repair of DNA DSBs. PMID:21880593

  2. 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.

  3. Genetic polymorphisms of DNA double-strand break repair pathway genes and glioma susceptibility

    International Nuclear Information System (INIS)

    Genetic variations in DNA double-strand break repair genes can influence the ability of a cell to repair damaged DNA and alter an individual’s susceptibility to cancer. We studied whether polymorphisms in DNA double-strand break repair genes are associated with an increased risk of glioma development. We genotyped 10 potentially functional single nucleotide polymorphisms (SNPs) in 7 DNA double-strand break repair pathway genes (XRCC3, BRCA2, RAG1, XRCC5, LIG4, XRCC4 and ATM) in a case–control study including 384 glioma patients and 384 cancer-free controls in a Chinese Han population. Genotypes were determined using the OpenArray platform. In the single-locus analysis there was a significant association between gliomas and the LIG4 rs1805388 (Ex2 +54C>T, Thr9Ile) TT genotype (adjusted OR, 3.27; 95% CI, 1.87-5.71), as well as the TC genotype (adjusted OR, 1.62; 95% CI, 1.20-2.18). We also found that the homozygous variant genotype (GG) of XRCC4 rs1805377 (IVS7-1A>G, splice-site) was associated with a significantly increased risk of gliomas (OR, 1.77; 95% CI, 1.12-2.80). Interestingly, we detected a significant additive and multiplicative interaction effect between the LIG4 rs1805388 and XRCC4 rs1805377 polymorphisms with an increasing risk of gliomas. When we stratified our analysis by smoking status, LIG4 rs1805388 was associated with an increased glioma risk among smokers. These results indicate for the first time that LIG4 rs1805388 and XRCC4 rs1805377, alone or in combination, are associated with a risk of gliomas

  4. Ser1778 of 53BP1 Plays a Role in DNA Double-strand Break Repairs

    OpenAIRE

    Lee, Jung-Hee; Cheong, Hyang-Min; Kang, Mi-Young; Kim, Sang-Young; Kang, Yoonsung

    2009-01-01

    53BP1 is an important genome stability regulator, which protects cells against double-strand breaks. Following DNA damage, 53BP1 is rapidly recruited to sites of DNA breakage, along with other DNA damage response proteins, including γ-H2AX, MDC1, and BRCA1. The recruitment of 53BP1 requires a tandem Tudor fold which associates with methylated histones H3 and H4. It has already been determined that the majority of DNA damage response proteins are phosphorylated by ATM and/or ATR after DNA dama...

  5. Homologous recombination repairs secondary replication induced DNA double-strand breaks after ionizing radiation

    OpenAIRE

    Groth, Petra; Orta, Manuel Luís; Elvers, Ingegerd; Majumder, Muntasir Mamun; Lagerqvist, Anne; Helleday, Thomas

    2012-01-01

    Ionizing radiation (IR) produces direct two-ended DNA double-strand breaks (DSBs) primarily repaired by non-homologous end joining (NHEJ). It is, however, well established that homologous recombination (HR) is induced and required for repair of a subset of DSBs formed following IR. Here, we find that HR induced by IR is drastically reduced when post-DNA damage replication is inhibited in mammalian cells. Both IR-induced RAD51 foci and HR events in the hprt gene are reduced in the presence of ...

  6. 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.

  7. Enhancement effect of asymmetry on the thermal conductivity of double-stranded chain systems

    International Nuclear Information System (INIS)

    Using nonequilibrium molecular dynamics simulations, we study the thermal conductivity of asymmetric double chains. We couple two different single chains through interchain coupling to build three kinds of asymmetric double-stranded chain system: intrachain interaction, external potential, and mass asymmetric double chains. It is reported that asymmetry is helpful in improving the thermal conductivity of the system. We first propose double-heat flux channels to explain the influence of asymmetric structures on the thermal conductivity. The phonon spectral behaviour and finite size effect are also included. (general)

  8. DNA double strand breaks in Ehrlich ascites tumour cells at low doses of X-rays

    International Nuclear Information System (INIS)

    DNA double strand breaks (dsb) were determined in Ehrlich ascites tumour cells at doses down to 5 Gy. Sedimentation profiles were analysed using a computer program and the number of dsb was determined by simulation of random breaks in the mass distribution of the control sample and by comparison of this simulated profile with that of the irradiated one. The number of dsb formed was proportional to X-ray dose in the range of 5 to 2000 Gy. The induction per dose was found to be nmsub(r)-1 D-1=(11.7+-2) x 10-12 Gy-1. (author)

  9. A critical role for topoisomerase IIb and DNA double strand breaks in transcription.

    Science.gov (United States)

    Calderwood, Stuart K

    2016-05-26

    Recent studies have indicated a novel role for topoisomerase IIb in transcription. Transcription of heat shock genes, serum-induced immediate early genes and nuclear receptor-activated genes, each required DNA double strands generated by topoisomerase IIb. Such strand breaks seemed both necessary and sufficient for transcriptional activation. In addition, such transcription was associated with initiation of the DNA damage response pathways, including the activation of the enzymes: ataxia-telangiectasia mutated (ATM), DNA-dependent protein kinase and poly (ADP ribose) polymerase 1. DNA damage response signaling was involved both in transcription and in repair of DNA breaks generated by topoisomerase IIb. PMID:27100743

  10. Interaction of double-stranded DNA with polymerized PprA protein from Deinococcus radiodurans

    OpenAIRE

    Adachi, Motoyasu; Hirayama, Hiroshi; Shimizu, Rumi; Satoh, Katsuya; Narumi, Issay; Kuroki, Ryota

    2014-01-01

    Pleiotropic protein promoting DNA repair A (PprA) is a key protein that facilitates the extreme radioresistance of Deinococcus radiodurans. To clarify the role of PprA in the radioresistance mechanism, the interaction between recombinant PprA expressed in Escherichia coli with several double-stranded DNAs (i.e., super coiled, linear, or nicked circular dsDNA) was investigated. In a gel-shift assay, the band shift of supercoiled pUC19 DNA caused by the binding of PprA showed a bimodal distribu...

  11. The response of the brain tissue to DNA double strand breaks

    International Nuclear Information System (INIS)

    Double-strand breaks (DSB) are the most deleterious form of DNA damage after ionizing radiation, the response of the brain tissue to DNA damage is related to the developmental dynamics of this system. Homology recombination is particularly important for proliferating cells, while non-homologous end joining is critical for differentiating cells. Defects in the related factors to DNA damage pathway underpin many human genopathy with neuropathology. Reviewed the signal conduction system involved in the DNA DSB response and human neuropathology genopathy related to DNA DSB factors deficiencies in the brain cells. (authors)

  12. 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...... integrity, as well as cell and organismal fitness....

  13. Sequence-specific DNA double-strand breaks induced by triplex forming 125I labeled oligonucleotides.

    OpenAIRE

    Panyutin, I G; Neumann, R D

    1994-01-01

    A triplex-forming oligonucleotide (TFO) complementary to the polypurine-polypyrimidine region of the nef gene of the Human Immunodeficiency Virus (HIV) was labeled with 125I at the C5 position of a single deoxycytosine residue. Labeled TFO was incubated with a plasmid containing a fragment of the nef gene. Decay of 125I was found to cause double-strand breaks (DSB) within the nef gene upon triplex formation in a sequence specific manner. No DSB were detected after incubation at ionic conditio...

  14. Single and double stranded DNA detection using locked nucleic acid (LNA) functionalized nanoparticles

    Science.gov (United States)

    McKenzie, Fiona; Stokes, Robert; Faulds, Karen; Graham, Duncan

    2008-08-01

    Gold and silver nanoparticles functionalized with oligonucleotides can be used for the detection of specific sequences of DNA. We show that gold nanoparticles modified with locked nucleic acid (LNA) form stronger duplexes with a single stranded DNA target and offer better discrimination against single base pair mismatches than analogous DNA probes. Our LNA nanoparticle probes have also been used to detect double stranded DNA through triplex formation, whilst still maintaining selectivity for only complementary targets. Nanoparticle conjugates embedded with suitable surface enhanced resonance Raman scattering (SERRS) labels have been synthesized enabling simultaneous detection and identification of multiple DNA targets.

  15. 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 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...

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

    OpenAIRE

    Gao, Min; Wei, Wei; Li, Ming-ming; Wu, Yong-Sheng; Ba, Zhaoqing; Jin, Kang-Xuan; Li, Miao-Miao; Liao, You-Qi; Adhikari, Samir; Chong, Zechen; Zhang, Ting; Guo, Cai-Xia; Tang, Tie-Shan; Zhu, Bing-Tao; Xu, Xing-Zhi

    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 h...

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

    OpenAIRE

    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, ...

  18. 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......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...

  19. Response of the electric conductivity of double-stranded DNA on moderate mechanical stretching stresses

    Science.gov (United States)

    Wolter, Mario; Woiczikowski, P. Benjamin; Elstner, Marcus; Kubař, Tomáš

    2012-02-01

    The response of charge transport in double-stranded DNA to mechanical pulling has been studied with a multiscale computational method using classical molecular dynamics simulation, approximative density-functional theory calculations and the Landauer-Büttiker theory. The effect depends on the exact nucleobase sequence notably, and this is explained in terms of structural changes of DNA upon stretching. The results of recent single-molecule experiments are interpreted on the basis of current results. Further, recommendations for the design of DNA sequences for nanoelectronic applications are formulated.

  20. Induction of double-strand breaks in DNA of prokaryotes and eukaryotes and their repair. 1. Application of elastoviscosimetry for studying double-strand breaks in DNA of Escherichia coli induced by γ-irradiation

    International Nuclear Information System (INIS)

    It is shown that the method of elastoviscosimetry gives a possibility to record the formation of DNA double-strand breaks in Escherichia coli cells induced by γ irradiation at doses close to D37. The dependence of changes of elastoviscosity parameter on the dose (tau0) passes through the maximum. It is shown that the ascending section of this curve (at minimum γ irradiation doses) characterizes the relaxation process of the superspiralised chromosome in nucleotide of the E. coli. This relaxation is observed due to γ induced damages which are not double-strand breaks. By the maximum position one can judge on a dose yield of the first DNA double-strand break, the descending part of the dose curve describes the kinetics of accumulation of breaks with the dose increase. The analysis of the data obtained gives the possibility to come to the conclusion that when applying a usual technique of irradiation and lysis of cells not providing for special measures on inhibition of endo-and exonuclease activity in γ irradiated cells, the dose yield of double-strand breaks noticeably increases (by 4.2 times). In the case of an essential, though incomplete, inhibition of nuclease activities in γ irradiated cells the dose yield of breaks approximately corresponds to the dose curve of inactivation of these cells (D3712.5+-3.0 krad, the first double-strand break -at 14.5+-2.4 krad)

  1. 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.

  2. 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. PMID:26682627

  3. Calibration of pulsed field gel electrophoresis for measurement of DNA double-strand breaks

    International Nuclear Information System (INIS)

    Pulsed field gel electrophoresis (PFGE) assay was calibrated for the measurement of X-ray induced DNA double-strand breaks in Chinese hamster ovary (CHO) cells. Calibration was conducted by incorporating [125I] deoxyuridine into DNA, which induces one double-strand break for every disintegration that occurs in frozen cells. Based on the percentage of DNA migrating into the gel, the number of breaks/dalton/Gy was estimated to be (9.3±1.0) x 10-12. This value is close to (10 to 12) x 10-12 determined by neutral filter elution using similar cell lysis procedures at 24oC and at pH8.0. The estimate is in good agreement with the value of (11.7±2) x 10-12 breaks/dalton/Gy as measured in Ehrlich ascites tumour cells using the neutral sucrose gradient method (Bloecher 1988), and (6 to 9) x 10-12 breaks/dalton/Gy as measured in mouse L and Chinese hamster V79 cells using neutral filter elution (Radford and Hodgson 1985). (author)

  4. DNA double-strand breaks measured in individual cells subjected to gel electrophoresis

    International Nuclear Information System (INIS)

    Microscopic examination of individual mammalian cells embedded in agarose, subjected to electrophoresis, and stained with a fluorescent DNA-binding dye provides a novel way of measuring DNA damage and more importantly, of assessing heterogeneity in DNA damage within a mixed population of cells. With this method, DNA double-strand breaks can be detected in populations of cells exposed to X-ray doses as low as 5 Gy. The radiation dose-response relationship for initial formation of double-strand breaks was identical for cell lines irradiated in G1, regardless of their sensitivity to killing by ionizing radiation. However, for cells irradiated in S phase, DNA migration was significantly reduced. For Chinese hamster V79 cells, Chinese hamster ovary cells, WiDr human colon carcinoma cells, and L5178Y-R mouse lymphoblastoid cells, S-phase DNA appeared to be about 3 times less sensitive to X-ray damage than DNA from other phases of the cell cycle. However, for the very radiosensitive L5178Y-S cells, the migration of replicating DNA was reduced only slightly. For Chinese hamster V79 and Chinese hamster ovary cells, damage was repaired at a similar rate in all cells of the population, and 85% of the breaks were rejoined within 2 h after irradiation. The radiosensitive L5178Y-S cells repaired damage more slowly than V79 or Chinese hamster ovary cells; 2 h after exposure to 50 Gy, approximately 50% of the damage was still present

  5. Molecular conductance of double-stranded DNA evaluated by electrochemical capacitance spectroscopy

    Science.gov (United States)

    Ribeiro, W. C.; Gonçalves, L. M.; Liébana, S.; Pividori, M. I.; Bueno, P. R.

    2016-04-01

    Conductance was measured in two different double stranded DNA (both with 20 bases), the more conducting poly(dG)-poly(dC) (ds-DNAc) and the less conducting poly(dA)-poly(dT) (ds-DNAi), by means of Electrochemical Capacitance Spectroscopy (ECS). The use of the ECS approach, exemplified herein with DNA nanowires, is equally a suitable and time-dependent advantageous alternative for conductance measurement of molecular systems, additionally allowing better understanding of the alignment existing between molecular scale conductance and electron transfer rate.Conductance was measured in two different double stranded DNA (both with 20 bases), the more conducting poly(dG)-poly(dC) (ds-DNAc) and the less conducting poly(dA)-poly(dT) (ds-DNAi), by means of Electrochemical Capacitance Spectroscopy (ECS). The use of the ECS approach, exemplified herein with DNA nanowires, is equally a suitable and time-dependent advantageous alternative for conductance measurement of molecular systems, additionally allowing better understanding of the alignment existing between molecular scale conductance and electron transfer rate. Electronic supplementary information (ESI) available. See DOI: 10.1039/c6nr01076h

  6. Merging Two Strategies for Mixed-Sequence Recognition of Double-Stranded DNA: Pseudocomplementary Invader Probes.

    Science.gov (United States)

    Anderson, Brooke A; Hrdlicka, Patrick J

    2016-04-15

    The development of molecular strategies that enable recognition of specific double-stranded DNA (dsDNA) regions has been a longstanding goal as evidenced by the emergence of triplex-forming oligonucleotides, peptide nucleic acids (PNAs), minor groove binding polyamides, and-more recently-engineered proteins such as CRISPR/Cas9. Despite this progress, an unmet need remains for simple hybridization-based probes that recognize specific mixed-sequence dsDNA regions under physiological conditions. Herein, we introduce pseudocomplementary Invader probes as a step in this direction. These double-stranded probes are chimeras between pseudocomplementary DNA (pcDNA) and Invader probes, which are activated for mixed-sequence dsDNA-recognition through the introduction of pseudocomplementary base pairs comprised of 2-thiothymine and 2,6-diaminopurine, and +1 interstrand zipper arrangements of intercalator-functionalized nucleotides, respectively. We demonstrate that certain pseudocomplementary Invader probe designs result in very efficient and specific recognition of model dsDNA targets in buffers of high ionic strength. These chimeric probes, therefore, present themselves as a promising strategy for mixed-sequence recognition of dsDNA targets for applications in molecular biology and nucleic acid diagnostics. PMID:26998918

  7. Interaction of fragmented double-stranded DNA with carbon nanotubes in aqueous solution

    Science.gov (United States)

    Gladchenko, G. O.; Karachevtsev, M. V.; Leontiev, V. S.; Valeev, V. A.; Glamazda, A. Yu.; Plokhotnichenko, A. M.; Stepanian, S. G.

    Aqueous suspensions of ultrasonically fragmented double-stranded (fds-) DNA and single-walled carbon nanotubes (SWNTs) have been investigated by UV- and IR-absorption, NIR-emission and Raman spectroscopy. According to gel-electrophoresis, the lengths of the polymer fragments were 100-500 base pairs. Analysis of IR and UV data indicates the presence of both double-stranded (ds) and single-stranded (ss)-regions in the fragments. SWNT complex with DNA was revealed by NIR-emission and Raman spectroscopy. It turned out that fds-DNA is less efficient in holding nanotubes in the aqueous solution than ss-DNA. From the UV-data, the character of the helix-coil transition is seen to be like that for fds-DNA off and on nanotube, however, DNA thermostability increased in this latter case. The effective charge density on the DNA sugar-phosphate backbone of the fds-DNA:SWNT hybrid was less than that of DNA alone. Spectroscopic data can be explained by a model in which the formation of hybrids starts due to the interaction between untwisted ss-regions of DNA and the nanotube: the strands wrap on the tube and thus create an 'anchor' for the whole polymer. The ds-part of the polymer is located close to the nanotube.

  8. 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.

  9. Protection against {sup 131}I-induced Double Strand DNA Breaks in Thyroid Cells

    Energy Technology Data Exchange (ETDEWEB)

    Hershman, J.M.; Okunyan, A.; Cannon, S.; Hogen, V. [Endocrinology, UCLA-VA, Los Angeles (United States); Rivina, Y. [Radiation Biology, UCLA, Los Angeles (United States)

    2012-07-01

    Radioiodine-131 (I{sup 131}) released from nuclear reactor accidents has dramatically increased the incidence of papillary thyroid cancer in exposed individuals, especially young children. The accepted measure for prevention of radiation-induced thyroid cancer is potassium iodide tablets that contain 100 mg iodide taken daily to block thyroid uptake of I{sup 131}. The deposition of ionizing radiation in cells results in double-strand DNA breaks (DSB) at fragile sites, and this early event can generate oncogenic rearrangements that eventually cause the cancer. We have developed a thyroid cell model to quantify the mitogenic effect of I{sup 131}. I{sup 131} causes double strand DNA breaks in FRTL-5 cells detected by 53BP1 or gamma H2AX and had no effect on cells that do not transport iodide. Perchlorate, iodide, and thiocyanate protect against DSB induced by I{sup 131}. Preincubation with the anion or radioprotective compounds prevents DSB; delayed addition of the anion is much less effective. These data provide a basis for studies of radioprotection against DSB induced by I{sup 131} in animals in order to refine the prevention of thyroid cancer resulting from nuclear fallout

  10. γ-ray dose rate effect in DNA double-strand break repair deficient murine cells

    International Nuclear Information System (INIS)

    Objective: To analyze the dose rate effect and potentially lethal damage repair in DNA double-strand break repair deficient murine cells (SCID) irradiated by γ-ray. Methods: The wild type (CB.17+/+) and SCID cells were exposed to γ-ray at high and low dose rates. The high dose rate exposure was fractionated into two equal doses at 24 h intervals. The survival rates of irradiated cells were calculated by clone-forming analysis. Results: When γ-ray was given to wild type (CB.17+/+) cells in two fractions at 24 h intervals, the survival rate was significantly higher than that when the same total dose was given singly. In contrast, there was no difference in the survival rates between the single and fractionated exposure in SCID cells. SCID cells were more sensitive than CB.17+/+ cells to both low and high dose rates γ-ray exposure for cell killing. The survival rate by low dose rate exposure was significantly higher than that by high dose rate exposure, not only in CB.17+/+ cells but also in SCID cells. Conclusions: SCID cells are deficient in repairing γ-ray induced double-strand breaks. There is dose rate effect in both SCID and CB.17+/+ cells

  11. 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. PMID:26895222

  12. 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.

  13. Survivin inhibition and DNA double-strand break repair: A molecular mechanism to overcome radioresistance in glioblastoma

    International Nuclear Information System (INIS)

    Background and purpose: Gliomas display prime examples of ionizing radiation (IR) resistant tumors. The IAP Survivin is reported to be critically involved in radiation resistance by anti-apoptotic and by caspase-independent mechanisms. The present study aimed to elucidate an interrelationship between Survivin’s cellular localization and DNA damage repair in glioma cells. Material and methods: Cellular distribution and nuclear complex formation were assayed by immunoblotting, immunofluorescence staining and co-immunoprecipitation of Survivin bound proteins in LN229 glioblastoma cells. Apoptosis induction, survival and DNA repair following IR were assayed by means of caspase3/7 activity, clonogenic assay, γ-H2AX/53BP1 foci formation, single cell gel electrophoresis assay, and DNA-PKcs kinase assay in the presence of Survivin siRNA or over expression of Survivin-GFP. Results: Following irradiation, we observed a nuclear accumulation and a direct interrelationship between Survivin, MDC1, γ-H2AX, 53BP1 and DNA-PKcs, which was confirmed by immunofluorescence co-localization. Survivin downregulation by siRNA resulted in an increased apoptotic fraction, decreased clonogenic survival and increased DNA-damage, as demonstrated by higher amount of DNA breaks and an increased amount of γ-H2AX/53BP1 foci post irradiation. Furthermore, we detected in Survivin-depleted LN229 cells a hampered S2056 (auto)phosphorylation and a significantly decreased DNA-PKcs kinase activity. Conclusion: Nuclear accumulation of Survivin and interaction with components of the DNA-double-strand break (DSB) repair machinery indicates Survivin to regulate DSB damage repair that leads to a significant improvement of survival of LN229 glioblastoma cells.

  14. Viroid-like RNAs from cherry trees affected by leaf scorch disease: further data supporting their association with mycoviral double-stranded RNAs.

    Science.gov (United States)

    Minoia, S; Navarro, B; Covelli, L; Barone, M; García-Becedas, M T; Ragozzino, A; Alioto, D; Flores, R; Di Serio, F

    2014-03-01

    Cherry trees from Spain affected by cherry leaf scorch (CLS), a fungal disease proposed to be caused by Apiognomonia erythrostoma, show symptoms (translucent-chlorotic leaf spots evolving into rusty areas) very similar to those of cherry chlorotic rusty spot disease (CCRS) and Amasya cherry disease, reported in Italy and Turkey, respectively. The three maladies are closely associated with 10-12 double-stranded viral RNAs, and CCRS is additionally associated with two cherry small circular RNAs (cscRNA1 and cscRNA2). Here, we report that a small viroid-like RNA similar to the CCRS-associated cscRNA1 is also present in CLS-affected trees, thus extending the link between the two diseases. Both CLS and CCRS cscRNA1 elements have common features, including sequence identity (88 %), a predicted quasi rod-like conformation with short bifurcations at both termini, and the presence of hammerhead ribozymes in the strands of both polarities. However, cscRNA2, apparently derived from cscRNA1 by deletion of a short hairpin, was not detected in CLS-affected material. Although the biological nature of cscRNAs is unknown, the identification of at least cscRNA1 in different cherry cultivars and in two distinct geographic areas (Spain and Italy), always in close association with the same mycoviral dsRNAs, supports that these viroid-like RNAs could be satellite RNAs. PMID:24757711

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

    OpenAIRE

    Fernet Marie; Giocanti Nicole; Noël Georges; Mégnin-Chanet Frédérique; Favaudon Vincent

    2003-01-01

    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...

  16. The mechanism of DNA double strand break and its repair induced by heavy ion irradiation

    International Nuclear Information System (INIS)

    In order to understand the relationship between DNA double strand break (DSB) repair proteins and the regulation of centrosome amplifications in cells irradiated with heavy ions, the behavior of centrosome was monitored by an immuno-fluorescence technique using γ-tublin antibodies. In AT cells, the amplification of centrosome clearly increased with X-irradiation compared with normal cells. Inhibition of ATM, DNA-PK and PI3K also increased the amplification of centrosome with X-irradiation in normal cells. In contrast, with heavy ion radiation, the inhibition of DNA-PK did not affects the amplification of centrosome in normal cells. Our results suggest a difference in the mechanism of centrosome amplification after X-irradiation and high linear energy transfer (LET) heavy ion irradiation. (author)

  17. The mechanism of DNA double strand break repair induced by heavy ion radiation

    International Nuclear Information System (INIS)

    In order to understand the unique molecular characteristics associated with DNA double strand break (DSB) repair in cells irradiated with high linear energy transfer (LET) heavy ion radiation, we have studied phosphorylation status of DNA-PKcs at Thr 2609 in human cells with various ATM status. After high LET carbon ion irradiation, this threonine site was not well phosphorylated in cells with ATM inhibitor, while the phosphorylation was always observed in cells exposed to X-rays irrespective of ATM status; this was demonstrated both by immune-staining and Western blotting. Another important finding is that ATM seems to play a precious role in complex DNA damage such as one induced by high LET irradiation; this kind of ATM property has only been shown with low LET radiation. (author)

  18. The mechanism of DNA double strand break repair induced by heavy ion radiation

    International Nuclear Information System (INIS)

    The combined effect of 17AAG (an Hsp90 inhibitor) and heavy ion irradiation was studied in vitro and in vivo. 17AAG was known to cause radio-sensitization in tumor cells irradiated with low linear energy transfer (LET) radiation by inhibiting DNA double strand break (DSB) repair. Our study shows the enhanced cell killing by the combination of 17AAG and carbon ion treatment, and this was also shown in in vivo mouse xenograft model. The cause of this may be S-phase related and involve non-DSB damage. The role played by DSB repair related critical proteins such as ATM, XLF and artemis was also investigated in cells exposed to heavy ions. Our study indicate that artemis plays a role in the slow component of DSB repair in cells with low LET, but this is not the case with high LET irradiation. (author)

  19. Double strand break repair: two mechanisms in competition but tightly linked to cell cycle

    International Nuclear Information System (INIS)

    DNA double strand breaks (DSB) are highly toxic damage although they can be induced to create genetic diversity. Two distinct pathways can repair DSB: Homologous Recombination (HR) and Non Homologous End Joining (NHEJ). If un- or mis-repaired, this damage can lead to cancer. Thus, it is essential to investigate how these two pathways are regulated for DSB repair. NHEJ inhibition leads to HR DSB repair stimulation. However, this channeling to HR is tightly linked to cell cycle since NHEJ and HR are active in G1/early S and late S/G2, respectively. Our results suggest that G1-unrepaired DSB go through S phase to be repaired by HR in G2. Those results allow a better understanding of DSB repair mechanisms regulation. (author)

  20. Biological defense mechanisms against DNA double-strand break and their possible medical applications

    International Nuclear Information System (INIS)

    Radiation is now widely used for clinical diagnosis and therapeutics. On the other hand, radiation influences various tissues represented by immunological and reproductive systems, and is also recognized as one of the cause of carcinogenesis. Such pleiotropic effects of radiation are mediated through generation of damages on DNA molecule, vitally important genetic macromolecule. Among various types of DNA damages, double-strand break (DSB) is considered most critical and, therefore, responsible for biological effects. DSB is repaired mainly through two pathways: non-homologous end joining (NHEJ) and homologous recombination (HR). Understanding of these mechanisms has been greatly deepened in past 20 years and is now providing a promising approach toward cancer therapy. We have studied the mechanisms of NHEJ, focusing especially on the role of phosphorylation and the assembly of machinery therein, which will be introduced below. (author)

  1. Ultrafast chemical repair of DNA single and double strand break precursors in irradiated V79 cells

    International Nuclear Information System (INIS)

    The fast kinetics of reactions of free radical precursors of DNA single strand breaks (ssb) and double strand breaks (dsb) have been determined in Chinese hamster V79 cells by fast mixing and irradiation methods using the alkaline unwinding technique to assay breaks. Fast chemical repair of oxygen-dependent ssb and dsb precursors was observed and approached completion within 10 to 20 ms of irradiation. Treatment of cells with the glutathione synthesis blocking agent, buthionine sulphoximine, showed that approximately half of the chemical repair was attributable to intracellular non-protein thiols. The nature of the residual repair is obscure, but it is apparently not attributable to non-protein thiols. Similar repair rates and thiol dependences were also found for cell kill. With all three endpoints, oxygen competes with and blocks the chemical repair. 36 refs., 6 figs., 1 tab

  2. Carbon ion induced DNA double-strand breaks in melanophore B16

    International Nuclear Information System (INIS)

    DNA double-strand breaks (DSBs) in melanophore B16 induced by plateau and extended Bragg peak of 75 MeV/u 12C6+ 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 B16. Percentage of DNA released from plug (PR) increased and trended towards a quasi-plateau ∝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.)

  3. APOBEC3 cytidine deaminases in double-strand DNA break repair and cancer promotion.

    Science.gov (United States)

    Nowarski, Roni; Kotler, Moshe

    2013-06-15

    High frequency of cytidine to thymidine conversions was identified in the genome of several types of cancer cells. In breast cancer cells, these mutations are clustered in long DNA regions associated with single-strand DNA (ssDNA), double-strand DNA breaks (DSB), and genomic rearrangements. The observed mutational pattern resembles the deamination signature of cytidine to uridine carried out by members of the APOBEC3 family of cellular deaminases. Consistently, APOBEC3B (A3B) was recently identified as the mutational source in breast cancer cells. A3G is another member of the cytidine deaminases family predominantly expressed in lymphoma cells, where it is involved in mutational DSB repair following ionizing radiation treatments. This activity provides us with a new paradigm for cancer cell survival and tumor promotion and a mechanistic link between ssDNA, DSBs, and clustered mutations. Cancer Res; 73(12); 3494-8. ©2013 AACR. PMID:23598277

  4. 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.

  5. Oxygen effect for DNA double-strand break induction determined by pulsed-field gel electrophoresis

    International Nuclear Information System (INIS)

    The induction of DNA double-strand breaks (dsb) following irradiation under oxygenated and hypoxic conditions with and without misonidazole was measured by pulsed-field gel electrophoresis (PFGE) in a human bladder carcinoma cell line. The dose-response curve for DNA dsb detection by PFGE was biphasic with an apparent reduction in rate of dsb induced with dose. Oxygen enhancement ratios (OER) for cell survival (at a surviving fraction of 0.1) and for DNA damage assessed by PFGE (at 80% retained) were 2.0 and 3.0 respectively. Dose-modifying factors for misonidazole (15 mM), of 1.9 (survival) and 2.4 (DNA damage) were found. (author)

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

    Science.gov (United States)

    Li, Jing; Xu, Xingzhi

    2016-07-01

    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. PMID:27217474

  7. [p53 activation by PI-3K family kinases after DNA double-strand breaks].

    Science.gov (United States)

    Pernin, D; Uhrhammer, N; Verrelle, P; Bignon, Y J; Bay, J O

    2000-09-01

    p53 plays a central role in the cellular response to DNA double-strand breaks (DSBs), and to DNA damage in general. The protein kinases ATM, ATR and DNA-PK detect DSBs and transmit this information to p53 by phosphorylation. This phosphorylation dissociates p53 from its negative regulator, mdm2. p53 then undergoes further modification and activates transcription of the genes responsible for cell cycle arrest. In certain circumstances, p53 also activates transcription of the genes responsible for apoptosis. The dysfunction of this cascade of events is oncogenic, with P53 itself being the most commonly mutated gene in malignant cells, although mutations in both the DNA damage sensors and cell cycle checkpoint and apoptosis effectors are frequent. A more complete understanding of p53 and the proteins it interacts with may allow the development of new cancer treatments. PMID:11038413

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

    DEFF Research Database (Denmark)

    Singh, Dharmendra Kumar; Karmakar, Parimal; Aamann, Maria Diget; Schurman, Shepherd H; May, Alfred; Croteau, Deborah L; Burks, Lynnette; Plon, Sharon E; Bohr, Vilhelm A

    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...

  9. Secondary structure of double-stranded DNA under stretching: elucidation of the stretched form.

    Science.gov (United States)

    Maaloum, M; Beker, A-F; Muller, P

    2011-03-01

    Almost two decades ago, measurements of force versus extension on isolated double-stranded DNA molecules revealed a force plateau. This unusual stretching phenomenon in DNA suggests that the long molecules may be extended from the usual B form into a new conformation. Different models have been proposed to describe the nature of DNA in its stretched form, S-DNA. Using atomic force microscopy combined with a molecular combing method, we identified the structure of λ-phage DNA for different stretching values. We provide strong evidence for the existence of a first-order transition between B form and S form. Beyond a certain extension of the natural length, DNA molecules adopt a new double-helix conformation characterized by a diameter of 1.2 nm and a helical pitch of 18 nm. PMID:21517521

  10. Ab initio bubble-driven denaturation of double-stranded DNA: Self-mechanical theory.

    Science.gov (United States)

    Kuetche, Victor K

    2016-07-21

    Among the different theoretical models of the open-site-driven DNA-denaturation found in the literature, very few interests are actually paid to the fundamental unzipping process of the double-stranded DNA within the vicinity of its ground state condensate. In this paper, we address an alternative to better understand the process of denaturation of such a macromolecule by investigating the onset of its dynamics around its equilibrium state. We show that from the initiation of the transcription bubble by the promoter to the termination state, the open-states of the strands evolve dynamically while generating some localized waveguide channels with elastic scattering properties. We properly discuss the nonlinear dynamics of these structures within the viewpoint of the self-mechanical theory while inferring to the physical structure of the findings and their potential issues. PMID:27113786

  11. Coupling end resection with the checkpoint response at DNA double-strand breaks.

    Science.gov (United States)

    Villa, Matteo; Cassani, Corinne; Gobbini, Elisa; Bonetti, Diego; Longhese, Maria Pia

    2016-10-01

    DNA double-strand breaks (DSBs) are a nasty form of damage that needs to be repaired to ensure genome stability. The DSB ends can undergo a strand-biased nucleolytic processing (resection) to generate 3'-ended single-stranded DNA (ssDNA) that channels DSB repair into homologous recombination. Generation of ssDNA also triggers the activation of the DNA damage checkpoint, which couples cell cycle progression with DSB repair. The checkpoint response is intimately linked to DSB resection, as some checkpoint proteins regulate the resection process. The present review will highlight recent works on the mechanism and regulation of DSB resection and its interplays with checkpoint activation/inactivation in budding yeast. PMID:27141941

  12. Inactivation, DNA double strand break induction and their rejoining in bacterial cells irradiated with heavy ions

    Science.gov (United States)

    Schaefer, M.; Zimmermann, H.; Schmitz, C.

    1994-01-01

    Besides inactivation one of the major interests in our experiments is to study the primary damage in the DNA double strand breaks (DSB) after heavy ion irradiation. These damages lead not only to cell death but also under repair activities to mutations. In further experiments we have investigated the inactivation with two different strains of Deinococcus radiodurans (R1, Rec 30) and the induction of DSB as well as the rejoining of DSB in stationary cells of E. coli (strain B/r) irradiated with radiations of different quality. In the latter case irradiations were done so that the cell survival was roughly at the same level. We measured the DSB using the pulse field gelelectrophoresis which allows to separate between intact (circular) and damaged (linear) DNA. The irradiated cells were transferred to NB medium and incubated for different times to allow rejoining.

  13. Functional significance of the interaction with Ku in DNA double-strand break recognition of XLF.

    Science.gov (United States)

    Yano, Ken-ichi; Morotomi-Yano, Keiko; Lee, Kyung-Jong; Chen, David J

    2011-03-23

    Ku heterodimer is essential for the repair of DNA double-strand breaks (DSBs) by non-homologous end-joining (NHEJ). Ku recruits XLF, also known as Cernunnos, to DSBs. Here we report domain analyses of Ku-XLF interaction. The heterodimeric domain of Ku was found to be sufficient for the recruitment of XLF to DSBs and for the interaction of Ku with XLF. A small C-terminal deletion of XLF completely abolished recruitment of XLF to DSBs and Ku-XLF interaction. This deletion also led to marked reduction of XLF-XRCC4 interaction although the XRCC4-binding site on the XLF N-terminal domain remained intact. These results demonstrate the significance of Ku-XLF interaction in the molecular assembly of NHEJ factors. PMID:21349273

  14. 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.

  15. Effects of the environment on the electric conductivity of double-stranded DNA molecules.

    Science.gov (United States)

    Malyshev, A V; Díaz, E; Domínguez-Adame, F; Malyshev, V A

    2009-08-19

    We present a theoretical analysis of the effects of the environment on charge transport in double-stranded synthetic poly(G)-poly(C) DNA molecules attached to two ideal leads. Coupling of the DNA to the environment results in two effects: (i) localization of carrier functions due to static disorder and (ii) phonon-induced scattering of the carriers between the localized states, resulting in hopping conductivity. A nonlinear Pauli master equation for populations of localized states is used to describe the hopping transport and calculate the electric current as a function of the applied bias. We demonstrate that, although the electronic gap in the density of states shrinks as the disorder increases, the voltage gap in the I-V characteristics becomes wider. A simple physical explanation of this effect is provided. PMID:21828599

  16. 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.

  17. RNF20-SNF2H Pathway of Chromatin Relaxation in DNA Double-Strand Break Repair

    Directory of Open Access Journals (Sweden)

    Akihiro Kato

    2015-07-01

    Full Text Available Rapid progress in the study on the association of histone modifications with chromatin remodeling factors has broadened our understanding of chromatin dynamics in DNA transactions. In DNA double-strand break (DSB repair, the well-known mark of histones is the phosphorylation of the H2A variant, H2AX, which has been used as a surrogate marker of DSBs. The ubiquitylation of histone H2B by RNF20 E3 ligase was recently found to be a DNA damage-induced histone modification. This modification is required for DSB repair and regulated by a distinctive pathway from that of histone H2AX phosphorylation. Moreover, the connection between H2B ubiquitylation and the chromatin remodeling activity of SNF2H has been elucidated. In this review, we summarize the current knowledge of RNF20-mediated processes and the molecular link to H2AX-mediated processes during DSB repair.

  18. Visualization of DNA Double-Strand Break Repair at the Single-Molecule Level

    Energy Technology Data Exchange (ETDEWEB)

    Dynan, William S.; Li, Shuyi; Mernaugh, Raymond; Wragg, Stephanie; Takeda, Yoshihiko

    2003-03-27

    Exposure to low doses of ionizing radiation is universal. The signature injury from ionizing radiation exposure is induction of DNA double-strand breaks (DSBs). The first line of defense against DSBs is direct ligation of broken DNA ends via the nonhomologous end-joining pathway. Because even a relatively high environmental exposure induces only a few DSBs per cell, our current understanding of the response to this exposure is limited by the ability to measure DSB repair events reliably in situ at a single-molecule level. To address this need, we have taken advantage of biological amplification, measuring relocalization of proteins and detection of protein phosphorylation as a surrogate for detection of broken ends themselves. We describe the use of specific antibodies to investigate the kinetics and mechanism of repair of very small numbers of DSBs in human cells by the nonhomologous end-joining pathway.

  19. 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.)

  20. 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. PMID:26159358

  1. Secondary structure of double-stranded DNA under stretching: Elucidation of the stretched form

    International Nuclear Information System (INIS)

    Almost two decades ago, measurements of force versus extension on isolated double-stranded DNA molecules revealed a force plateau. This unusual stretching phenomenon in DNA suggests that the long molecules may be extended from the usual B form into a new conformation. Different models have been proposed to describe the nature of DNA in its stretched form, S-DNA. Using atomic force microscopy combined with a molecular combing method, we identified the structure of λ-phage DNA for different stretching values. We provide strong evidence for the existence of a first-order transition between B form and S form. Beyond a certain extension of the natural length, DNA molecules adopt a new double-helix conformation characterized by a diameter of 1.2 nm and a helical pitch of18 nm.

  2. Correlation between residual level of DNA double-strand breaks and the radiosensitivity of cancer cells

    International Nuclear Information System (INIS)

    Objective: To understand the variation of the DNA double-strand break rejoining capacity among different cultured cancer cell lines and the primary cancer cells from brain cancer patients, and to explore the predictor of radiotherapy responses of cancers. Methods: DNA double-strand breaks (DSBs) were induced by 60Co γ-irradiation. Pulsed-field gel electrophoresis was used to analyze the initial production and rejoining of DNA DSBs. Radiosensitivity was determined by in vitro assay of clonogenic-forming capacity. Results: A wide variation of radiosensitivity, e.g. the survival parameter of Do varied from 0.65 to 2.15 Gy, was displayed among the eight cell lines derived from different type of cancers. Although differential level of initial DNA DSBs induced by 20 Gy γ-rays was observed among various cell lines, it was not correlated with the radiosensitivity. The deficiency of DNA DSB rejoining in radiosensitive cell lines was shown either in the early rapid-rejoining phase (SX-10 cells) or in the late slow-rejoining phase (A2780 cells). A significant relationship was observed between the residual level of DNA DSBs measured at 2 h post-20 Gy irradiation and the cellular radiosensitivity (D0 or SF2). The kinetic curves of rejoining DNA DSBs in the primary human brain tumor cells indicated a variation on DSB rejoining capacity among different individual tumor. The residual level of DNA DSBs after 2 h of rejoining post 20 Gy irradiation in primary human brain tumor cells is compatible to the results obtained in vitro culture cancer cell lines. Conclusions: The residual level of DNA DSBs is correlated with radioresistance of cancer cells, and the residual DNA damage is a useful parameter in predicting the response of tumor tissue to radiotherapy. (authors)

  3. Numt-mediated double-strand break repair mitigates deletions during primate genome evolution.

    Directory of Open Access Journals (Sweden)

    Einat Hazkani-Covo

    2008-10-01

    Full Text Available Non-homologous end joining (NHEJ is the major mechanism of double-strand break repair (DSBR in mammalian cells. NHEJ has traditionally been inferred from experimental systems involving induced double strand breaks (DSBs. Whether or not the spectrum of repair events observed in experimental NHEJ reflects the repair of natural breaks by NHEJ during chromosomal evolution is an unresolved issue. In primate phylogeny, nuclear DNA sequences of mitochondrial origin, numts, are inserted into naturally occurring chromosomal breaks via NHEJ. Thus, numt integration sites harbor evidence for the mechanisms that act on the genome over evolutionary timescales. We have identified 35 and 55 lineage-specific numts in the human and chimpanzee genomes, respectively, using the rhesus monkey genome as an outgroup. One hundred and fifty two numt-chromosome fusion points were classified based on their repair patterns. Repair involving microhomology and repair leading to nucleotide additions were detected. These repair patterns are within the experimentally determined spectrum of classical NHEJ, suggesting that information from experimental systems is representative of broader genetic loci and end configurations. However, in incompatible DSBR events, small deletions always occur, whereas in 54% of numt integration events examined, no deletions were detected. Numts show a statistically significant reduction in deletion frequency, even in comparison to DSBR involving filler DNA. Therefore, numts show a unique mechanism of integration via NHEJ. Since the deletion frequency during numt insertion is low, native overhangs of chromosome breaks are preserved, allowing us to determine that 24% of the analyzed breaks are cohesive with overhangs of up to 11 bases. These data represent, to the best of our knowledge, the most comprehensive description of the structure of naturally occurring DSBs. We suggest a model in which the sealing of DSBs by numts, and probably by other filler

  4. Endonucleolytic activity directed towards 8-(2-hydroxy-2-propyl) purines in double-stranded DNA.

    Science.gov (United States)

    Livneh, Z; Elad, D; Sperling, J

    1979-11-01

    Photoalkylation of circular covalently closed DNA from phage PM2 with isopropyl alcohol by using a free radical photoinitiator and UV light of lambda greater than 305 nm led to the specific 8-substitution of purine moieties in the DNA, yielding 8-(2-hydroxy-2-propyl)adenine and 8-(2-hydroxy-2-propyl)guanine as the only detectable damage in the DNA. Using this specifically photoalkylated DNA as a substrate, we discovered in extracts of Micrococcus luteus an endonucleolytic activity that is directed towards 8-(2-hydroxy-2-propyl) purines in DNA. The activity is not a combination of a DNA-glycosylase and an apurinic site endonuclease. It is not inhibited by single-stranded DNA, by UV- or gamma-irradiated single-stranded DNA, or by normal or depurinated double-stranded DNA. however, gamma- or UV-(254 nm) irradiated double-stranded DNAs to inhibit the activity, hinting at the possibility of a common type of lesion in these damaged DNAs. Divalent cations are not required for the incising activity, and it is fully active in 1 mM EDTA, whereas caffeine and ATP cause inhibition. Extracts of mutant M. luteus lacking pyrimidine-dimer-directed endonucleases were found to contain the endonucleolytic activity in levels comparable to those present in the wild type. After the incision, we could demonstrate the specific excision of the 8-alkylated purines from the damaged DNA. The special conformational consequences of the 8-alkylation of purines, at the nucleotide level, namely their nonregular syn conformation, suggest that it is the distortion in the DNA that is recognized by the endonuclease. PMID:293658

  5. An alternative mechanism for radioprotection by dimethyl sulfoxide. Possible facilitation of DNA double-strand break repair

    International Nuclear Information System (INIS)

    The radioprotective effects of dimethyl sulfoxide (DMSO) have been known for many years, and the suppression of hydroxyl (OH) radicals induced by ionizing radiation has been thought to be the main cause of this effect. However, the DMSO concentration used was very high, and might be toxic, in earlier studies. In the present study, we administered a lower, non-toxic concentration (0.5%, id est (i.e.), 64 mM) of DMSO before irradiation and examined its radioprotective effects. Colony formation assay and micronucleus assay showed significant radioprotective effects in Chinese hamster ovary (CHO), but not in xrs5, which is defective in the repair function of DNA double-strand breaks. The levels of phosphorylated H2AX and the formation of 53BP1 foci 15 minutes after irradiation, which might reflect initial DNA double-strand breaks, in DMSO-treated CHO cells were similar to those in non-treated cells, suggesting that the radioprotective effects were not attributable to the suppression of general indirect action in the lower concentration of DMSO. On the other hand, 2 hours after irradiation, the average number of 53BP1 foci, which might reflect residual DNA double-strand breaks, was significantly decreased in DMSO-treated CHO cells compared to non-treated cells. The results indicated that low concentration of DMSO exerts radioprotective effects through the facilitation of DNA double-strand break repair rather than through the suppression of indirect action. (author)

  6. Formation of radiation-induced DNA breaks: the ratio of double-strand breaks to single-strand breaks

    International Nuclear Information System (INIS)

    Ionizing radiation causes the formation of strand breaks in cellular DNA, as well as other types of lesions in the chromatin of cells. Some of the earliest investigations of the molecular basis of radiation-induced damage and the implications of enzymatic repair were done by Dr. H. S. Kaplan. Because it is difficult to assay for DNA lesions in the large mammalian genome, the authors have developed a method of assaying for DNA double-strand breaks in the supercoiled nucleosome-complexed Simian virus 40 (SV40) genome, irradiated intracellularly. In this communication they present their measurements of the DNA double-strand breaks (DSBs) to single-strand breaks (SSBs) ratio obtained from the intracellularly irradiated SV40 genome. After cobalt gamma ray and X ray irradiations, this ratio is about 1/10. Their methods and results are compared with pertinent data in the literature. If the DSBs/SSBs ratio of 1/10 for cellular chromatin is correct, a substantial number of DNA double-strand breaks are formed in a mammalian cell after moderate doses (1 Gy) of radiation. The implications of different types of DNA double-strand breaks are discussed

  7. 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 ...

  8. 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; Bekker-Jensen, Simon; Mailand, Niels

    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...

  9. 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.

  10. Modeling the yield of double-strand breaks due to formation of multiply damaged sites in irradiated plasmid DNA

    International Nuclear Information System (INIS)

    Although double-strand breaks have long been recognized as an important type of DNa lesion, it is well established that this broad class of damage does not correlate well with indicators of the effectiveness of radiation as the cellular level. Assays of double-strand breaks do not distinguish the degree of complexity or clustering of singly damaged sites produced in a single energy deposition event, which is currently hypothesized to be key to understanding cellular end points. As a step toward this understanding, double-strand breaks that are formed proportionally to dose in plasmid DNA are analyzed from the mechanistic aspect to evaluate the yield that arises from multiply damaged sites as hypothesized by Ward (Prog. Nucleic Acid Res. Mol. Biol. 35, 95-125, 1988) and Goodhead (Int. J. Radiat. Biol. 65, 7-17, 1994) as opposed to the yield that arises form single hydroxyl radicals as hypothesized by Siddiqi and Bothe (Radiat. Res. 112, 449-463, 1987). For low-LET radiation such as γ rays, the importance of multiply damaged sites is shown to increase with the solution's hydroxyl radical scavenging capacity. For moderately high-LET radiation such as 100 keV/μm helium ions, a much different behavior is observed. In this case, a large fraction of double-strand breaks are formed as a result of multiply damaged sties over a broad range of scavenging conditions. Results also indicate that the RBE for common cellular end points correlates more closely with the RBE for common cellular end points correlates more closely with the RBE for multiply damaged sites than with the RBE for total double-strand breaks over a range of LET up to at least 100 keV/μm. 22 refs., 3 figs., 2 tabs

  11. Repair of DNA double-strand breaks in Escherichia coli cells requires synthesis of proteins that can be induced by UV light

    International Nuclear Information System (INIS)

    The repair of DNA double-strand breaks in Escherichia coli cells irradiated with γ rays occurs only after new proteins are synthesized in response to damage introduced in the genome DNA. One protein whose synthesis is thus induced is the recA protein, and previous work has shown that recA- cells do not repair double-strand breaks. However, inducing recA protein by treating cells with nalidixic acid does not induce repair of double-strand breaks, so this repair requires more than the presence of the recA protein. When repair of double-strand breaks is blocked, the genome DNA is degraded by an endonuclease-like action. Evidence is presented to show that the inducible inhibition of DNA degradation after x-irradiation [Pollard, E.C. and Randall, E.P. (1973) Radiat. Res. 55, 265] is probably caused by the inducible repair of DNA double-strand breaks

  12. Hepatitis C Virus (HCV) NS5B Nonnucleoside Inhibitors Specifically Block Single-Stranded Viral RNA Synthesis Catalyzed by HCV Replication Complexes In Vitro▿

    OpenAIRE

    Yang, Wengang; Sun, Yongnian; Phadke, Avinash; Deshpande, Milind; Huang, Mingjun

    2006-01-01

    Replication complexes of hepatitis C virus synthesized two major species of viral RNA in vitro, double stranded and single stranded. NS5B nonnucleoside inhibitors inhibited dose dependently the synthesis of single-stranded RNA but not double-stranded RNA. Moreover, replication complexes carrying a mutation resistant to a nonnucleoside inhibitor lost their susceptibilities to the inhibitor.

  13. Genetic polymorphisms of DNA double strand break gene Ku70 and gastric cancer in Taiwan

    Directory of Open Access Journals (Sweden)

    Chang Wen-Shin

    2011-05-01

    Full Text Available Abstract Background and aim The DNA repair gene Ku70, an important member of non-homologous end-joining repair system, is thought to play an important role in the repairing of DNA double strand breaks. It is known that defects in double strand break repair capacity can lead to irreversible genomic instability. However, the polymorphic variants of Ku70, have never been reported about their association with gastric cancer susceptibility. Methods In this hospital-based case-control study, the associations of Ku70 promoter T-991C (rs5751129, promoter G-57C (rs2267437, promoter A-31G (rs132770, and intron 3 (rs132774 polymorphisms with gastric cancer risk in a Taiwanese population were investigated. In total, 136 patients with gastric cancer and 560 age- and gender-matched healthy controls recruited from the China Medical Hospital in Taiwan were genotyped. Results As for Ku70 promoter T-991C, the ORs after adjusted by age and gender of the people carrying TC and CC genotypes were 2.41 (95% CI = 1.53-3.88 and 3.21 (95% CI = 0.96-9.41 respectively, compared to those carrying TT wild-type genotype. The P for trend was significant (P Ku70 promoter T-991C polymorphism and the risk for gastric cancer was also significant (adjusted OR = 2.48, 95% CI = 1.74-3.92. When stratified by age and gender, the association was restricted to those at the age of 55 or elder of age (TC vs TT: adjusted OR = 2.52, 95% CI = 1.37-4.68, P = 0.0139 and male (TC vs TT: adjusted OR = 2.58, 95% CI = 1.33-4.47, P = 0.0085. As for the other three polymorphisms, there was no difference between both groups in the distributions of their genotype frequencies. Conclusion In conclusion, the Ku70 promoter T-991C (rs5751129, but not the Ku70 promoter C-57G (rs2267437, promoter A-31G (rs132770 or intron 3 (rs132774, is associated with gastric cancer susceptibility. This polymorphism may be a novel useful marker for gastric carcinogenesis.

  14. Molecular characterization of a complex site-specific radiation-induced DNA double-strand break

    International Nuclear Information System (INIS)

    Full text: Radiation lethality is a function of radiation-induced DNA double-strand breaks (DSB). Current models propose the lethality of a DSB to be a function of its structural complexity. We present here for the first time a map of damage associated with a site-specific double-strand break produced by decay of 125I in a plasmid bound by a 125I-labeled triplex forming oligonucleotide (125 I-TFO). The E. coli DNA repair enzymes, endonuclease IV (endo IV), endonuclease III (endo III), and formamidopyrimidine-DNA glycosylase (Fpg), which recognize AP sites, and pyrimidine and purine base damage respectively, were used as probes in this study. 125I-TFO bound plasmid was incubated with and without DMSO at -80 deg C for 1 month. No significant difference in DSB yield was observed under these conditions. A 32 base pair fragment from the upstream side of the decay site was isolated by restriction digestion and enzymatically probed to identify damage sites. Endo IV treatment of the 5'-end labeled upper strand indicated clustering of AP sites within 3 bases downstream and 7 bases upstream of the targeted base. Also, repeated experiments consistently detected an AP site 4 bases upstream of the 125 I target base. This was further supported by complementary results with the 3'-end labeled upper strand. Endo IV analysis of the lower strand also shows clustering of AP sites near the DSB end. Endo III and Fpg probing demonstrated that base damage is also clustered near the targeted break site. DSBs produced in the absence of DMSO displayed a different pattern of enzyme sensitive damage than those produced in the presence of DMSO. Identification of specific base damage types within the restriction fragment containing the DSB end was achieved with GC/MS. Base damage consisted of 8-hydroguanine, 8-hydroxyadenine, and 5-hydroxycytosine. These lesions were observed at relative yields of 8-hydroguanine and 5-hydroxycytosine to 8-hydroxyadenine of 7.4:1 and 4.7:1, respectively, in the

  15. Molecular characterization of a complex site-specific radiation-induced DNA double-strand break

    International Nuclear Information System (INIS)

    Radiation lethality is a function of radiation-induced DNA double-strand breaks (DSB). Current models propose the lethality of a DSB to be a function of its structural complexity. We present here for the first time a map of damage associated with a site-specific double-strand break produced by decay of 125I in a plasmid bound by a 125I-labeled triplex forming oligonucleotide ( 125I-TFO). The E. coli DNA repair enzymes, endonuclease IV (endo IV), endonuclease III (endo III), and formamidopyrimidine-DNA glycosylase (Fpg), which recognize AP sites, and pyrimidine and purine base damage respectively, were used as probes in this study. 125I-TFO bound plasmid was incubated with and without DMSO at -80 deg C for 1 month. No significant difference in DSB yield was observed under these conditions. A 32 base pair fragment from the upstream side of the decay site was isolated by restriction digestion and enzymatically probed to identify damage sites. Endo IV treatment of the 5'-end labeled upper strand indicated clustering of AP sites within 3 bases downstream and 7 bases upstream of the targeted base. Also, repeated experiments consistently detected an AP site 4 bases upstream of the 125Itarget base. This was further supported by complementary results with the 3'-end labeled upper strand. Endo IV analysis of the lower strand also shows clustering of AP sites near the DSB end. Endo III and Fpg probing demonstrated that base damage is also clustered near the targeted break site. DSBs produced in the absence of DMSO displayed a different pattern of enzyme sensitive damage than those produced in the presence of DMSO. Identification of specific base damage types within the restriction fragment containing the DSB end was achieved with GC/MS. Base damage consisted of 8-hydroguanine, 8-hydroxyadenine, and 5-hydroxycytosine. These lesions were observed at relative yields of 8-hydroguanine and 5-hydroxycytosine to 8-hydroxyadenine of 7.4:1 and 4.7:1, respectively, in the absence of

  16. Controlled DNA double-strand break induction in mice reveals post-damage transcriptome stability.

    Science.gov (United States)

    Kim, Jeongkyu; Sturgill, David; Tran, Andy D; Sinclair, David A; Oberdoerffer, Philipp

    2016-04-20

    DNA double-strand breaks (DSBs) and their repair can cause extensive epigenetic changes. As a result, DSBs have been proposed to promote transcriptional and, ultimately, physiological dysfunction via both cell-intrinsic and cell-non-autonomous pathways. Studying the consequences of DSBs in higher organisms has, however, been hindered by a scarcity of tools for controlled DSB induction. Here, we describe a mouse model that allows for both tissue-specific and temporally controlled DSB formation at ∼140 defined genomic loci. Using this model, we show that DSBs promote a DNA damage signaling-dependent decrease in gene expression in primary cells specifically at break-bearing genes, which is reversed upon DSB repair. Importantly, we demonstrate that restoration of gene expression can occur independently of cell cycle progression, underlining its relevance for normal tissue maintenance. Consistent with this, we observe no evidence for persistent transcriptional repression in response to a multi-day course of continuous DSB formation and repair in mouse lymphocytesin vivo Together, our findings reveal an unexpected capacity of primary cells to maintain transcriptome integrity in response to DSBs, pointing to a limited role for DNA damage as a mediator of cell-autonomous epigenetic dysfunction. PMID:26687720

  17. 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.

  18. Rapid pairing and resegregation of distant homologous loci enables double-strand break repair in bacteria.

    Science.gov (United States)

    Badrinarayanan, Anjana; Le, Tung B K; Laub, Michael T

    2015-08-01

    Double-strand breaks (DSBs) can lead to the loss of genetic information and cell death. Although DSB repair via homologous recombination has been well characterized, the spatial organization of this process inside cells remains poorly understood, and the mechanisms used for chromosome resegregation after repair are unclear. In this paper, we introduced site-specific DSBs in Caulobacter crescentus and then used time-lapse microscopy to visualize the ensuing chromosome dynamics. Damaged loci rapidly mobilized after a DSB, pairing with their homologous partner to enable repair, before being resegregated to their original cellular locations, independent of DNA replication. Origin-proximal regions were resegregated by the ParABS system with the ParA structure needed for resegregation assembling dynamically in response to the DSB-induced movement of an origin-associated ParB away from one cell pole. Origin-distal regions were resegregated in a ParABS-independent manner and instead likely rely on a physical, spring-like force to segregate repaired loci. Collectively, our results provide a mechanistic basis for the resegregation of chromosomes after a DSB. PMID:26240183

  19. Two pathways of DNA double-strand break repair in G1 cells of Saccharomyces cerevisiae

    International Nuclear Information System (INIS)

    The G1 cells of the diploid yeast Saccharomyces cerevislae are known to be capable of a slow repair of DNA double-strand breaks (DSB) during holding the cells in a non-nutrient medium. In the present paper, it has been shown that S. cerevislae cells γ-irradiated in the G1 phase of cell cycle are capable of fast repair of DNA DSB; this process is completed within 30-40 min of holding the cells in water at 28 deg C. For this reason, the kinetics of DNA DSB repair during holding the cells in a non-nutrient medium are biphasic, i.e., the first, ''fast'' phase is completed within 30-40 min; wheras the second, ''slow'' one, within 48 h. Mutations rad51, rad52, rad54 and rad55 inhibit the fast repair of DNA DSB, whereas mutations rad50, rad53 and rad57 do not practically influence this process. It has been shown that the observed fast and slow repair of DNA DSB in the G1 diploid cells of S, cerevislae are separate pathways of DNA DSB repair in yeast

  20. 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)

  1. 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.

  2. 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.

  3. 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.

  4. 1H NMR studies of selective interactions of norfloxacin with double-stranded DNA

    International Nuclear Information System (INIS)

    The interaction of the antibiotic drug norfloxacin with double-stranded DNA containing interior 5'-CpG-3', 5'-GpC-3', and 5'-GpG-3' steps was studied by 1H NMR. The drug is in fast exchange on the NMR timescale. A highly selective broadening of the imino proton resonances assigned to central CpG steps was observed after addition of drug, indicating an intercalation-like interaction. DNA sequences with central CpG steps also displayed broadening of non-hydrogen-bonded cytosine amino protons in the major groove upon addition of norfloxacin. Furthermore, a sequence-independent selective broadening of the adenine H2 resonance and an upfield shift of the guanine amino proton resonance, both protons located in the minor groove, was observed. Two-dimensional-NOESY spectra showed that no significant structural changes were induced in the DNA by the drug. The results suggest that the planar two-ring system of norfloxacin partially intercalates into CpG steps and that the drug also exhibits non-specific groove binding

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

    International Nuclear Information System (INIS)

    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

  6. The role of homologous recombination in radiation-induced double-strand break repair

    International Nuclear Information System (INIS)

    DNA double-strand breaks (DSBs) represent the most biologically significant lesions induced by ionizing radiation (IR). HR is the predominant pathway for repairing one-ended DSBs arising in S-phase when the replication fork encounters single-stranded breaks or base damages. Here, we discuss recent findings that two-ended DSBs directly induced by X- or γ-rays in late S- or G2-phase are repaired predominantly by NHEJ, with HR only repairing a sub-fraction of such DSBs. This sub-fraction represents DSBs which localize to heterochromatic DNA regions and, which in control cells, are repaired with slow kinetics over many hours post irradiation. The observation that defined DSB populations are repaired by either NHEJ or HR suggests an assignment of specific tasks for each of the two processes. Furthermore, heavy ion induced complex DSBs, which are in general more slowly repaired than X- or γ-ray induced breaks, are nearly always repaired by HR independent of chromatin localization suggesting that the speed of repair is an important factor determining the DSB repair pathway usage. Finally, NHEJ and HR can, under certain conditions, also compensate for each other such that DSBs normally repaired by one pathway can undergo repair by the other if genetic failures necessitate the pathway switch.

  7. A label-free electrochemical aptasensor based on graphene oxide/double-stranded DNA nanocomposite.

    Science.gov (United States)

    Li, Yu; Wang, Qi; Zhang, Yuting; Deng, Dongmei; He, Haibo; Luo, Liqiang; Wang, Zhenxin

    2016-09-01

    A novel label-free electrochemical impedance aptasensor based on a gold nanoparticles/double-stranded DNA-graphene (AuNPs/dsDNA-GO) nanocomposite modified glassy carbon electrode was presented for quantitative determination of thrombin. GO was covalently functionalized with dsDNA via a facile amidation process, and then AuNPs were electrodeposited onto the surface of dsDNA-GO. The morphology, conductivity and interaction of the as-prepared nanocomposites were characterized by scanning electron microscopy, cyclic voltammetry, electrochemical impedance spectroscopy (EIS), Raman and Fourier transform infrared spectroscopy. The thrombin-binding aptamer (TBA) was conjugated to AuNPs via gold-thiol chemistry to construct electrochemical aptasensing platform, and the specific recognition between TBA and thrombin was monitored by EIS. Under optimum conditions, thrombin could be quantified in a wide range of 0.1-100nM (R(2)=0.9960) with low detection limit of 0.06nM (S/N=3). PMID:27182650

  8. Detection of heavy ion induced DNA double-strand breaks using static-field gel electrophoresis

    International Nuclear Information System (INIS)

    Radiation induced DNA double-strand breaks (DSBs) were measured in Chinese hamster ovary cells (CHO-K1) using an experimental protocol involving static-field gel electrophoresis following exposure to various accelerated ions. Dose-effect curves were set up and relative biological efficiencies (RBEs) for DSB induction were determined for different radiation qualities. RBEs around 1 were obtained for low energy deuterons (6-7 keV/μm), while for high energy oxygen ions (20 keV/μm) an RBE value slightly greater than 1 was determined. Low energetic oxygen ions (LET ∼ 250 keV/μm) were found to show RBEs substantially below unity, and for higher LET particles (≥ 250 keV/μm) RBEs for DSB induction were generally found to be smaller than 1. The data presented here are in line with the generally accepted view that not induced DSBs, but misrepaired or unrepaired DNA-lesions are related to cellular inactivation. (orig.)

  9. DNA double strand break damage by radiation and behavioral imaging of DNA repair enzymes

    International Nuclear Information System (INIS)

    The theme in the title is described mainly on authors' studies. Finding of a jellyfish GFP (green fluorescent protein) and its genomic recombination technique with a target protein have made it possible to investigate the behavior of the protein (the repair enzymes in this review) within a cell by fluorescent microscopy. Double strand breaks (DSBs), the most severe damage of DNA leading to cell death and carcinogenesis, are induced by irradiation of ionizing radiation and/or ultraviolet light, and repair mechanisms of non homologous end-joining and homologous recombinant repair are known major in mammalian cells and in lower eukaryotes, respectively. Authors used UVA for inducing DSBs under the presence of benzo[a]pyrene in mammalian cells like Chinese hamster ovary (CHO)-K1 and xrs-5, the behaviors of Ku70/80 repair molecules tagged by GFP were imaged by confocal laser microscopy, and one of findings was that Ku80 moved to the level most intensely irradiated. Fluorescent molecular imaging technique will be employed widely in clinical diagnosis and new drug development as well as in basic bioscience. (S.I.)

  10. Repair pathways for heavy ion-induced complex DNA double strand breaks

    International Nuclear Information System (INIS)

    DNA double strand break (DSB) induced by ionizing radiation (IR) is a deleterious damage leading to cell death and genome instability if not properly repaired. It is well known that DSB is repaired by two major pathways, non-homologous end-joining (NHEJ) and homologous recombination (HR). It is also known that NHEJ is dominant throughout the cell cycle after X- or gamma-ray irradiation in mammalian cells, Meanwhile, it is thought that heavy-ion radiation (e.g., carbon-ions, iron-ions) gives rise to clustered DNA damages consisting of not only strand breaks but also aberrant bases in the vicinity of DSBs (complex DSBs). Our previous work suggested that the efficiency of NHEJ is diminished for repair of complex DSBs induced by heavy-ion radiation. We thought that this difficulty in NHEJ process associated with heavy ion induced complex DNA damage might be extended to HR process in cells exposed to heavy ions. In order to find out if this notion is true or not, exposed human cells to X-rays and heavy-ions, and studied HR associated processes at the molecular level. Our result indicates that complex DSBs induced by heavy ions effectively evoke DNA end resection activity during the HR process. Together with our results, a relevant recent progress in the field of DNA DSB repair will be discussed. (author)

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

    International Nuclear Information System (INIS)

    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)

  12. 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)

  13. Mixed-Sequence Recognition of Double-Stranded DNA Using Enzymatically Stable Phosphorothioate Invader Probes

    Directory of Open Access Journals (Sweden)

    Brooke A. Anderson

    2015-07-01

    Full Text Available Development of probes that allow for sequence-unrestricted recognition of double-stranded DNA (dsDNA continues to attract much attention due to the prospect for molecular tools that enable detection, regulation, and manipulation of genes. We have recently introduced so-called Invader probes as alternatives to more established approaches such as triplex-forming oligonucleotides, peptide nucleic acids and polyamides. These short DNA duplexes are activated for dsDNA recognition by installment of +1 interstrand zippers of intercalator-functionalized nucleotides such as 2′-N-(pyren-1-ylmethyl-2′-N-methyl-2′-aminouridine and 2′-O-(pyren-1-ylmethyluridine, which results in violation of the nearest neighbor exclusion principle and duplex destabilization. The individual probes strands have high affinity toward complementary DNA strands, which generates the driving force for recognition of mixed-sequence dsDNA regions. In the present article, we characterize Invader probes that are based on phosphorothioate backbones (PS-DNA Invaders. The change from the regular phosphodiester backbone furnishes Invader probes that are much more stable to nucleolytic degradation, while displaying acceptable dsDNA-recognition efficiency. PS-DNA Invader probes therefore present themselves as interesting probes for dsDNA-targeting applications in cellular environments and living organisms.

  14. Mixed-Sequence Recognition of Double-Stranded DNA Using Enzymatically Stable Phosphorothioate Invader Probes.

    Science.gov (United States)

    Anderson, Brooke A; Karmakar, Saswata; Hrdlicka, Patrick J

    2015-01-01

    Development of probes that allow for sequence-unrestricted recognition of double-stranded DNA (dsDNA) continues to attract much attention due to the prospect for molecular tools that enable detection, regulation, and manipulation of genes. We have recently introduced so-called Invader probes as alternatives to more established approaches such as triplex-forming oligonucleotides, peptide nucleic acids and polyamides. These short DNA duplexes are activated for dsDNA recognition by installment of +1 interstrand zippers of intercalator-functionalized nucleotides such as 2'-N-(pyren-1-yl)methyl-2'-N-methyl-2'-aminouridine and 2'-O-(pyren-1-yl)methyluridine, which results in violation of the nearest neighbor exclusion principle and duplex destabilization. The individual probes strands have high affinity toward complementary DNA strands, which generates the driving force for recognition of mixed-sequence dsDNA regions. In the present article, we characterize Invader probes that are based on phosphorothioate backbones (PS-DNA Invaders). The change from the regular phosphodiester backbone furnishes Invader probes that are much more stable to nucleolytic degradation, while displaying acceptable dsDNA-recognition efficiency. PS-DNA Invader probes therefore present themselves as interesting probes for dsDNA-targeting applications in cellular environments and living organisms. PMID:26230684

  15. PML nuclear body disruption impairs DNA double-strand break sensing and repair in APL

    Science.gov (United States)

    di Masi, A; Cilli, D; Berardinelli, F; Talarico, A; Pallavicini, I; Pennisi, R; Leone, S; Antoccia, A; Noguera, N I; Lo-Coco, F; Ascenzi, P; Minucci, S; Nervi, C

    2016-01-01

    Proteins involved in DNA double-strand break (DSB) repair localize within the promyelocytic leukemia nuclear bodies (PML-NBs), whose disruption is at the root of the acute promyelocytic leukemia (APL) pathogenesis. All-trans-retinoic acid (RA) treatment induces PML-RARα degradation, restores PML-NB functions, and causes terminal cell differentiation of APL blasts. However, the precise role of the APL-associated PML-RARα oncoprotein and PML-NB integrity in the DSB response in APL leukemogenesis and tumor suppression is still lacking. Primary leukemia blasts isolated from APL patients showed high phosphorylation levels of H2AX (γ-H2AX), an initial DSBs sensor. By addressing the consequences of ionizing radiation (IR)-induced DSB response in primary APL blasts and RA-responsive and -resistant myeloid cell lines carrying endogenous or ectopically expressed PML-RARα, before and after treatment with RA, we found that the disruption of PML-NBs is associated with delayed DSB response, as revealed by the impaired kinetic of disappearance of γ-H2AX and 53BP1 foci and activation of ATM and of its substrates H2AX, NBN, and CHK2. The disruption of PML-NB integrity by PML-RARα also affects the IR-induced DSB response in a preleukemic mouse model of APL in vivo. We propose the oncoprotein-dependent PML-NB disruption and DDR impairment as relevant early events in APL tumorigenesis. PMID:27468685

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

    International Nuclear Information System (INIS)

    DNA double strand breaks (DSBs) in living cells can be directly provoked by ionising radiation. DSBs can be visualized by immunostaining the phosphorylated histone γH2AX. Our concern was to test the feasibility of γH2AX 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 γH2AX 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 Si3N4 window showed a homogenous Hsp70 expression pattern

  17. Self-avoiding wormlike chain model for double-stranded-DNA loop formation

    Science.gov (United States)

    Pollak, Yaroslav; Goldberg, Sarah; Amit, Roee

    2014-11-01

    We compute the effects of excluded volume on the probability for double-stranded DNA to form a loop. We utilize a Monte Carlo algorithm for generation of large ensembles of self-avoiding wormlike chains, which are used to compute the J factor for varying length scales. In the entropic regime, we confirm the scaling-theory prediction of a power-law drop off of -1.92 , which is significantly stronger than the -1.5 power law predicted by the non-self-avoiding wormlike chain model. In the elastic regime, we find that the angle-independent end-to-end chain distribution is highly anisotropic. This anisotropy, combined with the excluded volume constraints, leads to an increase in the J factor of the self-avoiding wormlike chain by about half an order of magnitude relative to its non-self-avoiding counterpart. This increase could partially explain the anomalous results of recent cyclization experiments, in which short dsDNA molecules were found to have an increased propensity to form a loop.

  18. 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.

  19. 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

  20. 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

  1. Cernunnos/XLF: a new player in DNA double-strand break repair.

    Science.gov (United States)

    Yano, Ken-ichi; Morotomi-Yano, Keiko; Akiyama, Hidenori

    2009-06-01

    Non-homologous end-joining (NHEJ) is the predominant repair pathway for DNA double-strand breaks (DSBs) in vertebrates and also plays a crucial role in V(D)J recombination of immunoglobulin genes. Cernunnos/XLF is a newly identified core factor for NHEJ, and its defect causes a genetic disease characterized by neural disorders, immunodeficiency and increased radiosensitivity. Cernunnos/XLF has at least two distinct functions in NHEJ. Cernunnos/XLF interacts with and stimulates the XRCC4/DNA ligase IV complex, which acts at the final ligation step in NHEJ. In living cells, Cernunnos/XLF quickly responds to DSB induction and accumulates at damaged sites in a Ku-dependent but XRCC4-independent manner. These observations indicate that Cernunnos/XLF plays a unique role in bridging damage sensing and DSB rejoining steps of NHEJ. Recent crystallographic analyses of the homodimeric Cernunnos/XLF protein provide structural insights into the Cernunnos/XLF functions. These studies offer important clues toward understanding the molecular mechanism for NHEJ-defective diseases. PMID:18992362

  2. Primary immunodeficiency syndromes associated with defective DNA double-strand break repair.

    Science.gov (United States)

    Gennery, A R

    2006-01-01

    Damaging DNA double-strand breaks (DNA-DSBs) following ionizing radiation (IR) exposure, potentially lead to cell death or carcinogenesis. Non-homologous end-joining (NHEJ) is the main repair pathway employed by vertebrate cells to repair such damage. Many repair pathway proteins have been identified. The creation of many diverse lymphocyte receptors to identify potential pathogens has evolved by breaking and randomly re-sorting the gene segments coding for antigen receptors. Subsequent DNA-DSB repair utilizes the NHEJ proteins. Individuals with defective repair pathways are increasingly recognized with radiosensitivity and immunodeficiency. Patients with defects in ataxia-telangiectasia mutated, nibrin, MRE11, Rad50, Artemis, DNA ligase IV and Cernunnos-XRCC4-like factor have been identified. Most exhibit immunodeficiency, with a spectrum of presentation and overlap between conditions. Conventional treatment with immunoglobulin replacement or haematopoietic stem cell transplantation (HSCT) can be effective. A greater understanding of the molecular defect will enable better, tailored therapies to improve survival. PMID:16971555

  3. 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. PMID:26958920

  4. Pathway choice in DNA double strand break repair: observations of a balancing act

    Directory of Open Access Journals (Sweden)

    Brandsma Inger

    2012-11-01

    Full Text Available Abstract Proper repair of DNA double strand breaks (DSBs is vital for the preservation of genomic integrity. There are two main pathways that repair DSBs, Homologous recombination (HR and Non-homologous end-joining (NHEJ. HR is restricted to the S and G2 phases of the cell cycle due to the requirement for the sister chromatid as a template, while NHEJ is active throughout the cell cycle and does not rely on a template. The balance between both pathways is essential for genome stability and numerous assays have been developed to measure the efficiency of the two pathways. Several proteins are known to affect the balance between HR and NHEJ and the complexity of the break also plays a role. In this review we describe several repair assays to determine the efficiencies of both pathways. We discuss how disturbance of the balance between HR and NHEJ can lead to disease, but also how it can be exploited for cancer treatment.

  5. 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.

  6. DNA repair by thiols in air shows two radicals make a double-strand break

    International Nuclear Information System (INIS)

    Using agarose gel electrophoresis, we have measured the yields of DNA single- and double-strand breaks (SSBs and DSBs) for plasmid DNA γ-irradiated in aerobic aqueous solution. The presence during irradiation of either of the thiols cysteamine or N-(2-thioethyl)-1,3-diaminopropane (WR-1065) resulted in a concentration-dependent decrease in the yield of SSBs and a much greater decrease in the yield of DSBs. This large differential protective effect was not produced by thioethers or an alcohol of structural similarity to the two thiols, suggesting that repair of DSB radical precursors by thiols is more efficient than for SSB precursors. These observations suggest the existence of a diradical intermediate in the formation of DSBs. The results argue against a major contribution by a single radical mechanism involving interstrand radical transfer via hydrogen abstraction by a peroxyl intermediate, since the half-life of this radical transfer reaction appears to be significantly greater than the lifetime of the intermediate. 35 refs., 7 figs

  7. Preferred interaction of D-peptidyl-anthraquinones with double-stranded B-DNA.

    Science.gov (United States)

    Gatto, B; Zagotto, G; Sissi, C; Palumbo, M

    1997-12-01

    The quest for more specific drugs in antitumor chemotherapy led us to the design of anthraquinone-peptide conjugates capable of selective recognition of the nucleic acid. We present here the DNA binding characteristics, sequence specificity and geometry of interaction of a pair of enantiomers containing the lysine-glycine dipeptide in the side chains. The D enantiomer binds right handed double stranded DNA more efficiently than the L form under all conditions tested. The source of higher binding affinity is not electrostatic in nature and rests in the more favorable hydrophobic contacts of the D-lysyl side chains in the drug-DNA complex. Both derivatives exhibit preference for alternating GC base sequences and intercalate into DNA in a threading mode as suggested by chiroptical and theoretical studies. The D enantiomer, being a peptidyl derivative that contains a non-natural amino acid, has the considerable advantage of being less susceptible to enzymatic hydrolysis and could therefore represent a lead compound for further development. PMID:9493055

  8. Characteristics of γ-H2AX foci at DNA double-strand breaks sites

    International Nuclear Information System (INIS)

    Phosphorylated H2AX (γ-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 γ-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. H2AXΔ/Δ 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. γ-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 γ-H2AX formation. (author)

  9. FBH1 promotes DNA double-strand breakage and apoptosis in response to DNA replication stress.

    Science.gov (United States)

    Jeong, Yeon-Tae; Rossi, Mario; Cermak, Lukas; Saraf, Anita; Florens, Laurence; Washburn, Michael P; Sung, Patrick; Schildkraut, Carl L; Schildkraut, Carl; Pagano, Michele

    2013-01-21

    Proper resolution of stalled replication forks is essential for genome stability. Purification of FBH1, a UvrD DNA helicase, identified a physical interaction with replication protein A (RPA), the major cellular single-stranded DNA (ssDNA)-binding protein complex. Compared with control cells, FBH1-depleted cells responded to replication stress with considerably fewer double-strand breaks (DSBs), a dramatic reduction in the activation of ATM and DNA-PK and phosphorylation of RPA2 and p53, and a significantly increased rate of survival. A minor decrease in ssDNA levels was also observed. All these phenotypes were rescued by wild-type FBH1, but not a FBH1 mutant lacking helicase activity. FBH1 depletion had no effect on other forms of genotoxic stress in which DSBs form by means that do not require ssDNA intermediates. In response to catastrophic genotoxic stress, apoptosis prevents the persistence and propagation of DNA lesions. Our findings show that FBH1 helicase activity is required for the efficient induction of DSBs and apoptosis specifically in response to DNA replication stress. PMID:23319600

  10. High LET - induced H2AX phosphorylation at sites of DNA double strand breaks

    Science.gov (United States)

    Desai, N.; Cucinotta, F.; Wu, H.

    Within cell nuclei, traversing charged heavy ion particles lead to the accumulation of proteins related to DNA lesions and repair along the ion trajectories. Irradiation using a standard geometric setup with the beam path perpendicular to the cell monolayer generates discrete foci of several proteins known to localize at sites of DNA double strand breaks (DSBs). One such molecule is the histone protein H2AX (gamma-H2AX), which gets rapidly phosphorylated in response to ionizing radiation. Here we present data obtained with a modified irradiation geometry characterized by a beam path parallel to a monolayer of human fibroblast cells. This new irradiation geometry leads to the formation of gamma-H2AX aggregates in the shape of streaks stretching over several micrometers in the x/y plane, thus enabling the analysis of the fluorescence distributions along the particle trajectories. Qualitative analysis of these distributions presented insights into the DNA repair kinetics along the primary track structure and visualization of possible chromatin movement. We also present evidence of colocalization of gamma-H2AX with several other proteins in responses to ionizing radiation exposure. Analysis of gamma-H2AX has the potential to provide useful information on human cell responses to high LET radiation after exposure to space-like radiation.

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

    Science.gov (United States)

    Citterio, Elisabetta

    2015-01-01

    Ubiquitination is a reversible protein modification broadly implicated in cellular functions. Signaling processes mediated by ubiquitin (ub) 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 ub 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 processes. Here, we discuss the current knowledge of how ub-mediated signaling at DSBs is controlled by DUBs, with main focus on DUBs targeting histone H2A and on their recent implication in stem cell biology and cancer. PMID:26442100

  12. RNF4 regulates DNA double-strand break repair in a cell cycle-dependent manner.

    Science.gov (United States)

    Kuo, Ching-Ying; Li, Xu; Stark, Jeremy M; Shih, Hsiu-Ming; Ann, David K

    2016-03-18

    Both RNF4 and KAP1 play critical roles in the response to DNA double-strand breaks (DSBs), but the functional interplay of RNF4 and KAP1 in regulating DNA damage response remains unclear. We have previously demonstrated the recruitment and degradation of KAP1 by RNF4 require the phosphorylation of Ser824 (pS824) and SUMOylation of KAP1. In this report, we show the retention of DSB-induced pS824-KAP1 foci and RNF4 abundance are inversely correlated as cell cycle progresses. Following irradiation, pS824-KAP1 foci predominantly appear in the cyclin A (-) cells, whereas RNF4 level is suppressed in the G0-/G1-phases and then accumulates during S-/G2-phases. Notably, 53BP1 foci, but not BRCA1 foci, co-exist with pS824-KAP1 foci. Depletion of KAP1 yields opposite effect on the dynamics of 53BP1 and BRCA1 loading, favoring homologous recombination repair. In addition, we identify p97 is present in the RNF4-KAP1 interacting complex and the inhibition of p97 renders MCF7 breast cancer cells relatively more sensitive to DNA damage. Collectively, these findings suggest that combined effect of dynamic recruitment of RNF4 to KAP1 regulates the relative occupancy of 53BP1 and BRCA1 at DSB sites to direct DSB repair in a cell cycle-dependent manner. PMID:26766492

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

    Science.gov (United States)

    Vyas, R; Kumar, R; Clermont, F; Helfricht, A; Kalev, P; Sotiropoulou, P; Hendriks, I A; Radaelli, E; Hochepied, T; Blanpain, C; Sablina, A; van Attikum, H; Olsen, J V; Jochemsen, A G; Vertegaal, A C O; Marine, J-C

    2013-03-01

    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 signaling and repair proteins to the sites of DNA lesions. Coordinated protein SUMOylation and ubiquitylation have crucial roles in regulating the dynamic assembly of protein complexes at these sites. However, how SUMOylation influences protein ubiquitylation at DSBs is poorly understood. We show herein that Rnf4, an E3 ubiquitin ligase that targets SUMO-modified proteins, accumulates in DSB repair foci and is required 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, 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 in other key regulators of HR repair, Rnf4 deficiency leads to age-dependent impairment in spermatogenesis. These findings identify Rnf4 as a critical component of the DDR in vivo and support the possibility that Rnf4 controls protein localization at DNA damage sites by integrating SUMOylation and ubiquitylation events. PMID:23197296

  14. Small RNAs tackle large viruses: RNA interference-based antiviral defense against DNA viruses in insects

    OpenAIRE

    Bronkhorst, Alfred W.; Miesen, Pascal; Ronald P. van Rij

    2013-01-01

    The antiviral RNA interference (RNAi) pathway processes viral double-stranded RNA (dsRNA) into viral small interfering RNAs (vsiRNA) that guide the recognition and cleavage of complementary viral target RNAs. In RNA virus infections, viral replication intermediates, dsRNA genomes or viral structured RNAs have been implicated as Dicer-2 substrates. In a recent publication, we demonstrated that a double-stranded DNA virus, Invertebrate iridescent virus 6, is a target of the Drosophila RNAi mach...

  15. 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.

  16. Analysis of native cellular DNA after heavy ion irradiation: DNA double-strand breaks in CHO-K1 cells

    International Nuclear Information System (INIS)

    A fast assay for the detection of DNA double-strand breaks was developed involving constant field gel electrophoresis (Taucher-Scholz et al., 1994) and densitometric scanning of agarose gels stained with ethidium bromide. With this technique, DSB induction was investigated after irradiation of CHO cells with carbon ions with LET values between 14 keV/μm and 400 keV/μm. In parallel, a computer code was developed to simulate both the principle of the electrophoretic detection of DNA double-strand breaks and the action of radiations of different ionization density. The results of the experiments and the calculations are presented here and compared with each other. (orig./HSI)

  17. Microhomology-mediated end joining is the principal mediator of double-strand break repair during mitochondrial DNA lesions

    OpenAIRE

    Tadi, Satish Kumar; Sebastian, Robin; Dahal, Sumedha; Babu, Ravi K.; Choudhary, Bibha; Raghavan, Sathees C.

    2016-01-01

    Mitochondrial DNA (mtDNA) deletions are associated with various mitochondrial disorders. The deletions identified in humans are flanked by short, directly repeated mitochondrial DNA sequences; however, the mechanism of such DNA rearrangements has yet to be elucidated. In contrast to nuclear DNA (nDNA), mtDNA is more exposed to oxidative damage, which may result in double-strand breaks (DSBs). Although DSB repair in nDNA is well studied, repair mechanisms in mitochondria are not characterized....

  18. Adaptation of the neutral bacterial comet assay to assess antimicrobial-mediated DNA double-strand breaks in Escherichia coli

    OpenAIRE

    Solanky, Dipesh; Shelley E Haydel

    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 antimicro...

  19. Post-irradiation chemical processing of DNA damage generates double-strand breaks in cells already engaged in repair

    OpenAIRE

    Satyendra K Singh; 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, c...

  20. The logic of DNA replication in double-stranded DNA viruses: insights from global analysis of viral genomes

    OpenAIRE

    Kazlauskas, Darius; Krupovic, Mart; Venclovas, Česlovas

    2016-01-01

    Genomic DNA replication is a complex process that involves multiple proteins. Cellular DNA replication systems are broadly classified into only two types, bacterial and archaeo-eukaryotic. In contrast, double-stranded (ds) DNA viruses feature a much broader diversity of DNA replication machineries. Viruses differ greatly in both completeness and composition of their sets of DNA replication proteins. In this study, we explored whether there are common patterns underlying this extreme diversity...

  1. The opportunistic pathogen Pseudomonas aeruginosa activates the DNA double-strand break signaling and repair pathway in infected cells

    International Nuclear Information System (INIS)

    Highly hazardous DNA double-strand breaks can be induced in eukaryotic cells by a number of agents including pathogenic bacterial strains. We have investigated the genotoxic potential of Pseudomonas aeruginosa, an opportunistic pathogen causing devastating nosocomial infections in cystic fibrosis or immunocompromised patients. Our data revealed that infection of immune or epithelial cells by P. aeruginosa triggered DNA strand breaks and phosphorylation of histone H2AX (γH2AX), a marker of DNA double-strand breaks. Moreover, it induced formation of discrete nuclear repair foci similar to gamma-irradiation-induced foci, and containing γH2AX and 53BP1, an adaptor protein mediating the DNA-damage response pathway. Gene deletion, mutagenesis, and complementation in P. aeruginosa identified ExoS bacterial toxin as the major factor involved in γH2AX induction. Chemical inhibition of several kinases known to phosphorylate H2AX demonstrated that Ataxia Telangiectasia Mutated (ATM) was the principal kinase in P. aeruginosa-induced H2AX phosphorylation. Finally, infection led to ATM kinase activation by an auto-phosphorylation mechanism. Together, these data show for the first time that infection by P. aeruginosa activates the DNA double-strand break repair machinery of the host cells. This novel information sheds new light on the consequences of P. aeruginosa infection in mammalian cells. As pathogenic Escherichia coli or carcinogenic Helicobacter pylori can alter genome integrity through DNA double-strand breaks, leading to chromosomal instability and eventually cancer, our findings highlight possible new routes for further investigations of P. aeruginosa in cancer biology and they identify ATM as a potential target molecule for drug design. (authors)

  2. Nuclear organization and dynamics of the DSB-repair. Direct visualization of double strand breaks in functionally different chromatin domains

    Czech Academy of Sciences Publication Activity Database

    Falk, Martin; Lukášová, Emilie; Gabrielová, Barbora; Ondřej, Vladan; Kozubek, Stanislav; Kroupová, Iva

    La Jolla, 2007. s. 46-46. [Symposium on Biological Complexity: Diseases of Transcription. 11.01.2007-14.01.2007, La Jolla] R&D Projects: GA ČR(CZ) GP204/06/P349; GA MŠk(CZ) 1P05OC084 Institutional research plan: CEZ:AV0Z50040507 Keywords : double-strand breaks (DSB) * DSB-repair * higher-order chromatin structure Subject RIV: BO - Biophysics

  3. Double-Strand Break Repair by Interchromosomal Recombination: An In Vivo Repair Mechanism Utilized by Multiple Somatic Tissues in Mammals

    OpenAIRE

    White, Ryan R; Sung, Patricia; Vestal, C. Greer; Benedetto, Gregory; Cornelio, Noelle; Richardson, Christine

    2013-01-01

    Homologous recombination (HR) is essential for accurate genome duplication and maintenance of genome stability. In eukaryotes, chromosomal double strand breaks (DSBs) are central to HR during specialized developmental programs of meiosis and antigen receptor gene rearrangements, and form at unusual DNA structures and stalled replication forks. DSBs also result from exposure to ionizing radiation, reactive oxygen species, some anti-cancer agents, or inhibitors of topoisomerase II. Literature p...

  4. 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.

  5. A fluorescent aptasensor using double-stranded DNA/graphene oxide as the indicator probe.

    Science.gov (United States)

    Xing, Xiao-Jing; Xiao, Wan-Lu; Liu, Xue-Guo; Zhou, Ying; Pang, Dai-Wen; Tang, Hong-Wu

    2016-04-15

    We developed a fluorescent aptasensor based on the making use of double-stranded DNA (dsDNA)/graphene oxide (GO) as the signal probe and the activities of exonuclease I (Exo I). This method takes advantage of the stronger affinity of the aptamer to its target rather than to its complementary sequence (competitor), and the different interaction intensity of dsDNA, mononucleotides with GO. Specifically, in the absence of target, the competitor hybridizes with the aptamer, preventing the digestion of the competitor by Exo I, and thus the formed dsDNA is adsorbed on GO surface, allowing fluorescence quenching. When the target is introduced, the aptamer preferentially binds with its target. Thereby, the corresponding nuclease reaction takes place, and slight fluorescence change is obtained after the introduction of GO due to the weak affinity of the generated mononucleotides to GO. Adenosine (AD) was chosen as a model system and tested in detail. Under the optimized conditions, smaller dissociation constant (Kd, 311.0 µM) and lower detection limit (LOD, 3.1 µM) were obtained in contrast with traditional dye-labeled aptamer/GO based platform (Kd=688.8 µM, LOD=21.2 µM). Satisfying results were still obtained in the evaluation of the specificity and the detection of AD in human serum, making it a promising tool for the diagnosis of AD-relevant diseases. Moreover, we demonstrated the effect of the competitor on the LOD, and the results reveal that the sensitivity could be enhanced by using the rational competitor. The present design not only constructs a label-free aptamer based platform but also extends the application of dsDNA/GO complex in biochemical and biomedical studies. PMID:26655184

  6. Intrinsic radiosensitivity and DNA double-strand breaks in human cells

    International Nuclear Information System (INIS)

    Among the large spectrum of DNA damage induced by radiation, DNA double-strand breaks (DSBs) are considered, to date, as the key-lesions responsible for the cell killing. However, although it was always intuitive to radio-biologists, such a conclusion has only been reached after technical developments and conceptual advances and remains consensual rather than demonstrated formally. In this article, we have reviewed the results that have lead to the conclusion that the assessment of successful DSB repair can be the basis of reliable assays predictive of the clinical response to radiotherapy and some chemotherapeutic treatments. We have discussed a number of technical artifacts, the biases due to the extrapolation of data obtained in yeast and rodent model systems to the human situation and the variety of phenotypes observed in human cells and in particular: 1) the most recent techniques developed, based on immunofluorescence, which have revolutionized our understanding of the molecular events occurring early after irradiation but have also raised the crucial questions about the choice of techniques to. assess DSB repair and their specificity for different steps of the repair process; 2) While the homologous recombination repair pathway is predominant in yeasts, its importance in human cells appears less obvious, and raises the problem that the existence of randomized repair events may produce many more errors in human cells than in small genome organisms; 3) the impairment of DSB repair is observed in a plethora of genetic diseases, leading to radiosensitivity, immunodeficiency and sometimes cancer-proneness, but the low frequency and the pleiotropism of such diseases makes difficult the development of a single predictive assay. Therefore, although complete DSB repair appears to be crucial for cell survival, further research is still needed to provide innovative techniques for measuring repair which can be successfully transferred to the clinic and used to ensure

  7. 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.

  8. Repair response for DNA double-strand damage through ubiquitylation of chromatin

    International Nuclear Information System (INIS)

    The chromatin modulation (remodeling) via lysine63 (K63)-linked ubiquitin (U) has been found important in the repair response for DNA double-strand damage, and the sequential signaling events at the damage site are explained. As the first step of the repair, MRN (MRE11, RAD50 and nibrin) complex recognizes the damage site and binds to it followed by many linked reactions by recruited and activated enzymes of various protein kinases and phosphatases, which resulting in the enhanced early signaling. As well, gamma-H2AX (phosphorylated histone H2AX) is yielded by the process, to which phosphorylated MDC1 (mediator of DNA-damage checkpoint 1) binds to produce their complex. Then further binding of RNF8-HERC2-UBC13 (ring finger protein 8, hect domain and RCC1 (CHC1)-like domain, and U conjugating enzyme E2N, respectively) occurs for starting the cumulative ubiquitylation of H2AX via K63 as the middle phase response. Signaling in the late phase occurs on the U chain formed at the damage site by binding of RAP (receptor-associated protein) 80 and other recruited 5 proteins like BRCA1 (breast cancer 1, early onset) to repair DNA by the homologous recombination after 53BP1 (tumor protein p53 binding protein) binding followed by methylation of histone H4. In a case of human compound heterozygous RNF168 defect, RIDDLE syndrome (radiosensitivity, immunodeficiency, dysmorphic features and learning difficulties), cells have no and slight abnormality of G2/M and intra-S checkpoint, respectively. Another defecting case with homozygous nonsense mutation has high radiosensitivity, intra-S checkpoint abnormality and others. Abnormality of immuno-globulins observed in both cases is similar to that in the RNF8-knockout mouse. Many tasks in chromatin ubiquitylation in the repair are still remained to be solved for protection and treatment of related diseases. (T.T.)

  9. Identification of DNA double strand breaks at chromosome boundaries along the track of particle irradiation.

    Science.gov (United States)

    Niimi, Atsuko; Yamauchi, Motohiro; Limsirichaikul, Siripan; Sekine, Ryota; Oike, Takahiro; Sato, Hiro; Suzuki, Keiji; Held, Kathryn D; Nakano, Takashi; Shibata, Atsushi

    2016-08-01

    Chromosomal translocations arise from misrejoining of DNA double strand breaks (DSBs) between loci located on two chromosomes. One current model suggests that spatial proximity of potential chromosomal translocation partners influences translocation probability. Ionizing radiation (IR) is a potent inducer of translocations. Accumulating evidence demonstrates that particle irradiation more frequently causes translocations compared with X-ray irradiation. This observation has led to the hypothesis that the high frequency of translocations after particle irradiation may be due to the formation of DSBs at chromosome boundaries along the particle track, because such DSBs can be misrejoined between distinct chromosomes. In this study, we simultaneously visualized the site of IR-induced DSBs and chromosome position by combining Immunofluorescence and fluorescence in situ hybridization. Importantly, the frequency of γH2AX foci at the chromosome boundary of chromosome 1 after carbon-ion irradiation was >4-fold higher than that after X-ray irradiation. This observation is consistent with the idea that particle irradiation generates DSBs at the boundaries of two chromosomes along the track. Further, we showed that resolution of γH2AX foci at chromosome boundaries is prevented by inhibition of DNA-PKcs activity, indicating that the DSB repair is NHEJ-dependent. Finally, we found that γH2AX foci at chromosome boundaries after carbon-ion irradiation contain DSBs undergoing DNA-end resection, which promotes repair utilizing microhomology mediated end-joining during translocation. Taken together, our study suggests that the frequency of DSB formation at chromosome boundaries is associated with the incidence of chromosomal translocations, supporting the notion that the spatial proximity between breaks is an important factor in translocation formation. © 2016 Wiley Periodicals, Inc. PMID:27113385

  10. DNA polymerase θ (POLQ), double-strand break repair, and cancer.

    Science.gov (United States)

    Wood, Richard D; Doublié, Sylvie

    2016-08-01

    DNA polymerase theta (pol θ) is encoded in the genomes of many eukaryotes, though not in fungi. Pol θ is encoded by the POLQ gene in mammalian cells. The C-terminal third of the protein is a family A DNA polymerase with additional insertion elements relative to prokaryotic homologs. The N-terminal third is a helicase-like domain with DNA-dependent ATPase activity. Pol θ is important in the repair of genomic double-strand breaks (DSBs) from many sources. These include breaks formed by ionizing radiation and topoisomerase inhibitors, breaks arising at stalled DNA replication forks, breaks introduced during diversification steps of the mammalian immune system, and DSB induced by CRISPR-Cas9. Pol θ participates in a route of DSB repair termed "alternative end-joining" (altEJ). AltEJ is independent of the DNA binding Ku protein complex and requires DNA end resection. Pol θ is able to mediate joining of two resected 3' ends harboring DNA sequence microhomology. "Signatures" of Pol θ action during altEJ are the frequent utilization of longer microhomologies, and the insertion of additional sequences at joining sites. The mechanism of end-joining employs the ability of Pol θ to tightly grasp a 3' terminus through unique contacts in the active site, allowing extension from minimally paired primers. Pol θ is involved in controlling the frequency of chromosome translocations and preserves genome integrity by limiting large deletions. It may also play a backup role in DNA base excision repair. POLQ is a member of a cluster of similarly upregulated genes that are strongly correlated with poor clinical outcome for breast cancer, ovarian cancer and other cancer types. Inhibition of pol θ is a compelling approach for combination therapy of radiosensitization. PMID:27264557

  11. Analysis of proteins phosphorylated at the early response of DNA double-strand breaks

    International Nuclear Information System (INIS)

    The serine/threonine protein kinase, ATM, recognizes DNA double-strand breaks (DSBs) at a very early stage, and plays a central role in transmitting damage signals to the cell cycle control machinery. To further understand the early events in DSBs recognition, we are undertaking two approaches: 1) identification of novel substrates which are phosphorylated by ATM using phospho-specific antibody, and 2) analysis of phosphorylation profile employing the proteomics-based 2-DE gel electrophoresis technique. There is a well-known consensus sequence for phosphorylation by ATM characterized by serine followed by glutamine (SQ), and their surrounding amino acids. Using one of the consensus sequences, we synthesized a phosphorylated peptide for immunizing rabbit and generated a phospho-specific antibody. This novel antibody recognized phosphorylated proteins in mouse fibroblast cultures after 5 min of exposure to ionizing radiation. This high and constant level of phosphorylation persisted until the 2nd hour and started decreasing to basal levels from the fourth hour. This antibody also detected phospho-proteins in mouse fibroblast cultures exposed to chemical reagents that induce DSBs such as NCS, bleomycin and etoposide, and is thought to be specific for DNA DSBs-related proteins. Furthermore, this antibody did not recognize phospho-proteins in ATM-deficient cells, suggesting that this phosphorylation is dependent on ATM kinase or downstream kinases controlled by ATM. As a second approach, we identified several spots that are phosphorylated in ATM wild type cells but not in ATM-deficient cells by 2-DE proteomic analysis. We are conducting further studies to generate comprehensive, ATM-dependent phosphorylation profiles as a novel approach to understand early events in DNA damage recognition by ATM

  12. Radiation-induced heat-labile sites that convert into DNA double-strand breaks

    Science.gov (United States)

    Rydberg, B.; Chatterjee, A. (Principal Investigator)

    2000-01-01

    The yield of DNA double-strand breaks (DSBs) in SV40 DNA irradiated in aqueous solution was found to increase by more than a factor of two as a result of postirradiation incubation of the DNA at 50 degrees C and pH 8.0 for 24 h. This is in agreement with data from studies performed at 37 degrees C that were published previously. Importantly, similar results were also obtained from irradiation of mammalian DNA in agarose plugs. These results suggest that heat-labile sites within locally multiply damaged sites are produced by radiation and are subsequently transformed into DSBs. Since incubation at 50 degrees C is typically employed for lysis of cells in commonly used pulsed-field gel assays for detection of DSBs in mammalian cells, the possibility that heat-labile sites are present in irradiated cells was also studied. An increase in the apparent number of DSBs as a function of lysis time at 50 degrees C was found with kinetics that was similar to that for irradiated DNA, although the magnitude of the increase was smaller. This suggests that heat-labile sites are also formed in the cell. If this is the case, a proportion of DSBs measured by the pulsed-field gel assays may occur during the lysis step and may not be present in the cell as breaks but as heat-labile sites. It is suggested that such sites consist mainly of heat-labile sugar lesions within locally multiply damaged sites. Comparing rejoining of DSBs measured with short and long lysis procedure indicates that the heat-labile sites are repaired with fast kinetics in comparison with repair of the bulk of DSBs.

  13. 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.

  14. Interactions of Ku70/80 with Double-Strand DNA: Energetic, Dynamics, and Functional Implications

    Science.gov (United States)

    Hu, Shaowen; Cucinotta, Francis A.

    2010-01-01

    Space radiation is a proficient inducer of DNA damage leading to mutation, aberrant cell signaling, and cancer formation. Ku is among the first responding proteins in nucleus to recognize and bind the DNA double strand breaks (DSBs) whenever they are introduced. Once loaded Ku works as a scaffold to recruit other repair factors of non-homologous end joining and facilitates the following repair processes. The crystallographic study of the Ku70/80 heterodimer indicate the core structure of this protein shows virtually no conformational change after binding with DNA. To investigate the dynamical features as well as the energetic characteristics of Ku-DNA binding, we conduct multi-nanosecond molecular dynamics simulations of a modeled Ku70/80 structure and several complexes with two 24-bp DNA duplexes. Free energy calculations show significant energy differences between the complexes with Ku bound at DSBs and those with Ku associated at an internal site of a chromosome. The results also reveal detailed interactions between different nucleotides and the amino acids along the DNA-binding cradle of Ku, indicating subtle binding preference of Ku at specific DNA sequences. The covariance matrix analyses along the trajectories demonstrate the protein is stimulated to undergo correlated motions of different domains once bound to DNA ends. Additionally, principle component analyses identify these low frequency collective motions suitable for binding with and translocation along duplex DNA. It is proposed that the modification of dynamical properties of Ku upon binding with DSBs may provide a signal for the further recruitment of other repair factors such as DNA-PKcs, XLF, and XRCC4.

  15. 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.

  16. Tying the loose ends together in DNA double strand break repair with 53BP1.

    Science.gov (United States)

    Adams, Melissa M; Carpenter, Phillip B

    2006-01-01

    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. PMID:16945145

  17. 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.

  18. Smoking cessation reverses DNA double-strand breaks in human mononuclear cells.

    Directory of Open Access Journals (Sweden)

    Mari Ishida

    Full Text Available OBJECTIVE: Cigarette smoking is a major risk factor for atherosclerotic cardiovascular disease, which is responsible for a significant proportion of smoking-related deaths. However, the precise mechanism whereby smoking induces this pathology has not been fully delineated. Based on observation of DNA double-strand breaks (DSBs, the most harmful type of DNA damage, in atherosclerotic lesions, we hypothesized that there is a direct association between smoking and DSBs. The goal of this study was to investigate whether smoking induces DSBs and smoking cessation reverses DSBs in vivo through examination of peripheral mononuclear cells (MNCs. APPROACH AND RESULTS: Immunoreactivity of oxidative modification of DNA and DSBs were increased in human atherosclerotic lesions but not in the adjacent normal area. DSBs in human MNCs isolated from the blood of volunteers can be detected as cytologically visible "foci" using an antibody against the phosphorylated form of the histone H2AX (γ-H2AX. Young healthy active smokers (n = 15 showed increased γ-H2AX foci number when compared with non-smokers (n = 12 (foci number/cell: median, 0.37/cell; interquartile range [IQR], 0.31-0.58 vs. 4.36/cell; IQR, 3.09-7.39, p<0.0001. Smoking cessation for 1 month reduced the γ-H2AX foci number (median, 4.44/cell; IQR, 4.36-5.24 to 0.28/cell; IQR, 0.12-0.53, p<0.05. A positive correlation was noted between γ-H2AX foci number and exhaled carbon monoxide levels (r = 0.75, p<0.01. CONCLUSIONS: Smoking induces DSBs in human MNCs in vivo, and importantly, smoking cessation for 1 month resulted in a decrease in DSBs to a level comparable to that seen in non-smokers. These data reinforce the notion that the cigarette smoking induces DSBs and highlight the importance of smoking cessation.

  19. 125I-induced DNA double strand breaks: use in calibration of the neutral filter elution technique and comparison with X-ray induced breaks

    International Nuclear Information System (INIS)

    The neutral filter elution assay, for measurement of DNA double strand breakage, has been calibrated using mouse L cells and Chinese hamster V79 cells labelled with [125I]dUrd and then held at liquid nitrogen temperature to accumulate decays. The basis of the calibration is the observation that each 125I decay, occurring in DNA, produces a DNA double strand break. Linear relationships between 125I decays per cell and lethal lesions per cell (minus natural logarithm survival) and the level of elution, were found. Using the calibration data, it was calculated that the yield of DNA double strand breaks after X-irradiation of both cell types was from 6 to 9 x 10-12 DNA double strand breaks per Gy per dalton of DNA, for doses greater than 6 Gy. Neutral filter elution and survival data for X-irradiated and 125I-labelled cells suggested that the relationships between lethal lesions and DNA double strand breakage were significantly different for both cell types. An attempt was made to study the repair kinetics for 125I-induced DNA double strand breaks, but was frustrated by the rapid DNA degradation which occurs in cells that have been killed by the freezing-thawing process. (author)

  20. Formation and repair of DNA double-strand breaks in superinfecting phage lambda after ionizing irradiation of Escherichia coli host cells

    International Nuclear Information System (INIS)

    Cells of Escherichia coli containing superinfecting phage lambda DNA molecules were irradiated with 4-MeV electrons and lysed at neutral pH; the lysates were sedimented in neutral sucrose gradients to separate lambda DNA molecules containing no strand breaks, one or more single-strand breaks, or a double-strand break. About 40 single-strand breaks were induced for every double-strand break in both the presence and absence of oxygen. The single-strand breaks were rapidly repaired after irradiation in nitrogen anoxia and subsequent incubation in aerobic growth medium, but no net reduction in the number of double-strand breaks was observed. This is similar to observations after oxic irradiation. No quadratic dose dependence of double-strand break induction was found up to 500 krad in oxygen and up to 1800 krad in nitrogen anoxia. Also, no difference in double-strand breakage was observed when equal doses in the range of 60 to 80 krad were delivered either (i) acutely, with 1 sec. or (ii) intermittently, with time to repair most single-strand breaks before onset of the next radiation pulse. It is concluded that the proposed mechanism whereby two independently induced single-strand breaks pair to form a double-strand break is not significant in the biological dose range

  1. 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.

  2. Recombination-dependent DNA replication stimulated by double-strand breaks in bacteriophage T4.

    Science.gov (United States)

    Kreuzer, K N; Saunders, M; Weislo, L J; Kreuzer, H W

    1995-12-01

    We analyzed the mechanism of recombination-dependent DNA replication in bacteriophage T4-infected Escherichia coli using plasmids that have sequence homology to the infecting phage chromosome. Consistent with prior studies, a pBR322 plasmid, initially resident in the infected host cell, does not replicate following infection by T4. However, the resident plasmid can be induced to replicate when an integrated copy of pBR322 vector is present in the phage chromosome. As expected for recombination-dependent DNA replication, the induced replication of pBR322 required the phage-encoded UvsY protein. Therefore, recombination-dependent plasmid replication requires homology between the plasmid and phage genomes but does not depend on the presence of any particular T4 DNA sequence on the test plasmid. We next asked whether T4 recombination-dependent DNA replication can be triggered by a double-strand break (dsb). For these experiments, we generated a novel phage strain that cleaves its own genome within the nonessential frd gene by means of the I-TevI endonuclease (encoded within the intron of the wild-type td gene). The dsb within the phage chromosome substantially increased the replication of plasmids that carry T4 inserts homologous to the region of the dsb (the plasmids are not themselves cleaved by the endonuclease). The dsb stimulated replication when the plasmid was homologous to either or both sides of the break but did not stimulate the replication of plasmids with homology to distant regions of the phage chromosome. As expected for recombination-dependent replication, plasmid replication triggered by dsbs was dependent on T4-encoded recombination proteins. These results confirm two important predictions of the model for T4-encoded recombination-dependent DNA replication proposed by Gisela Mosig (p. 120-130, in C. K. Mathews, E. M. Kutter, G. Mosig, and P. B. Berget (ed.), Bacteriophage T4, 1983). In addition, replication stimulated by dsbs provides a site

  3. A role of NBS1 in genome stability after double-strand breaks

    International Nuclear Information System (INIS)

    DNA double-strand breaks (DSBs) represent the most serious damage in genome, and hence, the cells correctly repair one DSB generated in a cell. This efficiency of DNA repair could correspond to detect several bp out of 6x109 bps in genome. However, it is not yet characterized how DSBs are recognized and repair proteins are accurately recruited to the sites of DSBs. Here, we propose a two-step binding model led by NBS1, gene product defective in Nijmegen Breakage Syndrome. NBS1 physically interacts with histone, rather than damage DNA, by direct binding to γ -H2AX. We demonstrate that the NBS1-binding can occur in the absence of interaction with hMRE11 or BRCA1. NBS1 has no DNA binding region but carries a combination of the fork-head associated (FHA) and the BRCA1 C-terminal domains (BRCT). We show that the FHA/BRCT domain of NBS1 is essential for this physical interaction, since NBS1 lacking this domain failed to bind to γ -H2AX in cells, and a recombinant FHA/BRCT domain alone can bind to recombinant γ -H2AX. Thus, the interaction with γ -H2AX has a crucial role for re-localization of NBS1/hMRE11/hRAD50 nuclease complex to the vicinity of DNA damage. After conversion of this complex to binding to damage DNA, it will initiate DNA repair. When the ability of homologous recombination in chicken NBS cells was assayed by using SCneo reporter plasmid, the homologous recombination was about 100-fold decrease, compared to that of chicken wild cells. However, non-homologous end joining in chicken NBS cells is indistinguishable from those in wild type cells. NBS1 is also involved in S-phase checkpoint through the SMC1 phospohrylation when cells were irradiated with a low dose. As a result, this re-localization of NBS1/hMre11/ hRad50 complex through interaction of NBS1 with histone could be a key step in a two-step binding mechanism for homologous recombination repair and cell-cycle checkpoints

  4. 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

  5. Parp1-XRCC1 and the repair of DNA double strand breaks in mouse round spermatids

    International Nuclear Information System (INIS)

    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 γ-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.

  6. 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

  7. 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

    2016-07-01

    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 (reading frames (ORFs) encoding a putative coat protein (CP) and an RNA dependent RNA polymerase (RdRp), while one sequence (RPaTV9) showed similarity to another totivirus, Ustilago maydis virus H1 (UmV-H1) that encodes a single polyprotein (CP-RdRp fusion). Similar to yeast totiviruses, each ScV-L-A-like RPaTV contains a -1 ribosomal frameshift site downstream of a predicted pseudoknot structure in the overlapping region of these ORFs, suggesting that the RdRp is translated as a CP-RdRp fusion. Moreover, several ScV-L-A-like sequences were also found by searches of the transcriptome shotgun assembly (TSA) libraries from rust fungi, plants and insects. Phylogenetic analyses show that nine ScV-L-A-like RPaTVs along with ScV-L-A-like sequences derived from TSA libraries are clustered with most established members of the genus Totivirus, while one RPaTV forms a new distinct clade with UmV-H1, possibly establishing an additional genus in the family. Taken together, our results indicate the presence of diverse, novel totiviruses in the powdery mildew fungus populations infecting red clover plants in the field. PMID:27208846

  8. 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.

  9. Cell nonhomologous end joining capacity controls SAF-A phosphorylation by DNA-PK in response to DNA double-strand breaks inducers.

    Science.gov (United States)

    Britton, Sébastien; Froment, Carine; Frit, Philippe; Monsarrat, Bernard; Salles, Bernard; Calsou, Patrick

    2009-11-15

    Aiming to identify novel phosphorylation sites in response to DNA double-strand breaks (DSB) inducers, we have isolated a phosphorylation site on KU70. Unexpectedly, a rabbit antiserum raised against this site cross-reacted with a 120 kDa protein in cells treated by DNA DSB inducers. We identified this protein as SAF-A/hnRNP U, an abundant and essential nuclear protein containing regions binding DNA or RNA. The phosphorylation site was mapped at S59 position in a sequence context favoring a "S-hydrophobic" consensus model for DNA-PK phosphorylation site in vivo. This site was exclusively phosphorylated by DNA-PK in response to DNA DSB inducers. In addition, the extent and duration of this phosphorylation was in inverse correlation with the capacity of the cells to repair DSB by Nonhomologous End Joining. These results bring a new link between the hnRNP family and the DNA damage response. Addtionaly, the mapped phospho-site on SAF-A might serve as a potential bio-marker for DNA-PK activity in academic studies and clinical analyses of DNA-PK activators or inhibitors. PMID:19844162

  10. G9a inhibition potentiates the anti-tumour activity of DNA double-strand break inducing agents by impairing DNA repair independent of p53 status.

    Science.gov (United States)

    Agarwal, Pallavi; Jackson, Stephen P

    2016-10-01

    Cancer cells often exhibit altered epigenetic signatures that can misregulate genes involved in processes such as transcription, proliferation, apoptosis and DNA repair. As regulation of chromatin structure is crucial for DNA repair processes, and both DNA repair and epigenetic controls are deregulated in many cancers, we speculated that simultaneously targeting both might provide new opportunities for cancer therapy. Here, we describe a focused screen that profiled small-molecule inhibitors targeting epigenetic regulators in combination with DNA double-strand break (DSB) inducing agents. We identify UNC0638, a catalytic inhibitor of histone lysine N-methyl-transferase G9a, as hypersensitising tumour cells to low doses of DSB-inducing agents without affecting the growth of the non-tumorigenic cells tested. Similar effects are also observed with another, structurally distinct, G9a inhibitor A-366. We also show that small-molecule inhibition of G9a or siRNA-mediated G9a depletion induces tumour cell death under low DNA damage conditions by impairing DSB repair in a p53 independent manner. Furthermore, we establish that G9a promotes DNA non-homologous end-joining in response to DSB-inducing genotoxic stress. This study thus highlights the potential for using G9a inhibitors as anti-cancer therapeutic agents in combination with DSB-inducing chemotherapeutic drugs such as etoposide. PMID:27431310

  11. scid mutation in mice confers hypersensitivity to ionizing radiation and a deficiency in DNA double-strand break repair

    International Nuclear Information System (INIS)

    C.B-17 severe combined immunodeficient (scid) mice carry the scid mutation and are severely deficient in both T cell- and B cell-mediated immunity, apparently as a result of defective V(D)J joining of the immunoglobulin and T-cell receptor gene elements. In the present studies, we have defined the tissue, cellular, and molecular basis of another characteristic of these mice: their hypersensitivity to ionizing radiation. Bone marrow stem cells, intestinal crypt cells, and epithelial skin cells from scid mice are 2- to 3-fold more sensitive when irradiated in situ than are congenic BALB/c or C.B-17 controls. Two independently isolated embryo fibroblastic scid mouse cell lines display similar hypersensitivities to gamma-rays. In addition, these cell lines are sensitive to cell killing by bleomycin, which also produces DNA strand breaks, but not by the DNA crosslinking agent mitomycin C or UV irradiation. Measurement of the rejoining of gamma-ray-induced DNA double-strand breaks by pulsed-field gel electrophoresis indicates that these animals are defective in this repair system. This suggests that the gamma-ray sensitivity of the scid mouse fibroblasts could be the result of reduced repair of DNA double-strand breaks. Therefore, a common factor may participate in both the repair of DNA double-strand breaks as well as V(D)J rejoining during lymphocyte development. This murine autosomal recessive mutation should prove extremely useful in fundamental studies of radiation-induced DNA damage and repair

  12. Double-stranded cleavage of pBR322 by a diiron complex via a "hydrolytic" mechanism.

    OpenAIRE

    Schnaith, L M; Hanson, R S; Que, L

    1994-01-01

    Treatment of plasmid pBR322 with Fe2-(HPTB)(OH)(NO3)4(HPTB = N,N,N',N'-tetrakis(2-benzimidazolylmethyl)-2-hydroxy-1,3-diaminopr opane) and H2O2 or O2 and a reductant (dithiothreitol or ascorbate) results in double-stranded cleavage of the plasmid. The linearization of supercoiled pBR322 by this complex is not inhibited by hydroxyl radical scavengers. On the other hand, the linearized pBR322 is efficiently religated by T4 DNA ligase, and the presence of 3'-OH and 5'-OPO3 ends is corroborated b...

  13. Autophosphorylation of the DNA-dependent protein kinase catalytic subunit is required for rejoining of DNA double-strand breaks

    OpenAIRE

    Chan, Doug W.; Chen, Benjamin Ping-Chi; Prithivirajsingh, Sheela; Kurimasa, Akihiro; Story, Michael D.; Qin, Jun; Chen, David J.

    2002-01-01

    Nonhomologous end-joining (NHEJ) is the predominant pathway that repairs DNA double-strand breaks (DSBs) in mammalian cells. The DNA-dependent protein kinase (DNA-PK), consisting of Ku and DNA-PK catalytic subunit (DNA-PKcs), is activated by DNA in vitro and is required for NHEJ. We report that DNA-PKcs is autophosphorylated at Thr2609 in vivo in a Ku-dependent manner in response to ionizing radiation. Phosphorylated DNA-PKcs colocalizes with both γ-H2AX and 53BP1 after DNA damage. Mutation o...

  14. ATM and Artemis promote homologous recombination of radiation-induced DNA double-strand breaks in G2

    OpenAIRE

    Beucher, Andrea; Birraux, Julie; Tchouandong, Leopoldine; Barton, Olivia; Shibata, Atsushi; Conrad, Sandro; Goodarzi, Aaron A.; KREMPLER, ANDREA; Jeggo, Penny; Lo¨brich, Markus

    2009-01-01

    Homologous recombination (HR) and non-homologous end joining (NHEJ) represent distinct pathways for repairing DNA double-strand breaks (DSBs). Previous work implicated Artemis and ATM in an NHEJ-dependent process, which repairs a defined subset of radiation-induced DSBs in G1-phase. Here, we show that in G2, as in G1, NHEJ represents the major DSB-repair pathway whereas HR is only essential for repair of ∼15% of X- or γ-ray-induced DSBs. In addition to requiring the known HR proteins, Brca2, ...

  15. Mapping the double-strand breaks at the mating-type locus in fission yeast by genomic sequencing

    DEFF Research Database (Denmark)

    Nielsen, O; Egel, R; Nielsen, Olaf

    1989-01-01

    In fission yeast mating-type switching is initiated by the formation of a double-strand DNA break at the mating-type locus. A prerequisite for generation of the break is some 'imprinting' of the DNA in the previous cell cycle. We have used the technique of genomic sequencing to map the position of...... alleles the ends are probably masked by tightly bound proteins and therefore the precise nature of the break could not be determined. Since the break is stable throughout the cell cycle, these proteins may function in vivo to confer structural stability on the chromosomes having the break. The...

  16. The DNA replication priming protein, PriA, is required for homologous recombination and double-strand break repair.

    OpenAIRE

    Kogoma, T; Cadwell, G W; Barnard, K G; Asai, T

    1996-01-01

    The PriA protein, a component of the phiX174-type primosome, was previously shown to be essential for damage-inducible DNA replication in Escherichia coli, termed inducible stable DNA replication. Here, we show that priA::kan null mutants are defective in transductional and conjugational homologous recombination and are hypersensitive to mitomycin C and gamma rays, which cause double-strand breaks. The introduction of a plasmid carrying the priA300 allele, which encodes a mutant PriA protein ...

  17. Dynamics and Regulation of RecA Polymerization and De-Polymerization on Double-Stranded DNA

    OpenAIRE

    Fu, Hongxia; Le, Shimin; Muniyappa, Kalappa; Yan, Jie

    2013-01-01

    The RecA filament formed on double-stranded (ds) DNA is proposed to be a functional state analogous to that generated during the process of DNA strand exchange. RecA polymerization and de-polymerization on dsDNA is governed by multiple physiological factors. However, a comprehensive understanding of how these factors regulate the processes of polymerization and de-polymerization of RecA filament on dsDNA is still evolving. Here, we investigate the effects of temperature, pH, tensile force, an...

  18. Novel Smad proteins localize to IR-induced double-strand breaks: interplay between TGFβ and ATM pathways

    OpenAIRE

    Wang, Minli; Saha, Janapriya; Hada, Megumi; Anderson, Jennifer A.; Pluth, Janice M.; O’Neill, Peter; Cucinotta, Francis A.

    2012-01-01

    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...

  19. Double-Strand Break Repair in Bacteriophage T4: Recombination Effects of 3′–5′ Exonuclease Mutations

    OpenAIRE

    Shcherbakov, Victor P; Kudryashova, E. A.; Shcherbakova, T. S.; Sizova, S. T.; Plugina, L. A.

    2006-01-01

    The role of 3′–5′ exonucleases in double-strand break (DSB)-promoted recombination was studied in crosses of bacteriophage T4, in which DSBs were induced site specifically within the rIIB gene by SegC endonuclease in the DNA of only one of the parents. Frequency of rII+ recombinants was measured in two-factor crosses of the type i × ets1, where ets1 designates an insertion in the rIIB gene carrying the cleavage site for SegC and i's are rIIB or rIIA point mutations located at various distance...

  20. The Mismatch-Binding Factor MutSβ Can Mediate ATR Activation in Response to DNA Double-Strand Breaks

    Czech Academy of Sciences Publication Activity Database

    Burdová, Kamila; Mihaljevic, B.; Sturzenegger, A.; Chappidi, N.; Janščák, Pavel

    2015-01-01

    Roč. 59, č. 4 (2015), s. 603-614. ISSN 1097-2765 R&D Projects: GA ČR GAP305/10/0281; GA ČR(CZ) GA14-05743S Grant ostatní: Oncosuisse(CH) KLS-02344-02-2009; Swiss National Science Foundation(CH) 31003A_146206; Novartis Foundation for Medical and Biological Research(CH) 11A16 Institutional support: RVO:68378050 Keywords : Ataxia telangiectasia-mutated and Rad3-related (ATR) protein kinase * DNA-damage response * DNA Double-Strand Breaks Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 14.018, year: 2014

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

    Czech Academy of Sciences Publication Activity Database

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

    2014-01-01

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

  2. Detection and Repair of Ionizing Radiation-Induced DNA Double Strand Breaks: New Developments in Nonhomologous End Joining

    International Nuclear Information System (INIS)

    DNA damage can occur as a result of endogenous metabolic reactions and replication stress or from exogenous sources such as radiation therapy and chemotherapy. DNA double strand breaks are the most cytotoxic form of DNA damage, and defects in their repair can result in genome instability, a hallmark of cancer. The major pathway for the repair of ionizing radiation-induced DSBs in human cells is nonhomologous end joining. Here we review recent advances on the mechanism of nonhomologous end joining, as well as new findings on its component proteins and regulation

  3. Influence of a double-strand break in plasmid DNA on survival and formation of deletion mutations

    International Nuclear Information System (INIS)

    The fate of a DNA molecule carrying one double-strand break can easily be studied using enzymatic linearized plasmid DNA. The transformation of E. coli with such linearized plasmid molecules results in the formation of deletions in the plasmid DNA (Garev et al., 1982; Conley et al., 1986a; Bien and Schulte-Frohlinde, 1988). The authors examined the different behaviour of plasmid molecules linearized with various restriction enzymes and attempt to quantify the effect of short homologies for the deletion formation in E. coli. (author)

  4. Single -and double-strand breaks induced in plasmid DNA irradiated by ultra -soft X-ray

    International Nuclear Information System (INIS)

    In order to investigate the molecular consequences of a carbon K photo-ionization located on DNA, dry pBS plasmid samples were irradiated with ultra-soft X-rays at energies below and above the carbon K-threshold (Ek=278 eV). Single- and double-strand breaks (ssb and dsb) were quantified after resolution of the three plasmid forms (supercoiled, relaxed circular, linear) by gel electrophoresis. A factor of 1.2 was found between the doses required at 250 eV and 380 eV to induce the same number of dsb per plasmid. (authors)

  5. XLS (c9orf142) is a new component of mammalian DNA double-stranded break repair

    OpenAIRE

    Craxton, A; J. Somers; Munnur, D; Jukes-Jones, R; Cain, K.; Malewicz, M

    2015-01-01

    Repair of double-stranded DNA breaks (DSBs) in mammalian cells primarily occurs by the non-homologous end-joining (NHEJ) pathway, which requires seven core proteins (Ku70/Ku86, DNA-PKcs (DNA-dependent protein kinase catalytic subunit), Artemis, XRCC4-like factor (XLF), XRCC4 and DNA ligase IV). Here we show using combined affinity purification and mass spectrometry that DNA-PKcs co-purifies with all known core NHEJ factors. Furthermore, we have identified a novel evolutionary conserved protei...

  6. The spectrum of in vitro radiosensitivity in four human melanoma cell lines is not accounted for by differential induction or rejoining of DNA double strand breaks

    International Nuclear Information System (INIS)

    Purpose: Radioresistance is a significant clinical problem in advanced malignant melanoma and many melanoma cell lines show a radioresistant acute x-ray survival response in vitro. Given that the DNA double strand break is the lesion most closely correlated with x-ray induced cell lethality, differences in the induction and rejoining of these lesions may account for the radioresistance of some human melanoma cell lines. Methods and Materials: The above hypothesis was tested using pulsed field gel electrophoresis to measure x-ray induced DNA double strand break induction and rejoining in four human melanoma cell lines: MM138, MM170, MM96-L and HT 144. Results: The MM138, MM170 and MM96-L cell lines were characterized in vitro by low (α(β)) ratios and board x-ray survival curve shoulders. MM138 and MM170 were the most radioresistant and MM96-L had intermediate sensitivity. In contrast, HT144 was markedly x-ray sensitive, despite retaining a shoulder and like the other lines, having a low (α(β)) ratio. There were no significant differences in DNA double strand break induction between the cell lines, and thus no correlation existed between DNA double strand break induction and radiosensitivity. Consistent with the shoulders on the x-ray survival curves, all four cell lines showed efficient DNA double strand break rejoining. Highly efficient DNA double strand break rejoining could account for the radioresistance of one of the melanoma lines (MM138). For example, MM138 had rejoined 50% of the induced DNA double strand breaks by 5.5 min compared to 13-17 min for the other three cell lines. The development of postirradiation apoptosis was effectively excluded as the cause of the marked radiosensitivity of the HT144 cell line. Conclusion: Other factors (such as lesion repair fidelity or differential lesion tolerance) underlie the differences in the intrinsic radiosensitivity between these melanoma cell lines

  7. 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. PMID:26272087

  8. 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.)

  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

    International Nuclear Information System (INIS)

    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. Replication protein A and γ-H2AX foci assembly is triggered by cellular response to DNA double-strand breaks

    International Nuclear Information System (INIS)

    Human replication protein A (RPA p34), a crucial component of diverse DNA excision repair pathways, is implicated in DNA double-strand break (DSB) repair. To evaluate its role in DSB repair, the intranuclear dynamics of RPA was investigated after DNA damage and replication blockage in human cells. Using two different agents [ionizing radiation (IR) and hydroxyurea (HU)] to generate DSBs, we found that RPA relocated into distinct nuclear foci and colocalized with a well-known DSB binding factor, γ-H2AX, at the sites of DNA damage in a time-dependent manner. Colocalization of RPA and γ-H2AX foci peaked at 2 h after IR treatment and subsequently declined with increasing postrecovery times. The time course of RPA and γ-H2AX foci association correlated well with the DSB repair activity detected by a neutral comet assay. A phosphatidylinositol-3 (PI-3) kinase inhibitor, wortmannin, completely abolished both RPA and γ-H2AX foci formation triggered by IR. Additionally, radiosensitive ataxia telangiectasia (AT) cells harboring mutations in ATM gene product were found to be deficient in RPA and γ-H2AX colocalization after IR. Transfection of AT cells with ATM cDNA fully restored the association of RPA foci with γ-H2AX illustrating the requirement of ATM gene product for this process. The exact coincidence of RPA and γ-H2AX in response to HU specifically in S-phase cells supports their role in DNA replication checkpoint control. Depletion of RPA by small interfering RNA (SiRNA) substantially elevated the frequencies of IR-induced micronuclei (MN) and apoptosis in human cells suggestive of a role for RPA in DSB repair. We propose that RPA in association with γ-H2AX contributes to both DNA damage checkpoint control and repair in response to strand breaks and stalled replication forks in human cells

  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. Rejoining of DNA double-strand breaks in human fibroblasts and its impairment in one ataxia telangiectasia and two Fanconi strains

    International Nuclear Information System (INIS)

    Using the technique of neutral elution through polycarbonate filters as a measure of DNA length, and hence of the number of double-strand breaks incurred as a result of radiation damage, we found that normal human fibroblasts rejoin 50% of all breaks within only 3 min (37 degrees C). This fast rejoining was impaired in fibroblasts from one patient with Ataxia telangiectasia and in fibroblasts from two patients with Fanconi's anemia. Also the number of residual breaks after several hours of repair was higher than in control cells. Other cases with the same diseases were normal in their rejoining of double-strand breaks

  13. Synthetic double-stranded RNAs are adjuvants for the induction of T helper 1 and humoral immune responses to human papillomavirus in rhesus macaques.

    Directory of Open Access Journals (Sweden)

    Christiane Stahl-Hennig

    2009-04-01

    Full Text Available Toll-like receptor (TLR ligands are being considered as adjuvants for the induction of antigen-specific immune responses, as in the design of vaccines. Polyriboinosinic-polyribocytoidylic acid (poly I:C, a synthetic double-stranded RNA (dsRNA, is recognized by TLR3 and other intracellular receptors. Poly ICLC is a poly I:C analogue, which has been stabilized against the serum nucleases that are present in the plasma of primates. Poly I:C(12U, another analogue, is less toxic but also less stable in vivo than poly I:C, and TLR3 is essential for its recognition. To study the effects of these compounds on the induction of protein-specific immune responses in an animal model relevant to humans, rhesus macaques were immunized subcutaneously (s.c. with keyhole limpet hemocyanin (KLH or human papillomavirus (HPV16 capsomeres with or without dsRNA or a control adjuvant, the TLR9 ligand CpG-C. All dsRNA compounds served as adjuvants for KLH-specific cellular immune responses, with the highest proliferative responses being observed with 2 mg/animal poly ICLC (p = 0.002 or 6 mg/animal poly I:C(12U (p = 0.001 when compared with immunization with KLH alone. Notably, poly ICLC -- but not CpG-C given at the same dose -- also helped to induce HPV16-specific Th1 immune responses while both adjuvants supported the induction of strong anti-HPV16 L1 antibody responses as determined by ELISA and neutralization assay. In contrast, control animals injected with HPV16 capsomeres alone did not develop substantial HPV16-specific immune responses. Injection of dsRNA led to increased numbers of cells producing the T cell-activating chemokines CXCL9 and CXCL10 as detected by in situ hybridization in draining lymph nodes 18 hours after injections, and to increased serum levels of CXCL10 (p = 0.01. This was paralleled by the reduced production of the homeostatic T cell-attracting chemokine CCL21. Thus, synthetic dsRNAs induce an innate chemokine response and act as adjuvants

  14. DNA double strand breaks as the critical type of damage with regard to inactivation of cells through ionizing radiation

    International Nuclear Information System (INIS)

    This report presents the results of an investigation into the effects of ionizing radiation on eukaryotic cells, aimed at revealing the molecular mechanisms leading to cell inactivation as a result of ionizing radiation. The quantitative determination of radiation-induced double strand breaks (DSB) is done via sedimentation of the DNA released from the cells in a neutral saccharose gradient in a preparative ultracentrifuge. The 'experimental mass spectrum' of DNA molecules thus obtained, the mean number of DSB per cell is calculated using a special computer program which simulates the stochastic induction of DSB in the DNA of non-irradiated cells and links the 'simulated' mass spectrum with the 'experimental' one on the basis of the least square fit. The experimental and theoretical studies with the eukaryote yeast on the whole allow insight into the relation between energy absorption and the inactivation of irradiated cells. (orig./MG)

  15. Application of pulsed field gel electrophoresis to determine γ-ray-induced double-strand breaks in yeast chromosomal molecules

    International Nuclear Information System (INIS)

    The frequency of DNA double-strand breaks (dsb) was determined in yeast cells exposed to γ-rays under anoxic conditions. Genomic DNA of treated cells was separated by pulsed field gel electrophoresis, and two different approaches for the evaluation of the gels were employed: (1) The DNA mass distribution profile obtained by electrophoresis was compared to computed profiles, and the number of DSB per unit length was then derived in terms of a fitting procedure; (2) hybridization of selected chromosomes was performed, and a comparison of the hybridization signals in treated and untreated samples was then used to derive the frequency of dsb. The two assays gave similar results for the frequency of dsb ((1.07 ± 0.06) x 10-9 Gy-1 bp-1 and (0.93 ± 0.09) x 10-9 Gy-1 bp-1, respectively). The dsb frequency was found to be linearly dependent on dose. (author)

  16. Pulsed-field gel electrophoretic analysis of DNA double-strand breaks in mammalian cells using photostimulable storage phosphor imaging

    International Nuclear Information System (INIS)

    Double-strand break (dsb) induction and repair was determined in the human colon carcinoma cell line clone A using pulsed-field gel electrophoresis (PFGE) coupled with photostimulable storage phosphor imaging technology. Because 14C-radioactivity was measured in a dried agarose gel following electrophoresis, no laborious processing of the gel, cutting out regions of interest, liquid scintillation counting, etc., was necessary thereby saving labour, time and cost. The signal generated by phosphor screens was linear over 5 logs and sensitive to low levels of radionuclide exposure. Migration of broken DNA into the gel upon electrophoresis was determined to be log-linear as a function of dose, and dsb rejoining after irradiation could be measured for exposures as low as 5 Gy. (Author)

  17. Sibling rivalry: competition between Pol X family members in V(D)J recombination and general double strand break repair.

    Science.gov (United States)

    Nick McElhinny, Stephanie A; Ramsden, Dale A

    2004-08-01

    The nonhomologous end-joining pathway is a major means for repairing double-strand breaks (DSBs) in all mitotic cell types. This repair pathway is also the only efficient means for resolving DSB intermediates in V(D)J recombination, a lymphocyte-specific genome rearrangement required for assembly of antigen receptors. A role for polymerases in end-joining has been well established. They are a major factor in determining the character of repair junctions but, in contrast to 'core' end-joining factors, typically appear to have a subtle impact on the efficiency of end-joining. Recent work implicates several members of the Pol X family in end-joining and suggests surprising complexity in the control of how these different polymerases are employed in this pathway. PMID:15242403

  18. Kinetic study of the binding of triplex-forming oligonucleotides containing partial cationic modifications to double-stranded DNA.

    Science.gov (United States)

    Hari, Yoshiyuki; Ijitsu, Shin; Akabane-Nakata, Masaaki; Yoshida, Takuya; Obika, Satoshi

    2014-07-15

    Several triplex-forming oligonucleotides (TFOs) partially modified with 2'-O-(2-aminoethyl)- or 2'-O-(2-guanidinoethyl)-nucleotides were synthesized and their association rate constants (kon) with double-stranded DNA were estimated by UV spectrophotometry. Introduction of cationic modifications in the 5'-region of the TFOs significantly increased the kon values compared to that of natural TFO, while no enhancement in the rate of triplex DNA formation was observed when the modifications were in the middle and at the 3'-region. The kon value of a TFO with three adjacent cationic modifications at the 5'-region was found to be 3.4 times larger than that of a natural one. These results provide useful information for overcoming the inherent sluggishness of triplex DNA formation. PMID:24865415

  19. Combined Triplex/Duplex Invasion of Double-Stranded DNA by "Tail-Clamp" Peptide Nucleic Acid

    DEFF Research Database (Denmark)

    Bentin, Thomas; Larsen, H. J.; Nielsen, Peter E.

    2003-01-01

    "Tail-clamp" PNAs composed of a short (hexamer) homopyrimidine triplex forming domain and a (decamer) mixed sequence duplex forming extension have been designed. Tail-clamp PNAs display significantly increased binding to single-stranded DNA compared with PNAs lacking a duplex-forming extension as...... determined by T-m measurements. Binding to double-stranded (ds) DNA occurred by combined triplex and duplex invasion as analyzed by permanganate probing. Furthermore, C-50 measurements revealed that tail-clamp PNAs consistently bound the dsDNA target more efficiently, and kinetics experiments revealed that...... this was due to a dramatically reduced dissociation rate of such complexes. Increasing the PNA net charge also increased binding efficiency, but unexpectedly, this increase was much more pronounced for tailless-clamp PNAs than for tail-clamp PNAs. Finally, shortening the tail-clamp PNA triplex invasion...

  20. 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.

  1. Application of laser-accelerated protons to the demonstration of DNA double-strand breaks in human cancer cells

    Science.gov (United States)

    Yogo, A.; Sato, K.; Nishikino, M.; Mori, M.; Teshima, T.; Numasaki, H.; Murakami, M.; Demizu, Y.; Akagi, S.; Nagayama, S.; Ogura, K.; Sagisaka, A.; Orimo, S.; Nishiuchi, M.; Pirozhkov, A. S.; Ikegami, M.; Tampo, M.; Sakaki, H.; Suzuki, M.; Daito, I.; Oishi, Y.; Sugiyama, H.; Kiriyama, H.; Okada, H.; Kanazawa, S.; Kondo, S.; Shimomura, T.; Nakai, Y.; Tanoue, M.; Sasao, H.; Wakai, D.; Bolton, P. R.; Daido, H.

    2009-05-01

    We report the demonstrated irradiation effect of laser-accelerated protons on human cancer cells. In vitro (living) A549 cells are irradiated with quasimonoenergetic proton bunches of 0.8-2.4 MeV with a single bunch duration of 15 ns. Irradiation with the proton dose of 20 Gy results in a distinct formation of γ-H2AX foci as an indicator of DNA double-strand breaks generated in the cancer cells. This is a pioneering result that points to future investigations of the radiobiological effects of laser-driven ion beams. Unique high-current and short-bunch features make laser-driven proton bunches an excitation source for time-resolved determination of radical yields.

  2. 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.

  3. Repair of the double-strand breaks produced by 125I disintegrations in the DNA of micrococcus radiodurans

    International Nuclear Information System (INIS)

    Wild-type M. radiodurans and two radiosensitive mutants were used to study the lethal effects of 125I disintegrations in their DNA. The relative sensitivities of these three strains to inactivation by γ-radiation were reflected in their relative sensitivities to inactivation by 125I decay. The number of double-strand (ds) breaks in the DNA appeared to be similar at levels of γ-radiation and of 125I decay that reduced survival to 10%. All three strains of M. radiodurans rapidly repaired ds breaks produced in their DNA by either γ-radiation or 125I disintegrations. If one ds break per cell is a lethal event [Krisch. et al., 1975], cells of the three strains tested would die when they had left unrepaired one ds break out of an initial 45, 600 or 1800 ds breaks per single cell. (Auth.)

  4. Sensitive and direct electrochemical detection of double-stranded DNA utilizing alkaline phosphatase-labelled zinc finger proteins.

    Science.gov (United States)

    Noh, Soodong; Ha, Dat Thinh; Yang, Haesik; Kim, Moon-Soo

    2015-06-21

    Direct detection of double-stranded DNA (dsDNA) using zinc finger proteins (ZFPs) is of great importance in biomedical applications such as identifying pathogens and circulating DNAs. However, its sensitivity is still not sufficiently high because limited signalling labels can be conjugated or fused. Herein, we report sensitive and direct detection of dsDNA using (i) alkaline phosphatase (ALP) as a fast catalytic label conjugated to ZFPs along with (ii) electrochemical measurement of an ALP product (l-ascorbic acid) at the indium-tin oxide electrode with a high signal-to-background ratio. ALP is simply conjugated to a ZFP through lysine residues in a ZFP purification tag, a maltose binding protein (MBP). Sandwich-type electrochemical detection of dsDNA allows a detection limit of ca. 100 fM without using DNA amplification. PMID:25969923

  5. Influence of the sequence on the ab initio band structures of single and double stranded DNA models

    International Nuclear Information System (INIS)

    The solid state physical approach is widely used for the characterization of electronic properties of DNA. In the simplest case the helical symmetry is explicitly utilized with a repeat unit containing only a single nucleotide or nucleotide pair. This model provides a band structure that is easily interpretable and reflects the main characteristic features of the single nucleotide or a nucleotide pair chain, respectively. The chemical variability of the different DNA chains is, however, almost completely neglected in this way. In the present work we have investigated the effect of the different sequences on the band structure of periodic DNA models. For this purpose we have applied the Hartree–Fock crystal orbital method for single and double stranded DNA chains with two different subsequent nucleotides in the repeat unit of former and two different nucleotide pairs in the latter case, respectively. These results are compared to simple helical models with uniform sequences. The valence and conduction bands related to the stacked nucleotide bases of single stranded DNA built up only from guanidine as well as of double stranded DNA built up only from guanidine–cytidine pairs showed special properties different from the other cases. Namely, they had higher conduction and lower valence band positions and this way larger band gaps and smaller widths of these bands. With the introduction of non-uniform guanidine containing sequences band structures became more similar to each other and to the band structures of other sequences without guanidine. The maximal bandwidths of the non-uniform sequences are considerably smaller than in the case of uniform sequences implying smaller charge carrier mobilities both in the conduction and valence bands. - Highlights: • HF Energy bands in DNA. • The role of aperiodicity in the DNA band structure. • Hole mobilities in quasi-periodic DNA with broader valence bands

  6. 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.

  7. Triple Helical Recognition of RNA Using 2-Aminopyridine-Modified PNA at Physiologically Relevant Conditions**

    OpenAIRE

    Zengeya, Thomas; Gupta, Pankaj; Rozners, Eriks

    2012-01-01

    Peptide nucleic acids containing thymidine and 2-aminopyridine (M) nucleobases formed stable and sequence selective triple helices with double stranded RNA at physiologically relevant conditions. The M-modified PNA displayed unique RNA selectivity by having two orders of magnitude higher affinity for the double stranded RNAs than for the same DNA sequences. Preliminary results suggested that nucleobase-modified PNA could bind and recognize double helical precursors of microRNAs.

  8. Single-molecule manipulation of double-stranded DNA using optical tweezers: Interaction studies of DNA with RecA and YOYO-1

    NARCIS (Netherlands)

    Bennink, Martin L.; Scharer, Orlando D.; Kanaar, Ronald; Sakata-Sogawa, Kumiko; Schins, Juleon M.; Kanger, Johannes S.; Grooth, de Bart G.; Greve, Jan

    1999-01-01

    By using optical tweezers and a specially designed flow cell with an integrated glass micropipette, we constructed a setup similar to that of Smith et al. (Science 271:795-799, 1996) in which an individual double-stranded DNA (dsDNA) molecule can be captured between two polystyrene beads. The first

  9. Does the enzymatic conversion of DNA single-strand damage into double-strand breaks contribute to biological inactivation of γ-irradiated plasmid DNA?

    International Nuclear Information System (INIS)

    Incubation of γ-irradiated plasmid DNA (pBR322) in a protein extract of E. coli CMK wild-type cells leads to the formation of double-strand breaks which contribute to biological inactivation by ≥ 80%. (author)

  10. 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 ...

  11. 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 ...

  12. 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.

  13. Toll like receptor 3 & 4 responses of human turbinate derived mesenchymal stem cells: stimulation by double stranded RNA and lipopolysaccharide.

    Directory of Open Access Journals (Sweden)

    Se Hwan Hwang

    Full Text Available BACKGROUND AND OBJECTIVES: Multipotent mesenchymal stromal cells (MSCs represent a promising cell-based therapy for a number of inflammatory or autoimmune diseases. Herein, Toll like receptor (TLR expression by MSCs and their immune regulatory roles are investigated. In this study, we investigated the influence of TLR on the immune response, proliferation, and differentiation potential of human turbinated MSC (hTMSC cultures in vitro. SUBJECTS AND METHODS: After isolating hTMSCs from discarded inferior turbinate tissue, FACS analysis was used to assess the expression of TLRs such as TLR2, TLR3, TLR4, and TLR5 in hTMSCs and cell proliferation was assessed using a cell counting kit (CCK-8. Cytokine and chemokine secretions were analyzed with multiplex immunoassays for IL-1α, IL-1β, IL-4, IL-6, IL-8, IL-10, IL-12, IP-10 (CXCL10, RANTES (CCL5, TNF-a, GM-CSF, and IFN-γ. The differentiation potential of hTMSCs was evaluated in the osteogenic, chondogenic, and adipogeinc media and analyzed by histology and gene expression related to differentiation. RESULTS: FACS analysis revealed that TLR3 and TLR4 expression consisted of a relatively high percentage of the surface proteins expressed by hTMSCs. The proliferation of hTMSCs was influenced and significantly increased by the presence of TLR4 agonists. In particular, hTMSCs produced a set of cytokines and chemokines and the expression of IL-6, IL-8, IL-12, IP-10 (CXCL10, RANTES (CCL5, TNF-α, and GM-CSF were up-regulated in response to the TLR4 agonist LPS. The osteogenic and adipogeinc differentiation potential of hTMSCs was not affected by TLR agonists. CONCLUSIONS: We conclude that TLR4 stimulation affects TLR expression, proliferation, and the immunomodulation potential of hTMSCs. Understanding the mechanism behind TLR's influence on hTMSCs and their immunomodulating properties would be useful for providing a novel target to exploit in the improvement of stem cell-based therapeutic strategies.

  14. 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.

  15. 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....

  16. Synthesis of plus- and minus-strand RNA in rotavirus-infected cells.

    OpenAIRE

    Stacy-Phipps, S; Patton, J T

    1987-01-01

    The genomes of the rotaviruses consist of 11 segments of double-stranded RNA. During RNA replication, the viral plus-strand RNA serves as the template for minus-strand RNA synthesis. To characterize the kinetics of RNA replication, the synthesis and steady-state levels of viral plus- and minus-strand RNA and double-stranded RNA in simian rotavirus SA11-infected MA104 cells were analyzed by electrophoresis on 1.75% agarose gels containing 6 M urea (pH 3.0). Synthesis of viral plus-strand and m...

  17. The logic of DNA replication in double-stranded DNA viruses: insights from global analysis of viral genomes.

    Science.gov (United States)

    Kazlauskas, Darius; Krupovic, Mart; Venclovas, Česlovas

    2016-06-01

    Genomic DNA replication is a complex process that involves multiple proteins. Cellular DNA replication systems are broadly classified into only two types, bacterial and archaeo-eukaryotic. In contrast, double-stranded (ds) DNA viruses feature a much broader diversity of DNA replication machineries. Viruses differ greatly in both completeness and composition of their sets of DNA replication proteins. In this study, we explored whether there are common patterns underlying this extreme diversity. We identified and analyzed all major functional groups of DNA replication proteins in all available proteomes of dsDNA viruses. Our results show that some proteins are common to viruses infecting all domains of life and likely represent components of the ancestral core set. These include B-family polymerases, SF3 helicases, archaeo-eukaryotic primases, clamps and clamp loaders of the archaeo-eukaryotic type, RNase H and ATP-dependent DNA ligases. We also discovered a clear correlation between genome size and self-sufficiency of viral DNA replication, the unanticipated dominance of replicative helicases and pervasive functional associations among certain groups of DNA replication proteins. Altogether, our results provide a comprehensive view on the diversity and evolution of replication systems in the DNA virome and uncover fundamental principles underlying the orchestration of viral DNA replication. PMID:27112572

  18. 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.

  19. The location of DNA in complexes of recA protein with double-stranded DNA. A neutron scattering study

    International Nuclear Information System (INIS)

    Purified recA protein is found as rodlike homopolymers, and it forms filamentous complexes with double-stranded DNA that are stable in the presence of ATP gamma S, a nonhydrolyzable analogue of ATP. The structure of these filaments has been described in some detail by electron microscopy. Here we confirm the mass per length of 6.5 recA/100 A in solution by small-angle neutron scattering and extend the analysis to homopolymers of recA protein, finding a mass per length of about 7 recA/100 A and a radial mass distribution (cross-sectional radius of gyration) significantly different for the two filaments. The models proposed so far for the structure of the complex have placed the DNA in the center of the filament. Here we verify this assumption using small-angle neutron scattering to locate the DNA in the complexes, exploiting the contrast variation method in D2O/H2O mixtures. Model calculations show that the natural contrast difference between DNA and protein is not sufficient to locate the DNA (which accounts for only 4.7% of the mass in the complex). When deuterated DNA is used, the contrast difference is enhanced, and model calculations and experiment then converge, indicating that the DNA is indeed near the axis of the complex

  20. MEIOTIC F-BOX Is Essential for Male Meiotic DNA Double-Strand Break Repair in Rice[OPEN

    Science.gov (United States)

    Wang, Chong; Yu, Junping; Zong, Jie; Lu, Pingli

    2016-01-01

    F-box proteins constitute a large superfamily in plants and play important roles in controlling many biological processes, but the roles of F-box proteins in male meiosis in plants remain unclear. Here, we identify the rice (Oryza sativa) F-box gene MEIOTIC F-BOX (MOF), which is essential for male meiotic progression. MOF belongs to the FBX subfamily and is predominantly active during leptotene to pachytene of prophase I. mof meiocytes display disrupted telomere bouquet formation, impaired pairing and synapsis of homologous chromosomes, and arrested meiocytes at late prophase I, followed by apoptosis. Although normal, programmed double-stranded DNA breaks (DSBs) form in mof mutants, foci of the phosphorylated histone variant γH2AX, a marker for DSBs, persist in the mutant, indicating that many of the DSBs remained unrepaired. The recruitment of Completion of meiosis I (COM1) and Radiation sensitive51C (RAD51C) to DSBs is severely compromised in mutant meiocytes, indicating that MOF is crucial for DSB end-processing and repair. Further analyses showed that MOF could physically interact with the rice SKP1-like Protein1 (OSK1), indicating that MOF functions as a component of the SCF E3 ligase to regulate meiotic progression in rice. Thus, this study reveals the essential role of an F-box protein in plant meiosis and provides helpful information for elucidating the roles of the ubiquitin proteasome system in plant meiotic progression. PMID:27436711

  1. Hot spots of DNA double-strand breaks in human rDNA units are produced in vivo.

    Science.gov (United States)

    Tchurikov, Nickolai A; Yudkin, Dmitry V; Gorbacheva, Maria A; Kulemzina, Anastasia I; Grischenko, Irina V; Fedoseeva, Daria M; Sosin, Dmitri V; Kravatsky, Yuri V; Kretova, Olga V

    2016-01-01

    Endogenous hot spots of DNA double-strand breaks (DSBs) are tightly linked with transcription patterns and cancer genomics(1,2). There are nine hot spots of DSBs located in human rDNA units(3-6). Here we describe that the profiles of these hot spots coincide with the profiles of γ-H2AX or H2AX, strongly suggesting a high level of in vivo breakage inside rDNA genes. The data were confirmed by microscopic observation of the largest γ-H2AX foci inside nucleoli in interphase chromosomes. In metaphase chromosomes, we observed that only some portion of rDNA clusters possess γ-H2AX foci and that all γ-H2AX foci co-localize with UBF-1 binding sites, which strongly suggests that only active rDNA units possess the hot spots of DSBs. Both γ-H2AX and UBF-1 are epigenetically inherited and thus indicate the rDNA units that were active in the previous cell cycle. These results have implications for diverse fields, including epigenetics and cancer genomics. PMID:27160357

  2. The importance of becoming double-stranded: Innate immunity and the kinetic model of HIV-1 central plus strand synthesis

    International Nuclear Information System (INIS)

    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

  3. The importance of becoming double-stranded: Innate immunity and the kinetic model of HIV-1 central plus strand synthesis.

    Science.gov (United States)

    Poeschla, Eric

    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. PMID:23561461

  4. Influence of irradiation conditions on the measurement of DNA double-strand breaks by pulsed-field gel electrophoresis

    International Nuclear Information System (INIS)

    The induction of γ-ray induced DNA double-strand breaks (DSBs) determined by pulsed-field gel electrophoresis was compared in the DNA of intact Chinese hamster ovary (CHO) cells imbedded in agarose plugs, and in the isolated DNA of agarose-imbedded CHO cells that had been lysed before irradiation to determine whether these irradiation conditions would influence their measurement. DSB-induction in irradiated cells or isolated DNA was measured as the loss of DNA from the plug compared with the unirradiated control. Chromatin protein was completely digested by the lysis buffer and as such did not affect DNA migration upon electrophoresis, whereas concentrations of EDTA as low as 10-5 M affected the induction of DSBs in the isolated DNA. The gel plugs required several hours of washing with PBS to remove the contaminating EDTA from the lysis buffer. Once the residual EDTA was removed, DNA DSB induction as a function of dose was 70 times greater in isolated DNA than in the DNA of intact cells. (Author)

  5. Detection of heavy-ion-induced DNA double-strand breaks using static-field gel electrophoresis

    International Nuclear Information System (INIS)

    Radiation-induced DNA double-strand breaks (DSBs) were measured in Chinese hamster ovary cells (CHO-K1) using an experimental protocol involving static-field gel electrophoresis following exposure to various accelerated ions. Dose-effect curves were set up, and relative biological efficiencies (RBEs) for DSB induction were determined for different radiation qualities. RBEs around 1 were obtained for low energy deuterons (6-7 keV/μm), while for high energy oxygen ions (20 keV/μm) an RBE value slightly greater than 1 was determined. Low energetic oxygen ions (LET ∼ 250 keV/μm) were found to show RBEs substantially below unity, and for higher LET particles (≥ 250 keV/μm) RBEs for DSB induction were generally found to be smaller than 1. The data presented here are in line with the generally accepted view that not induced DSBs, but rather misrepaired or unrepaired DNA lesions are related to cellular inactivation. (orig.)

  6. The inflammatory response to double stranded DNA in endothelial cells is mediated by NFκB and TNFα.

    Directory of Open Access Journals (Sweden)

    Suraj J Patel

    Full Text Available Endothelial cells represent an important barrier between the intravascular compartment and extravascular tissues, and therefore serve as key sensors, communicators, and amplifiers of danger signals in innate immunity and inflammation. Double stranded DNA (dsDNA released from damaged host cells during injury or introduced by pathogens during infection, has emerged as a potent danger signal. While the dsDNA-mediated immune response has been extensively studied in immune cells, little is known about the direct and indirect effects of dsDNA on the vascular endothelium. In this study we show that direct dsDNA stimulation of endothelial cells induces a potent proinflammatory response as demonstrated by increased expression of ICAM1, E-selectin and VCAM1, and enhanced leukocyte adhesion. This response was dependent on the stress kinases JNK and p38 MAPK, required the activation of proinflammatory transcription factors NFκB and IRF3, and triggered the robust secretion of TNFα for sustained secondary activation of the endothelium. DNA-induced TNFα secretion proved to be essential in vivo, as mice deficient in the TNF receptor were unable to mount an acute inflammatory response to dsDNA. Our findings suggest that the endothelium plays an active role in mediating dsDNA-induced inflammatory responses, and implicate its importance in establishing an acute inflammatory response to sterile injury or systemic infection, where host or pathogen derived dsDNA may serve as a danger signal.

  7. 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.

  8. Gamma Irradiation Induces DNA Double-Strand Breaks in Fibroblasts: A Model Study for the Development of Biodosimetry

    International Nuclear Information System (INIS)

    Accidental exposure to ionizing radiation can immediately induce double-strand breaks (DSBs) of DNAs which later pose detrimental damage on organisms including genetic instability and cell death. The aim of this study is to simulate such incident by exposing a cell model to gamma radiation and the resulting DNA DSBs were immunofluorescently labeled and quantified to establish a dose response relationship. Human dermal fibroblasts were grown into monolayers before irradiated by gamma rays from a Co-60 source at doses 0, 0.2, 1, 2 and 4 Gy and a dose rate of 0.21 Gy/min. DNA DSBs, which appeared as foci inside the cells' nuclei, were evaluated by flow cytometry and confocal microscopy. Data showed that the foci intensity increased linearly in relation to the increase in irradiation dose within 1 h post exposure. These findings can be further developed to serve as a personal biodosimetry to assess the immediate extent and potential health risks of accidental exposure to ionizing radiation in individuals.

  9. 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.

  10. Radiation-induced DNA double strand breaks in Ehrlich ascites tumour cells and their possible effects on cell survival

    International Nuclear Information System (INIS)

    A method to prepare high-molecular, pure DNA with the aid of enzymes, detergents, and heat treatment is presented. A sedimentation technique with neutral density gradients has been introduced which permits mass separation and molecular mass analysis of high-molecular DNA (msub(r) 10). Using this method, the induction of DNA double strand breaks (DSB) in the dose range between 10 Gy <= D <= 100 Gy has been measured. Further, the induction of DSB in dependence of the radiation quality has been studied. A correlation between DSB induction and cell survival was not found for any type of radiation investigated. DNA repair was measured in a multitude of repair conditions. The repair kinetics shows that the ''apparent'' DNA fraction is negligible. A large number of DSB was found to be repaired. The effects of some repair inhibitors have been investigated. DSB repair after cell plating on agar was proved. This factor has been underestimated in all former estimates of DSB influence on cell survival. It has been shown that DSB in living cells cannot be tolerated. There was no indication of biologically irrelevant DSB. (orig./AJ)

  11. 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.

  12. 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. PMID:27148355

  13. A quantitative model of the major pathways for radiation-induced DNA double-strand break repair

    International Nuclear Information System (INIS)

    We have developed a model approach to simulate the major pathways of DNA double-strand break (DSB) repair in mammalian and human cells. The proposed model shows a possible mechanistic explanation of the basic regularities of DSB processing through the nonhomologous end-joining (NHEJ), homologous recombination (HR), and single-strand annealing (SSA). It reconstructs the time-courses of radiation-induced foci specific to particular repair processes including the major intermediate stages. The model is validated for ionizing radiations of a wide range of linear energy transfer (0.2-236 keV/μm) including a relatively broad spectrum of heavy ions. The appropriate set of reaction rate constants was suggested to satisfy the kinetics of DSB rejoining for the considered types of exposure. The simultaneous assessment of three repair pathways allows one to describe their possible biological relations in response to radiation. With the help of the proposed approach, we reproduce several experimental data sets on γ-H2AX foci remaining in different types of cells including those defective in NHEJ, HR, or SSA functions.

  14. 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.

  15. Nitric oxide mediated DNA double strand breaks induced in proliferating bystander cells after {alpha}-particle irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Han Wei [Department of Physics and Materials Science, City University of Hong Kong, Tat Chee Avenue, Kowloon Tong (Hong Kong); Chen Shaopeng [Department of Physics and Materials Science, City University of Hong Kong, Tat Chee Avenue, Kowloon Tong (Hong Kong); Key Laboratory of Ion Beam Bioengineering, Institute of Plasma Physics, Chinese Academy of Sciences, Hefei 230031 (China); Yu, K.N., E-mail: peter.yu@cityu.edu.hk [Department of Physics and Materials Science, City University of Hong Kong, Tat Chee Avenue, Kowloon Tong (Hong Kong); Wu Lijun [Key Laboratory of Ion Beam Bioengineering, Institute of Plasma Physics, Chinese Academy of Sciences, Hefei 230031 (China)

    2010-02-03

    Low-dose {alpha}-particle exposures comprise 55% of the environmental dose to the human population and have been shown to induce bystander responses. Previous studies showed that bystander effect could induce stimulated cell growth or genotoxicity, such as excessive DNA double strand breaks (DSBs), micronuclei (MN), mutation and decreased cell viability, in the bystander cell population. In the present study, the stimulated cell growth, detected with flow cytometry (FCM), and the increased MN and DSB, detected with p53 binding protein 1 (53BP1) immunofluorescence, were observed simultaneously in the bystander cell population, which were co-cultured with cells irradiated by low-dose {alpha}-particles (1-10 cGy) in a mixed system. Further studies indicated that nitric oxide (NO) and transforming growth factor {beta}1 (TGF-{beta}1) played very important roles in mediating cell proliferation and inducing MN and DSB in the bystander population through treatments with NO scavenger and TGF-{beta}1 antibody. Low-concentrations of NO, generated by spermidine, were proved to induce cell proliferation, DSB and MN simultaneously. The proliferation or shortened cell cycle in bystander cells gave them insufficient time to repair DSBs. The increased cell division might increase the probability of carcinogenesis in bystander cells since cell proliferation increased the probability of mutation from the mis-repaired or un-repaired DSBs.

  16. Photoactive, covalent attachment of deoxyribonucleic acid on gold with double-strand specificity using self-assembled monolayers containing psoralen

    International Nuclear Information System (INIS)

    Taking advantages of psoralen photochemistry, we have developed a new method of immobilizing DNA on gold substrate surfaces. A psoralen derivative having an alkylamine function was synthesized, and was self-assembled on gold substrate surfaces in a combined use of a thiol-derivatized molecule, 3,3'-dithiobis(succinimidyl propionate) forming amide bonds on the surface. We found that by irradiating with long wavelength ultraviolet light (320-400 nm), DNA molecules added in the solution phase were covalently immobilized on the monolayer surface through the photoadduct formation of the psoralen molecules with the DNA nucleobases. The present method has its advantage that is applicable to native DNAs, no chemically modifying DNAs, in spite of its covalent immobilization principle. We have examined 12 mer synthetic oligonucleotide immobilizations and have found that the surface concentration thus attained was to be 20 pmol cm-2, which is consistent with saturated surface coverage. Interestingly, the immobilization occurred double-stranded-DNA-preferentially; no immobilization for single-stranded DNAs. Characterization of the immobilization chemistry has been achieved using atomic force microscopic imaging, infrared absorption, X-ray photoelectron spectroscopy, electrochemistry, and quartz-crystal microbalance and their results were described

  17. 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.

  18. 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.

  19. Postreplication repair in ultraviolet-irradiated human fibroblasts: formation and repair of DNA double-strand breaks

    International Nuclear Information System (INIS)

    A neutral filter elution assay was used to determine if the post-replicational formation and repair of DNA double-strand breaks (DSB) occurs in u.v.-irradiated human cells. Excision-deficient XP12 cells were pulse-labeled with [3H]thymidine after u.v. irradiation (1.5-3J/m2), and the nascent DNA was followed during repair incubation. With increasing u.v. radiation fluences, an increasing fraction of DNA was eluted at a fast rate, indicating that DSB were produced. The maximum yield DSB was observed after about 24 h of post-irradiation incubation at 370C. Similar results were also obtained with repair-proficient VA13 cells when irradiated at much higher fluences (7.5-15J/m2). It is concluded that, at the u.v. radiation fluences used, the DSB produced in u.v.-irradiated human cells are the result of post-replication repair events, and at incubation times >24 h some of these DSB are repaired. (author)

  20. Ligase I and ligase III mediate the DNA double-strand break ligation in alternative end-joining.

    Science.gov (United States)

    Lu, Guangqing; Duan, Jinzhi; Shu, Sheng; Wang, Xuxiang; Gao, Linlin; Guo, Jing; Zhang, Yu

    2016-02-01

    In eukaryotes, DNA double-strand breaks (DSBs), one of the most harmful types of DNA damage, are repaired by homologous repair (HR) and nonhomologous end-joining (NHEJ). Surprisingly, in cells deficient for core classic NHEJ factors such as DNA ligase IV (Lig4), substantial end-joining activities have been observed in various situations, suggesting the existence of alternative end-joining (A-EJ) activities. Several putative A-EJ factors have been proposed, although results are mostly controversial. By using a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system, we generated mouse CH12F3 cell lines in which, in addition to Lig4, either Lig1 or nuclear Lig3, representing the cells containing a single DNA ligase (Lig3 or Lig1, respectively) in their nucleus, was completely ablated. Surprisingly, we found that both Lig1- and Lig3-containing complexes could efficiently catalyze A-EJ for class switching recombination (CSR) in the IgH locus and chromosomal deletions between DSBs generated by CRISPR/Cas9 in cis-chromosomes. However, only deletion of nuclear Lig3, but not Lig1, could significantly reduce the interchromosomal translocations in Lig4(-/-) cells, suggesting the unique role of Lig3 in catalyzing chromosome translocation. Additional sequence analysis of chromosome translocation junction microhomology revealed the specificity of different ligase-containing complexes. The data suggested the existence of multiple DNA ligase-containing complexes in A-EJ. PMID:26787905

  1. Inactivation of nuclear GSK3β by Ser(389) phosphorylation promotes lymphocyte fitness during DNA double-strand break response.

    Science.gov (United States)

    Thornton, Tina M; Delgado, Pilar; Chen, Liang; Salas, Beatriz; Krementsov, Dimitry; Fernandez, Miriam; Vernia, Santiago; Davis, Roger J; Heimann, Ruth; Teuscher, Cory; Krangel, Michael S; Ramiro, Almudena R; Rincón, Mercedes

    2016-01-01

    Variable, diversity and joining (V(D)J) recombination and immunoglobulin class switch recombination (CSR) are key processes in adaptive immune responses that naturally generate DNA double-strand breaks (DSBs) and trigger a DNA repair response. It is unclear whether this response is associated with distinct survival signals that protect T and B cells. Glycogen synthase kinase 3β (GSK3β) is a constitutively active kinase known to promote cell death. Here we show that phosphorylation of GSK3β on Ser(389) by p38 MAPK (mitogen-activated protein kinase) is induced selectively by DSBs through ATM (ataxia telangiectasia mutated) as a unique mechanism to attenuate the activity of nuclear GSK3β and promote survival of cells undergoing DSBs. Inability to inactivate GSK3β through Ser(389) phosphorylation in Ser(389)Ala knockin mice causes a decrease in the fitness of cells undergoing V(D)J recombination and CSR. Preselection-Tcrβ repertoire is impaired and antigen-specific IgG antibody responses following immunization are blunted in Ser(389)GSK3β knockin mice. Thus, GSK3β emerges as an important modulator of the adaptive immune response. PMID:26822034

  2. 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

  3. 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

  4. Repair of ionizing radiation-induced DNA double-strand breaks by non-homologous end-joining.

    Science.gov (United States)

    Mahaney, Brandi L; Meek, Katheryn; Lees-Miller, Susan P

    2009-02-01

    DNA DSBs (double-strand breaks) are considered the most cytotoxic type of DNA lesion. They can be introduced by external sources such as IR (ionizing radiation), by chemotherapeutic drugs such as topoisomerase poisons and by normal biological processes such as V(D)J recombination. If left unrepaired, DSBs can cause cell death. If misrepaired, DSBs may lead to chromosomal translocations and genomic instability. One of the major pathways for the repair of IR-induced DSBs in mammalian cells is NHEJ (non-homologous end-joining). The main proteins required for NHEJ in mammalian cells are the Ku heterodimer (Ku70/80 heterodimer), DNA-PKcs [the catalytic subunit of DNA-PK (DNA-dependent protein kinase)], Artemis, XRCC4 (X-ray-complementing Chinese hamster gene 4), DNA ligase IV and XLF (XRCC4-like factor; also called Cernunnos). Additional proteins, including DNA polymerases mu and lambda, PNK (polynucleotide kinase) and WRN (Werner's Syndrome helicase), may also play a role. In the present review, we will discuss our current understanding of the mechanism of NHEJ in mammalian cells and discuss the roles of DNA-PKcs and DNA-PK-mediated phosphorylation in NHEJ. PMID:19133841

  5. Nuclease-resistant double-stranded DNA controls or standards for hepatitis B virus nucleic acid amplification assays

    Directory of Open Access Journals (Sweden)

    Zhan Sien

    2009-12-01

    Full Text Available Abstract Background Identical blood samples tested using different kits can give markedly different hepatitis B virus (HBV DNA levels, which can cause difficulty in the interpretation of viral load. A universal double-stranded DNA control or standard that can be used in all commercial HBV DNA nucleic acid amplification assay kits is urgently needed. By aligning all HBV genotypes (A-H, we found that the surface antigen gene and precore-core gene regions of HBV are the most conserved regions among the different HBV genotypes. We constructed a chimeric fragment by overlapping extension polymerase chain reaction and obtained a 1,349-bp HBVC+S fragment. We then packaged the fragment into lambda phages using a traditional lambda phage cloning procedure. Results The obtained armored DNA was resistant to DNase I digestion and was stable, noninfectious to humans, and could be easily extracted using commercial kits. More importantly, the armored DNA may be used with all HBV DNA nucleic acid amplification assay kits. Conclusions The lambda phage packaging system can be used as an excellent expression platform for armored DNA. The obtained armored DNA possessed all characteristics of an excellent positive control or standard. In addition, this armored DNA is likely to be appropriate for all commercial HBV DNA nucleic acid amplification detection kits. Thus, the constructed armored DNA can probably be used as a universal positive control or standard in HBV DNA assays.

  6. Approach to the classical radiation biology. Ionizing radiation effects and repair mechanism of DNA double strand breaks

    International Nuclear Information System (INIS)

    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-/-, RAD54-/-, and KU70-/-/ RAD54-/- 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-/- 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 -/- 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)

  7. A 1D Double Stranded Chain Complex Based on Barium Ion and 1,3,5-Triazine

    Institute of Scientific and Technical Information of China (English)

    CAO Man-Li; ZHANG Xiu-Lian; YIN Wei

    2012-01-01

    A new coordination compound [Ba(OBPT)2(H2O)2]·H2O was obtained at room temperature by the reaction of 4,6-bis(2-pyridyl)-1,3,5-triazin-2-ol(HOBPT) with BaCl2.It was characterized by elemental analysis,FTIR,TG analysis,powder X-ray diffraction analysis and single-crystal X-ray diffraction analysis.The complex crystallizes in the monoclinic P21/n space group,with a = 16.325(1),b = 6.7977(5),c = 24.164(2) ,β = 104.009(1),V = 2601.8(3) 3,Z =4,C26H22BaN10O5,Mr = 691.88,Dc = 1.766 g/cm3,F(000) = 1376 and μ(MoKα) = 1.587 mm-1.The final R = 0.0282 and wR = 0.0724 for 5095 observed reflections with I 〉 2σ(I) and R = 0.0312 and wR = 0.0744 for all data.In the complex,the barium ion is ten-coordinated with six nitrogen atoms from two ligands,two deprotonated hydroxyl oxygen atoms from another two ligands and two coordinated water molecules to form a double stranded chain.The extensive supramolecular interac-tions lead to the formation of an infinite 2D framework.

  8. Tuning G-quadruplex vs double-stranded DNA recognition in regioisomeric lysyl-peptidyl-anthraquinone conjugates.

    Science.gov (United States)

    Zagotto, Giuseppe; Ricci, Antonio; Vasquez, Elena; Sandoli, Andrea; Benedetti, Silvia; Palumbo, Manlio; Sissi, Claudia

    2011-10-19

    Anthraquinone is a versatile scaffold to provide effective DNA binders. This planar system can be easily conjugated to protonable side chains: the nature of the lateral groups and their positions around the tricyclic moiety largely affect the DNA recognition process in terms of binding affinity and mode, as well as sequence and structure of the target nucleic acid. Starting from an anthracenedione system symmetrically functionalized with N-terminal lysyl residues, we incremented the length of side chains by introducing a Gly, Ala, or Phe spacer, characterized by different flexibility, lipophilicity, and bulkiness. Moreover, 2,6, 2,7, 1,8, and 1,5 regioisomers were examined to yield a small bis(lysyl-peptidyl) anthracenedione library. By merging spectroscopic, enzymatic, and cellular results, we showed that the proper combination of a basic aminoacid (Lys) with a more hydrophobic residue (Phe) can provide selective G-quadruplex recognition, in particular when side chains are located at positions 2,6 or 2,7. In fact, while these derivatives effectively bind G-quadruplex structures, they behave at the same time as rather poor double-stranded DNA intercalators. As a result, the Lys-Phe substituted anthraquinones are poorly cytotoxic but still able to promote a senescence mechanism in cancer cells. This combination of chemical and biological properties foresees potentially valuable applications in anticancer medicinal chemistry. PMID:21905741

  9. 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 Emilie; 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...... 390-490 and 787-846) are required to direct the protein to the DNA damage site. Our results reveal that protein domains in hEXO1 in conjunction with specific protein interactions control bi-directional routing of hEXO1 between on-going DNA replication and repair processes in living cells...

  10. The influence of bromodeoxyuridine on the induction and repair of DNA double-strand breaks in glioblastoma cells

    International Nuclear Information System (INIS)

    Aims: To examine the dose response of DNA damage and its modification by the radiosensitizer, 5-bromo-2'-deoxyuridine (BrdU). The sensitizing mechanism is analyzed with regard to its influence on the induction and repair of DNA double-strand breaks (DSBs). Material and Methods: Cells from three different human glioblastoma lines, A7, LH and U87MG, were X-irradiated with and without exposure to BrdU. DNA fragments were separated by field-inversion gel electrophoresis (FIGE) and quantified by fluorometry immediately and 24 h after irradiation. Results: In all cell lines, the dose response followed a linear-quadratic rather than a purely linear function. BrdU-treated cells exhibited a significantly higher amount of mobile DNA. In repair experiments with and without BrdU, the amount of mobile DNA fell close to control values within 24 h. Conclusions: The linear-quadratic model appropriately describes the X-ray induced fragmentation of DNA. BrdU sensitizing acts predominantly by increasing DNA fragility, and not by impairing damage repair. The amount of DSBs persistent after 24 h of repair is minimal, even after highly cytotoxic doses. However, it appears to depend on the extent of initial damage, causing sensitized cells to retain more DSBs than unsensitized cells. (orig.)

  11. The structure of ends determines the pathway choice and Mre11 nuclease dependency of DNA double-strand break repair

    Science.gov (United States)

    Liao, Shuren; Tammaro, Margaret; Yan, Hong

    2016-01-01

    The key event in the choice of repair pathways for DNA double-strand breaks (DSBs) is the initial processing of ends. Non-homologous end joining (NHEJ) involves limited processing, but homology-dependent repair (HDR) requires extensive resection of the 5′ strand. How cells decide if an end is channeled to resection or NHEJ is not well understood. We hypothesize that the structure of ends is a major determinant and tested this hypothesis with model DNA substrates in Xenopus egg extracts. While ends with normal nucleotides are efficiently channeled to NHEJ, ends with damaged nucleotides or bulky adducts are channeled to resection. Resection is dependent on Mre11, but its nuclease activity is critical only for ends with 5′ bulky adducts. CtIP is absolutely required for activating the nuclease-dependent mechanism of Mre11 but not the nuclease-independent mechanism. Together, these findings suggest that the structure of ends is a major determinant for the pathway choice of DSB repair and the Mre11 nuclease dependency of resection. PMID:27084932

  12. 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.

  13. Gastric cancer associated variant of DNA polymerase beta (Leu22Pro) promotes DNA replication associated double strand breaks.

    Science.gov (United States)

    Rozacky, Jenna; Nemec, Antoni A; Sweasy, Joann B; Kidane, Dawit

    2015-09-15

    DNA polymerase beta (Pol β) is a key enzyme for the protection against oxidative DNA lesions via its role in base excision repair (BER). Approximately 1/3 of tumors studied to date express Pol β variant proteins, and several tumors overexpress Pol β. Pol β possesses DNA polymerase and dRP lyase activities, both of which are known to be important for efficient BER. The dRP lyase activity resides within the 8kDa amino terminal domain of Pol β, is responsible for removal of the 5' phosphate group (5'-dRP). The DNA polymerase subsequently fills the gaps. Previously, we demonstrated that the human gastric cancer-associated variant of Pol β (Leu22Pro (L22P)) lacks dRP lyase function in vitro. Here, we report that L22P-expressing cells harbor significantly increased replication associated DNA double strand breaks (DSBs) and defective maintenance of the nascent DNA strand (NDS) during replication stress. Moreover, L22P-expressing cells are sensitive to PARP1 inhibitors, which suggests trapped PARP1 binds to the 5'-dRP group and blocks replications forks, resulting in fork collapse and DSBs. Our data suggest that the normal function of the dRP lyase is critical to maintain replication fork integrity and prevent replication fork collapse to DSBs and cellular transformation. PMID:26090616

  14. The foci of DNA double strand break-recognition proteins localize with γH2AX after heat treatment

    International Nuclear Information System (INIS)

    Recently, there have been many reports concerning proteins which can recognize DNA double strand break (DSBs), and such proteins include histone H2AX phosphorylated at serine 139 (γH2AX), ataxia telangiectasia mutated (ATM) phospho-serine 1981, DNA-dependent protein kinase catalytic subunit (DNA-PKcs) phospho-threonine 2609, Nijmegen breakage syndrome 1 (NBS1) phospho-serine 343, checkpoint kinase 2 (CHK2), phospho-threonine 68, and structural maintenance of chromosomes 1 (SMC1) phospho-serine 966. Thus, it should be possible to follow the formation of DSBs and their repair using immunohistochemical methods with multiple antibodies to detect these proteins. When normal human fibroblasts (AG1522 cells) were exposed to 3 Gy of X-rays as a control, clearly discernable foci for these proteins were detected, and these foci localized with γH2AX foci. After heat treatment at 45.5 deg C for 20 min, these proteins are partially localized with γH2AX foci. Here we show that there were slight differences in the localization pattern among these proteins, such as a disappearance from the nucleus (phospho-ATM) and translocation to the cytoplasm (phospho-NBS1) at 30 min after heat treatment, and some foci (phospho-DNA-PKcs and phospho-CHK2) appeared at 8 h after heat treatment. These results are discussed from perspectives of heat-induced denaturation of proteins and formation of DSBs. (author)

  15. Nucleolin participates in DNA double-strand break-induced damage response through MDC1-dependent pathway.

    Directory of Open Access Journals (Sweden)

    Junya Kobayashi

    Full Text Available H2AX is an important factor for chromatin remodeling to facilitate accumulation of DNA damage-related proteins at DNA double-strand break (DSB sites. In order to further understand the role of H2AX in the DNA damage response (DDR, we attempted to identify H2AX-interacting proteins by proteomics analysis. As a result, we identified nucleolin as one of candidates. Here, we show a novel role of a major nucleolar protein, nucleolin, in DDR. Nucleolin interacted with γ-H2AX and accumulated to laser micro-irradiated DSB damage sites. Chromatin Immunoprecipitation assay also displayed the accumulation of nucleolin around DSB sites. Nucleolin-depleted cells exhibited repression of both ATM-dependent phosphorylation following exposure to γ-ray and subsequent cell cycle checkpoint activation. Furthermore, nucleolin-knockdown reduced HR and NHEJ activity and showed decrease in IR-induced chromatin accumulation of HR/NHEJ factors, agreeing with the delayed kinetics of γ-H2AX focus. Moreover, nucleolin-knockdown decreased MDC1-related events such as focus formation of 53 BP1, RNF168, phosphorylated ATM, and H2A ubiquitination. Nucleolin also showed FACT-like activity for DSB damage-induced histone eviction from chromatin. Taken together, nucleolin could promote both ATM-dependent cell cycle checkpoint and DSB repair by functioning in an MDC1-related pathway through its FACT-like function.

  16. Depletion of tyrosyl DNA phosphodiesterase 2 activity enhances etoposide-mediated double-strand break formation and cell killing.

    Science.gov (United States)

    Kont, Yasemin Saygideger; Dutta, Arijit; Mallisetty, Apurva; Mathew, Jeena; Minas, Tsion; Kraus, Christina; Dhopeshwarkar, Priyanka; Kallakury, Bhaskar; Mitra, Sankar; Üren, Aykut; Adhikari, Sanjay

    2016-07-01

    DNA topoisomerase 2 (Top2) poisons, including common anticancer drugs etoposide and doxorubicin kill cancer cells by stabilizing covalent Top2-tyrosyl-DNA 5'-phosphodiester adducts and DNA double-strand breaks (DSBs). Proteolytic degradation of the covalently attached Top2 leaves a 5'-tyrosylated blocked termini which is removed by tyrosyl DNA phosphodiesterase 2 (TDP2), prior to DSB repair through non-homologous end joining (NHEJ). Thus, TDP2 confers resistance of tumor cells to Top2-poisons by repairing such covalent DNA-protein adducts, and its pharmacological inhibition could enhance the efficacy of Top2-poisons. We discovered NSC111041, a selective inhibitor of TDP2, by optimizing a high throughput screening (HTS) assay for TDP2's 5'-tyrosyl phosphodiesterase activity and subsequent validation studies. We found that NSC111041 inhibits TDP2's binding to DNA without getting intercalated into DNA and enhanced etoposide's cytotoxicity synergistically in TDP2-expressing cells but not in TDP2 depleted cells. Furthermore, NSC111041 enhanced formation of etoposide-induced γ-H2AX foci presumably by affecting DSB repair. Immuno-histochemical analysis showed higher TDP2 expression in a sub-set of different type of tumor tissues. These findings underscore the feasibility of clinical use of suitable TDP2 inhibitors in adjuvant therapy with Top2-poisons for a sub-set of cancer patients with high TDP2 expression. PMID:27235629

  17. Certain patterns of DNA double strand breaks in membrane-attached superstructure units cause cell killing: a radiation action model

    International Nuclear Information System (INIS)

    The discovery that the chromosomal DNA is arranged in membrane-attached superstructure units (MASSUs) is the key for the understanding of the action of ionizing radiations on mammalian cells. Concerning X-rays the following hypothesis is proved true: The appearance of K ≥ 2 double-strand breaks (DSBs) in any MASSU of a G1 cell, respectively, in both MASSUs of any sister MASSU pair of a S cell results in its inactivation (k = actual number of DSBs per MASSU). DSB patterns in the MASSUs characterized by less DSBs will be repaired by recombination with homologous MASSUs. In G1 cells it occurs by the recombination with the homologous MASSU of the homologous chromosome. In the replicated MASSUs of S cells it probably happens by the succession of the following mechanisms: recombination repair of MASSUs with one DSB using the sister MASSU as a matrix (sister chromatid exchange), establishment of 1 intact genome by the substitution of the heavily damaged MASSUs (k ≥ 2) by the intact or repaired sister MASSU at the common attachment point, and degradation of the heavily damaged or abundant MASSUs. Thus the dependence of the form and the steepness of the dose survival curves on the cell cycle stages is interpreted by a universally valid radiation action mechanism. (author)

  18. Radiation-induced DNA double-strand break frequencies in human squamous cell carcinoma cell lines of different radiation sensitivities

    International Nuclear Information System (INIS)

    DNA neutral (pH 9-6) filter elution was used to measure radiation-induced DNA double-strand break (dsb) frequencies in eight human squamous cell carcinoma cell lines with radiosensitivities (D0) ranging from 1.07 to 2.66 Gy and D-bar values ranging from 1.46 to 4.08 Gy. Elution profiles of unirradiated samples from more radiosensitive cell lines were all steeper in slope than profiles from resistant cells. The shapes of the dsb induction curves were curvilinear and there was some variability from cell line to cell line in the dose-response for the induction of DNA dsb after exposures to 5-100 Gy 60Co γ-rays. There was no relation between shapes of survival curves and shapes of the dose-responses for the induction of DNA dsb. At low doses (5-25 Gy), three out of four of the more sensitive cell lines (D-bar3.0 Gy). Although the low-dose (5-25 Gy) elution results were variable, they suggest that DNA neutral elution will detect differences between sensitive and resistant tumour cells in initial DNA dsb frequencies. (author)

  19. Action spectra for single- and double-strand break induction in plasmid DNA: studies using synchrotron radiation

    International Nuclear Information System (INIS)

    Ionizing radiations deposit a wide range of energies in and around DNA and this leads to a corresponding spectrum of complexity of the lesions induced. The relationships between the amount of energy deposited and the yields and types of damage induced are important in modelling the physical and chemical stages of radiation effect and linking them to biological outcome. To study these relationships experimentally, plasmids were mounted as a monolayer and exposed in vacuum to near-monoenergetic photons from the Daresbury Synchrotron. After irradiation, the DNA was washed off and assayed for single-(ssb) and double-strand breaks (dsb) using agarose gel electrophoresis. Dose-effect relationships for ssb and dsb induction were obtained at various energies in the range 8-25 eV. The initial responses in the low-dose region allowed damage yields to be estimated. The data appear to show that the yields of ssb and dsb increase only slowly with photon energies > 10 eV, with a suggestion of similar threshold energies for both lesions. In the energy range covered, the yield of ssb is 12-20-fold greater than that of dsb. The data indicate that ssb and dsb may have a common precursor in this system. Earlier work with low-energy electrons showed that at 25 eV ssb were induced but no dsb were detected. (author)

  20. 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.

  1. Processing by MRE11 is involved in the sensitivity of subtelomeric regions to DNA double-strand breaks.

    Science.gov (United States)

    Muraki, Keiko; Han, Limei; Miller, Douglas; Murnane, John P

    2015-09-18

    The caps on the ends of chromosomes, called telomeres, keep the ends of chromosomes from appearing as DNA double-strand breaks (DSBs) and prevent chromosome fusion. However, subtelomeric regions are sensitive to DSBs, which in normal cells is responsible for ionizing radiation-induced cell senescence and protection against oncogene-induced replication stress, but promotes chromosome instability in cancer cells that lack cell cycle checkpoints. We have previously reported that I-SceI endonuclease-induced DSBs near telomeres in a human cancer cell line are much more likely to generate large deletions and gross chromosome rearrangements (GCRs) than interstitial DSBs, but found no difference in the frequency of I-SceI-induced small deletions at interstitial and subtelomeric DSBs. We now show that inhibition of MRE11 3'-5' exonuclease activity with Mirin reduces the frequency of large deletions and GCRs at both interstitial and subtelomeric DSBs, but has little effect on the frequency of small deletions. We conclude that large deletions and GCRs are due to excessive processing of DSBs, while most small deletions occur during classical nonhomologous end joining (C-NHEJ). The sensitivity of subtelomeric regions to DSBs is therefore because they are prone to undergo excessive processing, and not because of a deficiency in C-NHEJ in subtelomeric regions. PMID:26209132

  2. XLS (c9orf142) is a new component of mammalian DNA double-stranded break repair.

    Science.gov (United States)

    Craxton, A; Somers, J; Munnur, D; Jukes-Jones, R; Cain, K; Malewicz, M

    2015-06-01

    Repair of double-stranded DNA breaks (DSBs) in mammalian cells primarily occurs by the non-homologous end-joining (NHEJ) pathway, which requires seven core proteins (Ku70/Ku86, DNA-PKcs (DNA-dependent protein kinase catalytic subunit), Artemis, XRCC4-like factor (XLF), XRCC4 and DNA ligase IV). Here we show using combined affinity purification and mass spectrometry that DNA-PKcs co-purifies with all known core NHEJ factors. Furthermore, we have identified a novel evolutionary conserved protein associated with DNA-PKcs-c9orf142. Computer-based modelling of c9orf142 predicted a structure very similar to XRCC4, hence we have named c9orf142-XLS (XRCC4-like small protein). Depletion of c9orf142/XLS in cells impaired DSB repair consistent with a defect in NHEJ. Furthermore, c9orf142/XLS interacted with other core NHEJ factors. These results demonstrate the existence of a new component of the NHEJ DNA repair pathway in mammalian cells. PMID:25941166

  3. DNA Ligase IV and Artemis Act Cooperatively to Suppress Homologous Recombination in Human Cells: Implications for DNA Double-Strand Break Repair

    OpenAIRE

    Aya Kurosawa; Shinta Saito; Sairei So; Mitsumasa Hashimoto; Kuniyoshi Iwabuchi; Haruka Watabe; Noritaka Adachi

    2013-01-01

    Nonhomologous end-joining (NHEJ) and homologous recombination (HR) are two major pathways for repairing DNA double-strand breaks (DSBs); however, their respective roles in human somatic cells remain to be elucidated. Here we show using a series of human gene-knockout cell lines that NHEJ repairs nearly all of the topoisomerase II- and low-dose radiation-induced DNA damage, while it negatively affects survival of cells harbouring replication-associated DSBs. Intriguingly, we find that loss of ...

  4. The Vitamin A Derivative All-Trans Retinoic Acid Repairs Amyloid-β-Induced Double-Strand Breaks in Neural Cells and in the Murine Neocortex

    OpenAIRE

    Emmanuelle Gruz-Gibelli; Natacha Chessel; Clélia Allioux; Pascale Marin; Françoise Piotton; Geneviève Leuba; Herrmann, François R.; Armand Savioz

    2016-01-01

    The amyloid-β peptide or Aβ is the key player in the amyloid-cascade hypothesis of Alzheimer's disease. Aβ appears to trigger cell death but also production of double-strand breaks (DSBs) in aging and Alzheimer's disease. All-trans retinoic acid (RA), a derivative of vitamin A, was already known for its neuroprotective effects against the amyloid cascade. It diminishes, for instance, the production of Aβ peptides and their oligomerisation. In the present work we investigat...

  5. RSC Facilitates Rad59-Dependent Homologous Recombination between Sister Chromatids by Promoting Cohesin Loading at DNA Double-Strand Breaks ▿

    OpenAIRE

    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

    2011-01-01

    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 cont...

  6. Double-stranded DNA breaks hidden in the neutral Comet assay suggest a role of the sperm nuclear matrix in DNA integrity maintenance

    OpenAIRE

    Ribas-Maynou, J.; Gawecka, J.E.; Benet, J.; Ward, W.S.

    2013-01-01

    We used a mouse model in which sperm DNA damage was induced to understand the relationship of double-stranded DNA (dsDNA) breaks to sperm chromatin structure and to the Comet assay. Sperm chromatin fragmentation (SCF) produces dsDNA breaks located on the matrix attachment regions, between protamine toroids. In this model, epididymal sperm induced to undergo SCF can religate dsDNA breaks while vas deferens sperm cannot. Here, we demonstrated that the conventional neutral Comet assay underestim...

  7. Strand-specific contacts and divalent metal ion regulate double-strand break formation by the GIY-YIG homing endonuclease I-BmoI.

    Science.gov (United States)

    Carter, Jonathan M; Friedrich, Nancy C; Kleinstiver, Benjamin; Edgell, David R

    2007-11-23

    GIY-YIG homing endonucleases are modular enzymes consisting of a well-defined N-terminal catalytic domain connected to a variable C-terminal DNA-binding domain. Previous studies have revealed that the role of the DNA-binding domain is to recognize and bind intronless DNA substrate, positioning the N-terminal catalytic domain such that it is poised to generate a staggered double-strand break by an unknown mechanism. Interactions of the N-terminal catalytic domain with intronless substrate are therefore a critical step in the reaction pathway but have been difficult to define. Here, we have taken advantage of the reduced activity of I-BmoI, an isoschizomer of the well-studied bacteriophage T4 homing endonuclease I-TevI, to examine double-strand break formation by I-BmoI. We present evidence demonstrating that I-BmoI generates a double-strand break by two sequential but chemically independent nicking reactions where divalent metal ion is a limiting factor in top-strand nicking. We also show by in-gel footprinting that contacts by the I-BmoI catalytic domain induce significant minor groove DNA distortions that occur independently of bottom-strand nicking. Bottom-strand contacts are critical for accurate top-strand nicking, whereas top-strand contacts have little influence on the accuracy of bottom-strand nicking. We discuss our results in the context of current models of GIY-YIG endonuclease function, with emphasis on the role of divalent metal ion and strand-specific contacts in regulating the activity of a single active site to generate a staggered double-strand break. PMID:17936302

  8. Aberrant Double-Strand Break Repair Resulting in Half Crossovers in Mutants Defective for Rad51 or the DNA Polymerase δ Complex▿

    OpenAIRE

    Smith, Catherine E.; Lam, Alicia F.; Symington, Lorraine S

    2009-01-01

    Homologous recombination is an error-free mechanism for the repair of DNA double-strand breaks (DSBs). Most DSB repair events occur by gene conversion limiting loss of heterozygosity (LOH) for markers downstream of the site of repair and restricting deleterious chromosome rearrangements. DSBs with only one end available for repair undergo strand invasion into a homologous duplex DNA, followed by replication to the chromosome end (break-induced replication [BIR]), leading to LOH for all marker...

  9. Effects of camptothecin on double-strand break repair by non-homologous end-joining in DNA mismatch repair-deficient human colorectal cancer cell lines

    OpenAIRE

    Jacob, Sandrine; Miquel, Catherine; Sarasin, Alain; Praz, Françoise

    2005-01-01

    Loss of a functional mismatch repair (MMR) system in colorectal cancer (CRC) cells is associated with microsatellite instability and increased sensitivity to topoisomerase inhibitors. In this study, we have investigated whether a defect in double-strand break (DSB) repair by non-homologous end-joining (NHEJ) could explain why MMR-deficient CRC cells are hypersensitive to camptothecin (CPT), a topoisomerase I inhibitor. To evaluate the efficiency and the fidelity of DSB repair, we have transie...

  10. Efficient Rejoining of DNA Double-Strand Breaks despite Increased Cell-Killing Effectiveness following Spread-Out Bragg Peak Carbon-Ion Irradiation

    OpenAIRE

    Averbeck, Nicole B.; Topsch, Jana; Scholz, Michael; Kraft-Weyrather, Wilma; Durante, Marco; Taucher-Scholz, Gisela

    2016-01-01

    Radiotherapy of solid tumors with charged particles holds several advantages in comparison to photon therapy; among them conformal dose distribution in the tumor, improved sparing of tumor-surrounding healthy tissue, and an increased relative biological effectiveness (RBE) in the tumor target volume in the case of ions heavier than protons. A crucial factor of the biological effects is DNA damage, of which DNA double-strand breaks (DSBs) are the most deleterious. The reparability of these les...

  11. Synthesis of 5-[3-(2-aminopyrimidin-4-yl)aminopropyn-1-yl]uracil derivative that recognizes Ade-Thy base pairs in double-stranded DNA.

    Science.gov (United States)

    Ito, Yu; Masaki, Yoshiaki; Kanamori, Takashi; Ohkubo, Akihiro; Seio, Kohji; Sekine, Mitsuo

    2016-01-01

    5-[3-(2-Aminopyrimidin-4-yl)aminopropyn-1-yl]uracil (Ura(Pyr)) was designed as a new nucleobase to recognize Ade-Thy base pair in double-stranded DNA. We successfully synthesized the dexoynucleoside phosphoramidite having Ura(Pyr) and incorporated it into triplex forming oligonucleotides (TFOs). Melting temperature analysis revealed that introduction of Ura(Pyr) into TFOs could effectively stabilize their triplex structures without loss of base recognition capabilities. PMID:26602276

  12. Bloom’s syndrome helicase and Mus81 are required to induce transient double-strand DNA breaks in response to DNA replication stress

    OpenAIRE

    Shimura, Tsutomu; Torres, Michael J.; Melvenia M Martin; Rao, V. Ashutosh; Pommier, Yves; Katsura, Mari; Miyagawa, Kiyoshi; Aladjem, Mirit I

    2007-01-01

    Perturbed DNA replication either activates a cell cycle checkpoint, which halts DNA replication, or decreases the rate of DNA synthesis without activating a checkpoint. Here we report that at low doses, replication inhibitors did not activate a cell cycle checkpoint, but they did activate a process that required functional Bloom’s syndrome-associated (BLM) helicase, Mus81 nuclease and ATR kinase to induce transient double stranded DNA breaks. The induction of transient DNA breaks was accompan...

  13. Life forms employ different repair strategies of repair single- and double strand DNA breaks caused by different qualities of radiation: criticality of RecA mediated repair system

    International Nuclear Information System (INIS)

    Different qualities of radiation, either through direct or indirect pathway, induce qualitative different spectrum of damages in DNA, which are also different in in vitro and in vivo systems. The single- and double strand breaks of DNA are of special interest as they lead to serious biological consequences. The implications of such damage to DNA and their processing by various inherent repair pathways together decide the fate of the living form

  14. Ionizing radiation damage to the folded chromosome of Escherichia coli K-12: repair of double-strand breaks in deoxyribonucleic acid

    International Nuclear Information System (INIS)

    The extremely gentle lysis and unfolding procedures that have been developed for the isolation of nucleoid deoxyribonucleic acid yield undamaged, replicating genomes, thus permitting direct measurement of the formation and repair of DNA double-strand breaks at biologically significant doses of ionizing radiation. Repair of ionizing radiation damage to folded chromosomes of Escherichia coli K-12 strain AB2497 was observed within 2 to 3 h of post-irradiation incubation in growth medium. Such behavior was not observed after post-irradiation incubation in growth medium of a recA13 strain (strain AB2487). A model based on recombinational repair is proposed to explain the formation of 2,200 to 2,300S material during early stages of incubation and to explain subsequent changes in the gradient profiles. Association of unrepaired DNA with the plasma membrane is proposed to explain the formation of a peak of rapidly sedimenting material (greater than 3,100S) during the later stage of repair. Direct evidence of repair of double-strand breaks during post-irradiation incubation in growth medium was obtained from gradient profiles of DNA from ribonuclease-digested chromosomes. The sedimentation coefficient of broken molecules was restored to the value of unirradiated DNA after 2 to 3 h of incubation, and the fraction of the DNA repaired in this fashion was equal to the fraction of cells that survived at the same dose. An average of 2.7 double-strand breaks per genome per lethal event was observed, suggesting that one to two double-strand breaks per genome are repairable in E. coli K-12 strain AB2497

  15. In vivo expression of a single viral DNA-binding protein generates systemic lupus erythematosus-related autoimmunity to double-stranded DNA and histones.

    OpenAIRE

    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 ...

  16. Structural stability versus conformational sampling in biomolecular systems: Why is the charge transfer efficiency in G4-DNA better than in double-stranded DNA?

    OpenAIRE

    Woiczikowski, P. Benjamin; Kubař, Tomáš; Gutiérrez, Rafael; Cuniberti, Gianaurelio; Elstner, Marcus

    2010-01-01

    The electrical conduction properties of G4-DNA are investigated using a hybrid approach, which combines electronic structure calculations, molecular dynamics (MD) simulations, and the formulation of an effective tight-binding model Hamiltonian. Charge transport is studied by computing transmission functions along the MD trajectories. Though G4-DNA is structurally more stable than double-stranded DNA (dsDNA), our results strongly suggest that the potential improvement of the electrical transpo...

  17. Production of DNA Double Strand Breaks in Human Cells due to Acute Exposure to Tritiated Water (HTO)

    International Nuclear Information System (INIS)

    The average and maximum energies of the beta emission from 3H are 5.69 keV and 18.6 keV respectively. The average range in water (or soft tissues), around 0.5 1/4m (500 nm), is considerably less than the typical diameter of a cell (10-30 1/4m), and even of a cell nucleus (5-10 1/4m), thus the micro-location of the tritium atom may well be crucial in determining its biochemical consequences. Due to the high ionization density of the beta particles emitted by tritium (about 400 ion pairs/1/4m) possible interaction of tritium beta radiation with DNA may play a significant role. Tritiated water (HTO) is the main chemical form in which tritium is found in the environment. In the body it may be retained as organically bound tritium (OBT), binding to biological molecules or remaining as OBT with various degrees of solubility. OBT can be retained in the human body much longer than HTO and therefore the dose arising from OBT can reach 50% of the total tritium dose . Histones are major protein components of chromatin. They function as spools around which DNA winds and play an important role in the regulation of gene expression. In the absence of histones, the DNA in chromosomes would be unmanageably long, as human cells each have about 1.8 m of DNA. During mitosis, DNA is duplicated and condensed, resulting in about 120 1/4m of chromosomes. It was recently reported that the phosphorylation of histone H2AX on serine residue 139 (D3-H2AX) is associated with Double Strand Breaks (DSB) sites in DNA), which indicates the possibility of research based on the detection of DSBs in DNA. The phosphorylated megabase chromatin domain surrounding the DSB can be immunostained and visualized as discrete foci by fluorescence microscopy, as each DNA DSB formed produces a visible D3-H2AX focus. Since 1 Gy of radiation produces approximately 60 DSBs/cell, doses of a few mGy should be distinguishable from the background, and it was recently shown that the exposure to 1 mGy of X-rays induces a

  18. Double-strand break formation by the RAG complex at the bcl-2 major breakpoint region and at other non-B DNA structures in vitro.

    Science.gov (United States)

    Raghavan, Sathees C; Swanson, Patrick C; Ma, Yunmei; Lieber, Michael R

    2005-07-01

    The most common chromosomal translocation in cancer, t(14;18) at the 150-bp bcl-2 major breakpoint region (Mbr), occurs in follicular lymphomas. The bcl-2 Mbr assumes a non-B DNA conformation, thus explaining its distinctive fragility. This non-B DNA structure is a target of the RAG complex in vivo, but not because of its primary sequence. Here we report that the RAG complex generates at least two independent nicks that lead to double-strand breaks in vitro, and this requires the non-B DNA structure at the bcl-2 Mbr. A 3-bp mutation is capable of abolishing the non-B structure formation and the double-strand breaks. The observations on the bcl-2 Mbr reflect more general properties of the RAG complex, which can bind and nick at duplex-single-strand transitions of other non-B DNA structures, resulting in double-strand breaks in vitro. Hence, the present study reveals novel insight into a third mechanism of action of RAGs on DNA, besides the standard heptamer/nonamer-mediated cleavage in V(D)J recombination and the in vitro transposase activity. PMID:15988007

  19. DNA double strand breaks in fibroblast cell lines from non-Hodgkin's lymphoma patients showing increased sensitivity to chronic gamma irradiation

    International Nuclear Information System (INIS)

    Cultured skin fibroblast cell lines from two non-Hodgkin's lymphoma patients (NHL) and a normal subject were studied for cell killing, chromosomal aberrations (breaks, translocations, dicentrics and rings) and DNA double strand breaks (dsbs) following chronic gamma irradiation. Compared to the cell line from the normal donor, the NHL patients' fibroblasts showed enhanced radiosensitivity for both cell survival and chromosomal aberrations. While spontaneous breaks were observed in both normal and patients' cells, spontaneous translocations and radiation-induced dicentrics and rings were found only in the latter. Radiation-induced DNA double-strand breaks (dsb) were determined by CHEF electrophoresis. After chronic irradiation with gamma rays the fraction of residual dsb was significantly increased from 1.4% in controls to 1.9% in the NHL cell lines. These data, thus suggest that the cellular and chromosomal sensitivity to chronic irradiation observed in NHL patients may be due to a deficiency in the repair of a small fraction of DNA double strand breaks. (author)

  20. Concerted control of DNA double strand break repair and cell cycle progression in X-irradiated mammalian cells

    International Nuclear Information System (INIS)

    Upon examination of cell cycle regulation in a damaged cell, relations were discovered of the cell cycle control mechanisms with a complicated web of signalling pathways, eventually called the genome surveillance system. After infliction of DNA double strand breaks (DSB), the signalling is initiated by sensor proteins and transducer protein kinase ATM. This kinase phosphorylates downstream effector proteins, such as checkpoint kinases CHK1 and CHK2, which initiate the pathways leading to cell cycle arrest. In contrast with the older model of linear transmission of signals in a certain sequence, it is now accepted that the damage signalling system is branched and contains feedback loops. DSB's presence is signalled by sensor proteins (MRE11-RAD50-nibrin complex, MRN) to ATM and the signal is amplified through adaptor proteins, MDC1/NFBD1 or 53BP1 (Tp53 binding protein). MRN contains a forkheadassociated (FHA) domain and BRCA1 carboxyl-terminal (BRCT) domain. The combination of the FHA/BRCT domains has a crucial role for the binding of nibrin to the H2AX histone, assembling the components of repair foci. These domains also are important for interaction of other proteins localised in the foci. For example, MDC1/NFBD1 contains a FHA domain and two BRCT domains which are involved in protein interactions. The signal generated at DSBs is amplified and transduced to recruit components of DNA repair systems. In a concerted way with the sequential recruitment of components of repair foci, activation of transcription of genes takes place, that is necessary for blocking progression through the cell cycle, for DNA repair or apoptosis. (author)

  1. Investigation of double strand breaks induced by alpha particle irradiation using C.N.B.G. microbeam in human keratinocytes

    International Nuclear Information System (INIS)

    To understand the mechanisms of interaction of ionizing radiation with living tissues exposed to low and protracted doses remains a major issue for risk evaluation. The response cannot be found in epidemiological studies because the only available data concern accidental exposures to high doses of radiation. The natural exposure represents the main source of exposure in the daily life, just before the medical sources (radiology, radiotherapy). In addition, this kind of exposure is very difficult to reproduce in vitro by irradiating cell lines. The method per preference is based on random irradiation of cell populations. The mean number of particles having traversed cells is then calculated on the basis of Poisson statistics. In addition to inevitable multiple impacts, the numerous potential intracellular targets (nuclei, cytoplasm), the indirect effects induced by the impact of particles on neighbouring cells or simply the extracellular targets, constitute phenomena that make more complex the interpretation of experimental data. A charged particle microbeam was developed at C.E.N.B.G. to perform the targeted irradiation of individual cells with a targeting precision of a few microns. It is possible to deliver a counted number of alpha particles down to the ultimate dose of one alpha per cell, to target predetermined cells and then to observe the response of the neighbouring cells. This facility has been validated during this work on human keratinocyte cells expressing a recombinant nuclear fluorescent protein (histone H2B-GFP). The combination of ion micro-beams with confocal microscopy and numeric quantitative analysis allowed the measurement of DNA double strand breaks via the phosphorylation of the histone H2A.X in individual cells. The mechanisms of DNA reparation and apoptosis induction were also in the scope of those studies. The experimental results obtained during this thesis validate the methodology we have developed by demonstrating the targeting

  2. 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.

  3. 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.

  4. 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)

  5. Influence of Double-Strand Break Repair on Radiation Therapy-Induced Acute Skin Reactions in Breast Cancer Patients

    International Nuclear Information System (INIS)

    Purpose: Curative radiation therapy (RT)-induced toxicity poses strong limitations for efficient RT and worsens the quality of life. The parameter that explains when and to what extent normal tissue toxicity in RT evolves would be of clinical relevance because of its predictive value and may provide an opportunity for personalized treatment approach. Methods and Materials: DNA double-strand breaks and repair were analyzed by microscopic γ-H2AX foci analysis in peripheral lymphocytes from 38 healthy donors and 80 breast cancer patients before RT, a 2 Gy challenge dose of x-ray exposed in vitro. Results: The actual damage (AD) at 0.25, 3, and 6 hours and percentage residual damage (PRD) at 3 and 6 hours were used as parameters to measure cellular radiosensitivity and correlated with RT-induced acute skin reactions in patients stratified as non-overresponders (NOR) (Radiation Therapy Oncology Group [RTOG] grade <2) and overresponders (OR) (RTOG grade ≥2). The results indicated that the basal and induced (at 0.25 and 3 hours) γ-H2AX foci numbers were nonsignificant (P>.05) between healthy control donors and the NOR and OR groups, whereas it was significant between ORs and healthy donors at 6 hours (P<.001). There was a significantly higher PRD in OR versus NOR (P<.05), OR versus healthy donors (P<.001) and NOR versus healthy donors (P<.01), supported further by the trend analysis (r=.2392; P=.0326 at 6 hours). Conclusions: Our findings strongly suggest that the measurement of PRD by performing γ-H2AX foci analysis has the potential to be developed into a clinically useful predictive assay

  6. 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.

  7. DNA double strand breaks but not interstrand crosslinks prevent progress through meiosis in fully grown mouse oocytes.

    Directory of Open Access Journals (Sweden)

    Wai Shan Yuen

    Full Text Available There is some interest in how mammalian oocytes respond to different types of DNA damage because of the increasing expectation of fertility preservation in women undergoing chemotherapy. Double strand breaks (DSBs induced by ionizing radiation and agents such as neocarzinostatin (NCS, and interstrand crosslinks (ICLs induced by alkylating agents such as mitomycin C (MMC, are toxic DNA lesions that need to be repaired for cell survival. Here we examined the effects of NCS and MMC treatment on oocytes collected from antral follicles in mice, because potentially such oocytes are readily collected from ovaries and do not need to be in vitro grown to achieve meiotic competency. We found that oocytes were sensitive to NCS, such that this ionizing radiation mimetic blocked meiosis I and caused fragmented DNA. In contrast, MMC had no impact on the completion of either meiosis I or II, even at extremely high doses. However, oocytes treated with MMC did show γ-H2AX foci and following their in vitro maturation and parthenogenetic activation the development of the subsequent embryos was severely compromised. Addition of MMC to 1-cell embryos caused a similarly poor level of development, demonstrating oocytes have eventual sensitivity to this ICL-inducing agent but this does not occur during their meiotic division. In oocytes, the association of Fanconi Anemia protein, FANCD2, with sites of ICL lesions was not apparent until entry into the embryonic cell cycle. In conclusion, meiotic maturation of oocytes is sensitive to DSBs but not ICLs. The ability of oocytes to tolerate severe ICL damage and yet complete meiosis, means that this type of DNA lesion goes unrepaired in oocytes but impacts on subsequent embryo quality.

  8. 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.

  9. 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

  10. Effects of chemopreventive natural products on non-homologous end-joining DNA double-strand break repair.

    Science.gov (United States)

    Charles, Catherine; Nachtergael, Amandine; Ouedraogo, Moustapha; Belayew, Alexandra; Duez, Pierre

    2014-07-01

    Double-strand breaks (DSBs) may result from endogenous (e.g., reactive oxygen species, variable (diversity) joining, meiotic exchanges, collapsed replication forks, nucleases) or exogenous (e.g., ionizing radiation, chemotherapeutic agents, radiomimetic compounds) events. DSBs disrupt the integrity of DNA and failed or improper DSBs repair may lead to genomic instability and, eventually, mutations, cancer, or cell death. Non-homologous end-joining (NHEJ) is the major pathway used by higher eukaryotic cells to repair these lesions. Given the complexity of NHEJ and the number of proteins and cofactors involved, secondary metabolites from medicinal or food plants might interfere with the process, activating or inhibiting repair. Twelve natural products, arbutin, curcumin, indole-3-carbinol, and nine flavonoids (apigenin, baicalein, chalcone, epicatechin, genistein, myricetin, naringenin, quercetin, sakuranetin) were chosen for their postulated roles in cancer chemoprevention and/or treatment. The effects of these compounds on NHEJ were investigated with an in vitro protocol based on plasmid substrates. Plasmids were linearized by a restriction enzyme, generating cohesive ends, or by a combination of enzymes, generating incompatible ends; plasmids were then incubated with a nuclear extract prepared from normal human small-intestinal cells (FHS 74 Int), either treated with these natural products or untreated (controls). The NHEJ repair complex from nuclear extracts ligates linearized plasmids, resulting in plasmid oligomers that can be separated and quantified by on-chip microelectrophoresis. Some compounds (chalcone, epicatechin, myricetin, sakuranetin and arbutin) clearly activated NHEJ, whereas others (apigenin, baicalein and curcumin) significantly reduced the repair rate of both types of plasmid substrates. Although this in vitro protocol is only partly representative of the in vivo situation, the natural products appear to interfere with NHEJ repair and warrant

  11. Nonhomologous end joining of complex DNA double-strand breaks with proximal thymine glycol and interplay with base excision repair.

    Science.gov (United States)

    Almohaini, Mohammed; Chalasani, Sri Lakshmi; Bafail, Duaa; Akopiants, Konstantin; Zhou, Tong; Yannone, Steven M; Ramsden, Dale A; Hartman, Matthew C T; Povirk, Lawrence F

    2016-05-01

    DNA double-strand breaks induced by ionizing radiation are often accompanied by ancillary oxidative base damage that may prevent or delay their repair. In order to better define the features that make some DSBs repair-resistant, XLF-dependent nonhomologous end joining of blunt-ended DSB substrates having the oxidatively modified nonplanar base thymine glycol at the first (Tg1), second (Tg2), third (Tg3) or fifth (Tg5) positions from one 3' terminus, was examined in human whole-cell extracts. Tg at the third position had little effect on end-joining even when present on both ends of the break. However, Tg as the terminal or penultimate base was a major barrier to end joining (>10-fold reduction in ligated products) and an absolute barrier when present at both ends. Dideoxy trapping of base excision repair intermediates indicated that Tg was excised from Tg1, Tg2 and Tg3 largely if not exclusively after DSB ligation. However, Tg was rapidly excised from the Tg5 substrate, resulting in a reduced level of DSB ligation, as well as slow concomitant resection of the opposite strand. Ligase reactions containing only purified Ku, XRCC4, ligase IV and XLF showed that ligation of Tg3 and Tg5 was efficient and only partially XLF-dependent, whereas ligation of Tg1 and Tg2 was inefficient and only detectable in the presence of XLF. Overall, the results suggest that promoting ligation of DSBs with proximal base damage may be an important function of XLF, but that Tg can still be a major impediment to repair, being relatively resistant to both trimming and ligation. Moreover, it appears that base excision repair of Tg can sometimes interfere with repair of DSBs that would otherwise be readily rejoined. PMID:27049455

  12. Variations in the Processing of DNA Double-Strand Breaks Along 60-MeV Therapeutic Proton Beams

    Science.gov (United States)

    Chaudhary, Pankaj; Marshall, Thomas I.; Currell, Frederick J.; Kacperek, Andrzej; Schettino, Giuseppe; Prise, Kevin M.

    2016-01-01

    Purpose To investigate the variations in induction and repair of DNA damage along the proton path, after a previous report on the increasing biological effectiveness along clinically modulated 60-MeV proton beams. Methods and Materials Human skin fibroblast (AG01522) cells were irradiated along a monoenergetic and a modulated spread-out Bragg peak (SOBP) proton beam used for treating ocular melanoma at the Douglas Cyclotron, Clatterbridge Centre for Oncology, Wirral, Liverpool, United Kingdom. The DNA damage response was studied using the 53BP1 foci formation assay. The linear energy transfer (LET) dependence was studied by irradiating the cells at depths corresponding to entrance, proximal, middle, and distal positions of SOBP and the entrance and peak position for the pristine beam. Results A significant amount of persistent foci was observed at the distal end of the SOBP, suggesting complex residual DNA double-strand break damage induction corresponding to the highest LET values achievable by modulated proton beams. Unlike the directly irradiated, medium-sharing bystander cells did not show any significant increase in residual foci. Conclusions The DNA damage response along the proton beam path was similar to the response of X rays, confirming the low-LET quality of the proton exposure. However, at the distal end of SOBP our data indicate an increased complexity of DNA lesions and slower repair kinetics. A lack of significant induction of 53BP1 foci in the bystander cells suggests a minor role of cell signaling for DNA damage under these conditions. PMID:26452569

  13. Identification of defective illegitimate recombinational repair of oxidatively-induced DNA double-strand breaks in ataxia-telangiectasia cells

    Science.gov (United States)

    Dar, M. E.; Winters, T. A.; Jorgensen, T. J.

    1997-01-01

    Ataxia-telangiectasia (A-T) is an autosomal-recessive lethal human disease. Homozygotes suffer from a number of neurological disorders, as well as very high cancer incidence. Heterozygotes may also have a higher than normal risk of cancer, particularly for the breast. The gene responsible for the disease (ATM) has been cloned, but its role in mechanisms of the disease remain unknown. Cellular A-T phenotypes, such as radiosensitivity and genomic instability, suggest that a deficiency in the repair of DNA double-strand breaks (DSBs) may be the primary defect; however, overall levels of DSB rejoining appear normal. We used the shuttle vector, pZ189, containing an oxidatively-induced DSB, to compare the integrity of DSB rejoining in one normal and two A-T fibroblast cells lines. Mutation frequencies were two-fold higher in A-T cells, and the mutational spectrum was different. The majority of the mutations found in all three cell lines were deletions (44-63%). The DNA sequence analysis indicated that 17 of the 17 plasmids with deletion mutations in normal cells occurred between short direct-repeat sequences (removing one of the repeats plus the intervening sequences), implicating illegitimate recombination in DSB rejoining. The combined data from both A-T cell lines showed that 21 of 24 deletions did not involve direct-repeats sequences, implicating a defect in the illegitimate recombination pathway. These findings suggest that the A-T gene product may either directly participate in illegitimate recombination or modulate the pathway. Regardless, this defect is likely to be important to a mechanistic understanding of this lethal disease.

  14. The repair of environmentally relevant DNA double strand breaks caused by high linear energy transfer irradiation--no simple task.

    Science.gov (United States)

    Moore, Shaun; Stanley, Fintan K T; Goodarzi, Aaron A

    2014-05-01

    High linear energy transfer (LET) ionising radiation (IR) such as radon-derived alpha particles and high mass, high energy (HZE) particles of cosmic radiation are the predominant forms of IR to which humanity is exposed throughout life. High-LET forms of IR are established carcinogens relevant to human cancer, and their potent mutagenicity is believed, in part, to be due to a greater incidence of clustered DNA double strand breaks (DSBs) and associated lesions, as ionization events occur within a more confined genomic space. The repair of such DNA damage is now well-documented to occur with slower kinetics relative to that induced by low-LET IR, and to be more reliant upon homology-directed repair pathways. Underlying these phenomena is the relative inability of non-homologous end-joining (NHEJ) to adequately resolve high-LET IR-induced DSBs. Current findings suggest that the functionality of the DNA-dependent protein kinase (DNA-PK), comprised of the Ku70-Ku80 heterodimer and the DNA-PK catalytic subunit (DNA-PKcs), is particularly perturbed by high-LET IR-induced clustered DSBs, rendering DNA-PK dependent NHEJ less relevant to resolving these lesions. By contrast, the NHEJ-associated DNA processing endonuclease Artemis shows a greater relevance to high-LET IR-induced DSB repair. Here, we will review the cellular response to high-LET irradiation, the implications of the chronic, low-dose modality of this exposure and molecular pathways that respond to high-LET irradiation induced DSBs, with particular emphasis on NHEJ factors. PMID:24565812

  15. 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.

  16. Reduced contribution of thermally labile sugar lesions to DNA double strand break formation after exposure to heavy ions

    International Nuclear Information System (INIS)

    In cells exposed to low linear energy transfer (LET) ionizing-radiation (IR), double-strand-breaks (DSBs) form within clustered-damage-sites (CDSs) from lesions disrupting the DNA sugar-phosphate backbone. It is commonly assumed that all DSBs form promptly and are immediately detected by the cellular DNA-damage-response (DDR) apparatus. However, there is evidence that the pool of DSBs detected by physical methods, such as pulsed-field gel electrophoresis (PFGE), comprises not only promptly forming DSBs (prDSBs) but also DSBs developing during lysis at high temperatures from thermally-labile sugar-lesions (TLSLs). We recently demonstrated that conversion of TLSLs to DNA breaks and ultimately to DSBs also occurs in cells during the first hour of post-irradiation incubation at physiological temperatures. Thus, TLSL-dependent DSBs (tlDSBs) are not an avoidable technique-related artifact, but a reality the cell always faces. The biological consequences of tlDSBs and the dependence of their formation on LET require in-depth investigation. Heavy-ions (HI) are a promising high-LET radiation modality used in cancer treatment. HI are also encountered in space and generate serious radiation protection problems to prolonged space missions. Here, we study, therefore, the effect of HI on the yields of tlDSBs and prDSBs. We report a reduction in the yield of tlDBSs stronger than that earlier reported for neutrons, and with pronounced cell line dependence. We conclude that with increasing LET the complexity of CDSs increases resulting in a commensurate increase in the yield prDSBs and a decrease in tlDSBs. The consequences of these effects to the relative biological effectiveness are discussed

  17. Double-strand break repair by interchromosomal recombination: an in vivo repair mechanism utilized by multiple somatic tissues in mammals.

    Directory of Open Access Journals (Sweden)

    Ryan R White

    Full Text Available Homologous recombination (HR is essential for accurate genome duplication and maintenance of genome stability. In eukaryotes, chromosomal double strand breaks (DSBs are central to HR during specialized developmental programs of meiosis and antigen receptor gene rearrangements, and form at unusual DNA structures and stalled replication forks. DSBs also result from exposure to ionizing radiation, reactive oxygen species, some anti-cancer agents, or inhibitors of topoisomerase II. Literature predicts that repair of such breaks normally will occur by non-homologous end-joining (in G1, intrachromosomal HR (all phases, or sister chromatid HR (in S/G(2. However, no in vivo model is in place to directly determine the potential for DSB repair in somatic cells of mammals to occur by HR between repeated sequences on heterologs (i.e., interchromosomal HR. To test this, we developed a mouse model with three transgenes-two nonfunctional green fluorescent protein (GFP transgenes each containing a recognition site for the I-SceI endonuclease, and a tetracycline-inducible I-SceI endonuclease transgene. If interchromosomal HR can be utilized for DSB repair in somatic cells, then I-SceI expression and induction of DSBs within the GFP reporters may result in a functional GFP+ gene. Strikingly, GFP+ recombinant cells were observed in multiple organs with highest numbers in thymus, kidney, and lung. Additionally, bone marrow cultures demonstrated interchromosomal HR within multiple hematopoietic subpopulations including multi-lineage colony forming unit-granulocyte-erythrocyte-monocyte-megakaryocte (CFU-GEMM colonies. This is a direct demonstration that somatic cells in vivo search genome-wide for homologous sequences suitable for DSB repair, and this type of repair can occur within early developmental populations capable of multi-lineage differentiation.

  18. RNF4, a SUMO-targeted ubiquitin E3 ligase, promotes DNA double-strand break repair.

    Science.gov (United States)

    Galanty, Yaron; Belotserkovskaya, Rimma; Coates, Julia; Jackson, Stephen P

    2012-06-01

    Protein ubiquitylation and sumoylation play key roles in regulating cellular responses to DNA double-strand breaks (DSBs). Here, we show that human RNF4, a small ubiquitin-like modifier (SUMO)-targeted ubiquitin E3 ligase, is recruited to DSBs in a manner requiring its SUMO interaction motifs, the SUMO E3 ligases PIAS1 and PIAS4, and various DSB-responsive proteins. Furthermore, we reveal that RNF4 depletion impairs ubiquitin adduct formation at DSB sites and causes persistent histone H2AX phosphorylation (γH2AX) associated with defective DSB repair, hypersensitivity toward DSB-inducing agents, and delayed recovery from radiation-induced cell cycle arrest. We establish that RNF4 regulates turnover of the DSB-responsive factors MDC1 and replication protein A (RPA) at DNA damage sites and that RNF4-depleted cells fail to effectively replace RPA by the homologous recombination factors BRCA2 and RAD51 on resected DNA. Consistent with previous data showing that RNF4 targets proteins to the proteasome, we show that the proteasome component PSMD4 is recruited to DNA damage sites in a manner requiring its ubiquitin-interacting domains, RNF4 and RNF8. Finally, we establish that PSMD4 binds MDC1 and RPA1 in a DNA damage-induced, RNF4-dependent manner and that PSMD4 depletion cause MDC1 and γH2AX persistence in irradiated cells. RNF4 thus operates as a DSB response factor at the crossroads between the SUMO and ubiquitin systems. PMID:22661229

  19. 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.

  20. The construction of a recombinant cDNA library representative of the poly(A)+ mRNA population from normal human lymphocytes.

    OpenAIRE

    Woods, D.; Crampton, J.; Clarke, B.; Williamson, R

    1980-01-01

    A recombinant library has been constructed using the plasmid pAT153 and double stranded cDNA prepared from normal human lymphocyte poly(A)+ RNA. Transformation conditions were optimized to yield approximately 200,000 recombinants per microgram of double stranded cDNA. Statistical analysis as well as sequence complexity analysis of the inserted sequences indicates that the cDNA library is representative of > 99% of the poly(A)+ RNA present in the normal human lymphocyte.

  1. SiRNA Mediated Gene Silencing: A Mini Review

    OpenAIRE

    M.V Jeevitha; S.U Ajisha; Baby Joseph

    2012-01-01

    RNA interference (RNAi) technology has become a novel tool for silencing gene expression in cells or organisms. RNA interference is the process that double-stranded RNA induces the homology-dependent degradation of cognate mRNA mediated by 21-23 nucleotide short interfering RNA (siRNA). RNA interference is a powerful mechanism of gene silencing that underlies many aspects of eukaryotic biology. On the molecular level, RNAi is mediated by a family of ribonucleoprotein (RNP) complexes called R...

  2. Induction of DNA double-strand breaks by ionizing radiation of different quality and their relevance for cell inactivation

    International Nuclear Information System (INIS)

    By investigation of the production of DNA strand breaks and of DNA release from the nuclear membrane complex in Chinese hamster cells using different radiation qualities from 1 to 360 keV/μm, partly also under hypoxic conditions, and by relating the results to the induction of chromosome aberrations and to cell inactivation it has become possible to find connections between the induction of molecular lesions and the expression of this damage on the cellular level. From the studies follows that DNA pieces are cut off from the nuclear membrane complex by DNA double-strand breaks (DSB). The share and size of the released pieces depends on radiation dose and quality as well as on the oxygen conditions. The lesions can partly be repaired. In connection with the DSB rates the results of the DNA release studies led to the conclusion that the DNA in the cells must be organized in superstructure units (MASSUs) with a DNA mass of about 2 x 109 g/mol, which are associated to the nuclear membrane in attachment points. The numerical relations show that for a 37% survival probability about 90 DSB per genome are required with sparsely ionizing radiation; this number declines to about 40 by use of more densely ionizing radiation up to 150 keV/μm, and increases again with further rise of the ionization density. Hence, for cell inactivation not simply a certain number of DSB per cell is required but rather seems their cooperation within a small structure section of the DNA to be relevant. These critical structures are with high probability the MASSUs. An irrepairable release of DNA from such a structure unit can bring about a chromosome break detectable in the metaphase and finally lead to cell inactivation. DSB turned out to be the essential lethal events in bacteria as well. The relatively small differences to the eukaryotic cells in the position of the maximum of radiation sensitivity on the LET scale and in the lesion sensitivity towards DSB let suggest that a common critical

  3. 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

  4. Enzymatic synthesis of long double-stranded DNA labeled with haloderivatives of nucleobases in a precisely pre-determined sequence

    Directory of Open Access Journals (Sweden)

    Rak Janusz

    2011-08-01

    Full Text Available Abstract Background Restriction endonucleases are widely applied in recombinant DNA technology. Among them, enzymes of class IIS, which cleave DNA beyond recognition sites, are especially useful. We use BsaI enzyme for the pinpoint introduction of halogen nucleobases into DNA. This has been done for the purpose of anticancer radio- and phototherapy that is our long-term objective. Results An enzymatic method for synthesizing long double-stranded DNA labeled with the halogen derivatives of nucleobases (Hal-NBs with 1-bp accuracy has been put forward and successfully tested on three different DNA fragments containing the 5-bromouracil (5-BrU residue. The protocol assumes enzymatic cleavage of two Polymerase-Chain-Reaction (PCR fragments containing two recognition sequences for the same or different class IIS restriction endonucleases, where each PCR fragment has a partially complementary cleavage site. These sites are introduced using synthetic DNA primers or are naturally present in the sequence used. The cleavage sites are not compatible, and therefore not susceptible to ligation until they are partially filled with a Hal-NB or original nucleobase, resulting in complementary cohesive end formation. Ligation of these fragments ultimately leads to the required Hal-NB-labeled DNA duplex. With this approach, a synthetic, extremely long DNA fragment can be obtained by means of a multiple assembly reaction (n × maximum PCR product length: n × app. 50 kb. Conclusions The long, precisely labeled DNA duplexes obtained behave in very much the same manner as natural DNA and are beyond the range of chemical synthesis. Moreover, the conditions of synthesis closely resemble the natural ones, and all the artifacts accompanying the chemical synthesis of DNA are thus eliminated. The approach proposed seems to be completely general and could be used to label DNA at multiple pre-determined sites and with halogen derivatives of any nucleobase. Access to DNAs

  5. Rapid MCNP simulation of DNA double strand break (DSB) relative biological effectiveness (RBE) for photons, neutrons, and light ions

    International Nuclear Information System (INIS)

    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. (paper)

  6. 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.

  7. 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, (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. PMID:26449929

  8. 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

  9. DNA double-strand break induction in Ku80-deficient CHO cells following Boron Neutron Capture Reaction

    International Nuclear Information System (INIS)

    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. 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. 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. 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 an advantage over gamma-ray sensitive cells in BNCR

  10. 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

  11. ATM protein is indispensable to repair complex-type DNA double strand breaks induced by high LET heavy ion irradiation.

    Science.gov (United States)

    Sekine, Emiko; Yu, Dong; Fujimori, Akira; Anzai, Kazunori; Okayasu, Ryuichi

    ATM (ataxia telangiectasia-mutated) protein responsible for a rare genetic disease with hyperradiosensitivity, is the one of the earliest repair proteins sensing DNA double-strand breaks (DSB). ATM is known to phosphorylate DNA repair proteins such as MRN complex (Mre11, Rad50 and NBS1), 53BP1, Artemis, Brca1, gamma-H2AX, and MDC. We studied the interactions between ATM and DNA-PKcs, a crucial NHEJ repair protein, after cells exposure to high and low LET irradiation. Normal human (HFL III, MRC5VA) and AT homozygote (AT2KY, AT5BIVA, AT3BIVA) cells were irradiated with X-rays and high LET radiation (carbon ions: 290MeV/n initial energy at 70 keV/um, and iron ions: 500MeV/n initial energy at 200KeV/um), and several critical end points were examined. AT cells with high LET irradiation showed a significantly higher radiosensitivity when compared with normal cells. The behavior of DNA DSB repair was monitored by immuno-fluorescence techniques using DNA-PKcs (pThr2609, pSer2056) and ATM (pSer1981) antibodies. In normal cells, the phosphorylation of DNA-PKcs was clearly detected after high LET irradiation, though the peak of phosphorylation was delayed when compared to X-irradiation. In contrast, almost no DNA-PKcs phosphorylation foci were detected in AT cells irradiated with high LET radiation. A similar result was also observed in normal cells treated with 10 uM ATM kinase specific inhibitor (KU55933) one hour before irradiation. These data suggest that the phosphorylation of DNA-PKcs with low LET X-rays is mostly ATM-independent, and the phosphorylation of DNA-PKcs with high LET radiation seems to require ATM probably due to its complex nature of DSB induced. Our study indicates that high LET heavy ion irradiation which we can observe in the space environment would provide a useful tool to study the fundamental mechanism associated with DNA DSB repair.

  12. RNA Binding Specificity of Drosophila Muscleblind†

    OpenAIRE

    Goers, Emily S.; Voelker, Rodger B.; Gates, Devika P.; Berglund, J. Andrew

    2008-01-01

    Members of the muscleblind family of RNA binding proteins found in Drosophila and mammals are key players in both the human disease myotonic dystrophy and the regulation of alternative splicing. Recently, the mammalian muscleblind-like protein, MBNL1, has been shown to have interesting RNA binding properties with both endogenous and disease-related RNA targets. Here we report the characterization of RNA binding properties of the Drosophila muscleblind protein Mbl. Mutagenesis of double-strand...

  13. RNA-Dependent RNA Polymerase 6 Is Required for Efficient hpRNA-Induced Gene Silencing in Plants

    OpenAIRE

    Harmoko, Rikno; Fanata, Wahyu Indra Duwi; Yoo, Jae Yong; Ko, Ki Seong; Rim, Yeong Gil; Uddin, Mohammad Nazim; Siswoyo, Tri Agus; Lee, Seung Sik; Kim, Dool Yi; Lee, Sang Yeol; Lee, Kyun Oh

    2013-01-01

    In plants, transgenes with inverted repeats are used to induce efficient RNA silencing, which is also frequently induced by highly transcribed sense transgenes. RNA silencing induced by sense transgenes is dependent on RNA-dependent RNA polymerase 6 (RDR6), which converts single-stranded (ss) RNA into double-stranded (ds) RNA. By contrast, it has been proposed that RNA silencing induced by self-complementary hairpin RNA (hpRNA) does not require RDR6, because the hpRNA can directly fold back o...

  14. Assessment of DNA double-strand breaks induced by intravascular iodinated contrast media following in vitro irradiation and in vivo, during paediatric cardiac catheterization.

    Science.gov (United States)

    Gould, Richard; McFadden, Sonyia L; Horn, Simon; Prise, Kevin M; Doyle, Philip; Hughes, Ciara M

    2016-01-01

    Paediatric cardiac catheterizations may result in the administration of substantial amounts of iodinated contrast media and ionizing radiation. The aim of this work was to investigate the effect of iodinated contrast media in combination with in vitro and in vivo X-ray radiation on lymphocyte DNA. Six concentrations of iodine (15, 17.5, 30, 35, 45, and 52.5 mg of iodine per mL blood) represented volumes of iodinated contrast media used in the clinical setting. Blood obtained from healthy volunteers was mixed with iodinated contrast media and exposed to radiation doses commonly used in paediatric cardiac catheterizations (0 mGy, 70 mGy, 140 mGy, 250 mGy and 450 mGy). Control samples contained no iodine. For in vivo experimentation, pre and post blood samples were collected from children undergoing cardiac catheterization, receiving iodine concentrations of up to 51 mg of iodine per mL blood and radiation doses of up to 400 mGy. Fluorescence microscopy was performed to assess γH2AX-foci induction, which corresponded to the number of DNA double-strand breaks. The presence of iodine in vitro resulted in significant increases of DNA double-strand breaks beyond that induced by radiation for ≥ 17.5 mg/mL iodine to blood. The in vivo effects of contrast media on children undergoing cardiac catheterization resulted in a 19% increase in DNA double-strand breaks in children receiving an average concentration of 19 mg/mL iodine to blood. A larger investigation is required to provide further information of the potential benefit of lowering the amount of iodinated contrast media received during X-ray radiation investigations. PMID:26549792

  15. DNA double-strand breaks induced by high-energy neon and iron ions in human fibroblasts. I. Pulsed-field gel electrophoresis method

    International Nuclear Information System (INIS)

    The relative effectiveness of high-energy neon and iron ions for the production of DNA double-strand breaks was measured in one transformed and one nontransformed human fibroblast cell line using pulsed-field gel electrophoresis. The DNA released from the gel plug (fraction of activity released: FAR) as well as the size distribution of the DNA entering the gel were used to compare the effects of the heavy-ion exposure with X-ray exposure. Both methods gave similar results, indicating similar distributions of breaks over megabase-pair distances for the heavy ions and the X rays. The relative biological effectiveness (RBE) compared to 225 kVp X rays of initially induced DNA double-strand breaks was found to be 0.85 for 425 MeV/u neon ions (LET 32 keV/μm) and 0.42-0.55 for 250-600 MeV/u iron ions (LET 190-350 keV/μm). Postirradiation incubation showed less efficient repair of breaks induced by the neon ions and the 600 MeV/u iron ions compared to X rays. Survival experiments demonstrated RBE values larger than one for cell killing by the heavy ions in parallel experiments (neon: RBE = 1.2, iron: RBE = 2.3-3.0, based on D10 values). It is concluded that either the initial yield of DNA double-strand breaks induced by the high-energy particles is lower than the yield for X rays, or the breaks induced by heavy ions are present in clusters that cannot be resolved with the technique used. These results are confirmed in the accompanying paper. 48 refs., 5 figs., 2 tabs

  16. Arising and elimination of single- and double-strand DNA breaks in xeroderma pigmentosum fibroblasts under effect of γ-irradiation

    International Nuclear Information System (INIS)

    Defect on recombine ability of DNA, having gamma-induced single-strand breaks, detected earlier in blood lymphocytes of a patient with xeroederma pigmentosum KhR2LE is shown also during irradiation of skin fibrcblasts in a culture. This DNA reparation deficiency is more obvious in early transitions (passages) of cultivated fibroblasts; with the cell passivation recombine ability gradually grows. Double-strand DNA breaks are liquidated in KhR2LE fibroblasts with the same completeness and rate as in the cells of patients with classical xeroderma pigmentosum and of healthy donors

  17. Double Strand Break Unwinding and Resection by the Mycobacterial Helicase-Nuclease AdnAB in the Presence of Single Strand DNA-binding Protein (SSB)*

    OpenAIRE

    Unciuleac, Mihaela-Carmen; Shuman, Stewart

    2010-01-01

    Mycobacterial AdnAB is a heterodimeric DNA helicase-nuclease and 3′ to 5′ DNA translocase implicated in the repair of double strand breaks (DSBs). The AdnA and AdnB subunits are each composed of an N-terminal motor domain and a C-terminal nuclease domain. Inclusion of mycobacterial single strand DNA-binding protein (SSB) in reactions containing linear plasmid dsDNA allowed us to study the AdnAB helicase under conditions in which the unwound single strands are coated by SSB and thereby prevent...

  18. Repair of DNA double-strand breaks induced in Saccharomyces cerevisiae using different γ-ray dose rates: a pulsed-field gel electrophoresis analysis

    International Nuclear Information System (INIS)

    We investigated the effects of γ-ray exposures at high dose-rate (HDR, 23·2 Gy/min) and low dose-rate (LDR, 0·47 Gy/min) on survival and the induction of DNA double-strand breaks (dsb) in a diploid wild-type (D7) and the repair-deficient mutant strain rad52/rad52 of Saccharomyces cerevisiae. The relationship observed between γ-ray survival and dsb repair clearly indicates that increases in survival of wild-type cells, during LDR as compared with HDR exposures and after LHR, are strongly related to the repair of dsb. (author)

  19. Human chromosome 5 complements the DNA double-strand break-repair deficiency and gamma-ray sensitivity of the XR-1 hamster variant.

    OpenAIRE

    Giaccia, A. J.; Denko, N; MAcLAREN, R.; Mirman, D; Waldren, C; Hart, I; Stamato, T D

    1990-01-01

    XR-1 is a Chinese hamster ovary (CHO) cell mutant which is unusually sensitive to killing by gamma rays in the G1 portion of the cell cycle but has nearly normal resistance to gamma-ray damage in late S phase. The cell-cycle sensitivity correlates with the mutant's inability to repair DNA double-strand breaks (DSBs) produced by ionizing radiation and restriction enzymes. We have previously shown in somatic cell hybrids of XR-1 cells and human fibroblasts that the XR-1 mutation is a recessive ...

  20. 60Co gamma-rays induce predominantly C/G to G/C transversions in double-stranded M13 DNA.

    OpenAIRE

    Hoebee, B.; J. Brouwer; van de Putte, P; Loman, H; Retèl, J

    1988-01-01

    Upon irradiation with gamma rays of an oxygenated aqueous solution of double-stranded M13 DNA, a very specific mutation spectrum was found with respect to both the type and the positions in the DNA sequence. Of the 23 mutations, which were sequenced, 16 represent a C/G to G/C transversion. A C/G to T/A transition was found once and a G/C to T/A transversion twice. The remaining 4 mutations are frameshifts, 2 are identical and formed by the insertion of a G/C basepair; the other 2 mutations ar...