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Sample records for double-stranded rna-activated protein

  1. Double-stranded RNA-activated protein kinase regulates early innate immune responses during respiratory syncytial virus infection.

    PubMed

    Minor, Radiah A Corn; Limmon, Gino V; Miller-DeGraff, Laura; Dixon, Darlene; Andrews, Danica M K; Kaufman, Randal J; Imani, Farhad

    2010-04-01

    Respiratory syncytial virus (RSV) is the most common cause of childhood viral bronchiolitis and lung injury. Inflammatory responses significantly contribute to lung pathologies during RSV infections and bronchiolitis but the exact mechanisms have not been completely defined. The double-stranded RNA-activated protein kinase (PKR) functions to inhibit viral replication and participates in several signaling pathways associated with innate inflammatory immune responses. Using a functionally defective PKR (PKR(-/-)) mouse model, we investigated the role of this kinase in early events of RSV-induced inflammation. Our data showed that bronchoalveolar lavage (BAL) fluid from infected PKR(-/-) mice had significantly lower levels of several innate inflammatory cytokines and chemokines. Histological examinations revealed that there was less lung injury in infected PKR(-/-) mice as compared to the wild type. A genome-wide analysis showed that several early antiviral and immune regulatory genes were affected by PKR activation. These data suggest that PKR is a signaling molecule for immune responses during RSV infections.

  2. Specificity of the double-stranded RNA-binding domain from the RNA-activated protein kinase PKR for double-stranded RNA: insights from thermodynamics and small-angle X-ray scattering.

    PubMed

    Patel, Sunita; Blose, Joshua M; Sokoloski, Joshua E; Pollack, Lois; Bevilacqua, Philip C

    2012-11-20

    The interferon-inducible, double-stranded (ds) RNA-activated protein kinase (PKR) contains a dsRNA-binding domain (dsRBD) and plays key roles in viral pathogenesis and innate immunity. Activation of PKR is typically mediated by long dsRNA, and regulation of PKR is disfavored by most RNA imperfections, including bulges and internal loops. Herein, we combine isothermal titration calorimetry (ITC), electrophoretic mobility shift assays, and small-angle X-ray scattering (SAXS) to dissect the thermodynamic basis for the specificity of the dsRBD termed "p20" for various RNAs and to detect any RNA conformational changes induced upon protein binding. We monitor binding of p20 to chimeric duplexes containing terminal RNA-DNA hybrid segments and a central dsRNA segment, which was either unbulged ("perfect") or bulged. The ITC data reveal strong binding of p20 to the perfect duplex (K(d) ~ 30 nM) and weaker binding to the bulged duplex (K(d) ~ 2-5 μM). SAXS reconstructions and p(r) distance distribution functions further uncover that p20 induces no significant conformational change in perfect dsRNA but largely straightens bulged dsRNA. Together, these observations support the dsRBD's ability to tightly bind to only A-form RNA and suggest that in a noninfected cell, PKR may be buffered via weak interactions with various bulged and looped RNAs, which it may straighten. This work suggests that PKR-regulating RNAs with complex secondary and tertiary structures likely mimic dsRNA and/or engage portions of PKR outside of the dsRBD.

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

    PubMed

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

    2016-02-01

    In vivo, ectopic accumulation of fatty acids in muscles leads to alterations in insulin signaling at both the IRS1 and Akt steps. However, in vitro treatments with saturated fatty acids or their derivative ceramide demonstrate an effect only at the Akt step. In this study, we adapted our experimental procedures to mimic the in vivo situation and show that the double-stranded RNA-dependent protein kinase (PKR) is involved in the long-term effects of saturated fatty acids on IRS1. C2C12 or human muscle cells were incubated with palmitate or directly with ceramide for short or long periods, and insulin signaling pathway activity was evaluated. PKR involvement was assessed through pharmacological and genetic studies. Short-term treatments of myotubes with palmitate, a ceramide precursor, or directly with ceramide induce an inhibition of Akt, whereas prolonged periods of treatment show an additive inhibition of insulin signaling through increased IRS1 serine 307 phosphorylation. PKR mRNA, protein, and phosphorylation are increased in insulin-resistant muscles. When PKR activity is reduced (siRNA or a pharmacological inhibitor), serine phosphorylation of IRS1 is reduced, and insulin-induced phosphorylation of Akt is improved. Finally, we show that JNK mediates ceramide-activated PKR inhibitory action on IRS1. Together, in the long term, our results show that ceramide acts at two distinct levels of the insulin signaling pathway (IRS1 and Akt). PKR, which is induced by both inflammation signals and ceramide, could play a major role in the development of insulin resistance in muscle cells. PMID:26698173

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

    NASA Astrophysics Data System (ADS)

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

    2009-11-01

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

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

    PubMed

    Lemaître, Charlène; Soutoglou, Evi

    2014-07-01

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

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

    PubMed

    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

  7. Standard atomic volumes in double-stranded DNA and packing in protein--DNA interfaces.

    PubMed

    Nadassy, K; Tomás-Oliveira, I; Alberts, I; Janin, J; Wodak, S J

    2001-08-15

    Standard volumes for atoms in double-stranded B-DNA are derived using high resolution crystal structures from the Nucleic Acid Database (NDB) and compared with corresponding values derived from crystal structures of small organic compounds in the Cambridge Structural Database (CSD). Two different methods are used to compute these volumes: the classical Voronoi method, which does not depend on the size of atoms, and the related Radical Planes method which does. Results show that atomic groups buried in the interior of double-stranded DNA are, on average, more tightly packed than in related small molecules in the CSD. The packing efficiency of DNA atoms at the interfaces of 25 high resolution protein-DNA complexes is determined by computing the ratios between the volumes of interfacial DNA atoms and the corresponding standard volumes. These ratios are found to be close to unity, indicating that the DNA atoms at protein-DNA interfaces are as closely packed as in crystals of B-DNA. Analogous volume ratios, computed for buried protein atoms, are also near unity, confirming our earlier conclusions that the packing efficiency of these atoms is similar to that in the protein interior. In addition, we examine the number, volume and solvent occupation of cavities located at the protein-DNA interfaces and compared them with those in the protein interior. Cavities are found to be ubiquitous in the interfaces as well as inside the protein moieties. The frequency of solvent occupation of cavities is however higher in the interfaces, indicating that those are more hydrated than protein interiors. Lastly, we compare our results with those obtained using two different measures of shape complementarity of the analysed interfaces, and find that the correlation between our volume ratios and these measures, as well as between the measures themselves, is weak. Our results indicate that a tightly packed environment made up of DNA, protein and solvent atoms plays a significant role in

  8. Chromosome Scaffold is a Double-Stranded Assembly of Scaffold Proteins

    PubMed Central

    Poonperm, Rawin; Takata, Hideaki; Hamano, Tohru; Matsuda, Atsushi; Uchiyama, Susumu; Hiraoka, Yasushi; Fukui, Kiichi

    2015-01-01

    Chromosome higher order structure has been an enigma for over a century. The most important structural finding has been the presence of a chromosome scaffold composed of non-histone proteins; so-called scaffold proteins. However, the organization and function of the scaffold are still controversial. Here, we use three dimensional-structured illumination microscopy (3D-SIM) and focused ion beam/scanning electron microscopy (FIB/SEM) to reveal the axial distributions of scaffold proteins in metaphase chromosomes comprising two strands. We also find that scaffold protein can adaptably recover its original localization after chromosome reversion in the presence of cations. This reversion to the original morphology underscores the role of the scaffold for intrinsic structural integrity of chromosomes. We therefore propose a new structural model of the chromosome scaffold that includes twisted double strands, consistent with the physical properties of chromosomal bending flexibility and rigidity. Our model provides new insights into chromosome higher order structure. PMID:26132639

  9. The Drosophila Zinc Finger Protein Trade Embargo Is Required for Double Strand Break Formation in Meiosis

    PubMed Central

    Lake, Cathleen M.; Nielsen, Rachel J.; Hawley, R. Scott

    2011-01-01

    Homologous recombination in meiosis is initiated by the programmed induction of double strand breaks (DSBs). Although the Drosophila Spo11 ortholog Mei-W68 is required for the induction of DSBs during meiotic prophase, only one other protein (Mei-P22) has been shown to be required for Mei-W68 to exert this function. We show here that the chromatin-associated protein Trade Embargo (Trem), a C2H2 zinc finger protein, is required to localize Mei-P22 to discrete foci on meiotic chromosomes, and thus to promote the formation of DSBs, making Trem the earliest known function in the process of DSB formation in Drosophila oocytes. We speculate that Trem may act by either directing the binding of Mei-P22 to preferred sites of DSB formation or by altering chromatin structure in a manner that allows Mei-P22 to form foci. PMID:21383963

  10. Deficient signaling in mice devoid of double-stranded RNA-dependent protein kinase.

    PubMed Central

    Yang, Y L; Reis, L F; Pavlovic, J; Aguzzi, A; Schäfer, R; Kumar, A; Williams, B R; Aguet, M; Weissmann, C

    1995-01-01

    Double-stranded RNA-dependent protein kinase (PKR) has been implicated in interferon (IFN) induction, antiviral response and tumor suppression. We have generated mice devoid of functional PKR (Pkr%). Although the mice are physically normal and the induction of type I IFN genes by poly(I).poly(C) (pIC) and virus is unimpaired, the antiviral response induced by IFN-gamma and pIC was diminished. However, in embryo fibroblasts from Pkr knockout mice, the induction of type I IFN as well as the activation of NF-kappa B by pIC, were strongly impaired but restored by priming with IFN. Thus, PKR is not directly essential for responses to pIC, and a pIC-responsive system independent of PKR is induced by IFN. No evidence of the tumor suppressor activity of PKR was demonstrated. Images PMID:8557029

  11. The Regulatory and Kinase Domains but Not the Interdomain Linker Determine Human Double-stranded RNA-activated Kinase (PKR) Sensitivity to Inhibition by Viral Non-coding RNAs.

    PubMed

    Sunita, S; Schwartz, Samantha L; Conn, Graeme L

    2015-11-20

    Double-stranded RNA (dsRNA)-activated protein kinase (PKR) is an important component of the innate immune system that presents a crucial first line of defense against viral infection. PKR has a modular architecture comprising a regulatory N-terminal dsRNA binding domain and a C-terminal kinase domain interposed by an unstructured ∼80-residue interdomain linker (IDL). Guided by sequence alignment, we created IDL deletions in human PKR (hPKR) and regulatory/kinase domain swap human-rat chimeric PKRs to assess the contributions of each domain and the IDL to regulation of the kinase activity by RNA. Using circular dichroism spectroscopy, limited proteolysis, kinase assays, and isothermal titration calorimetry, we show that each PKR protein is properly folded with similar domain boundaries and that each exhibits comparable polyinosinic-cytidylic (poly(rI:rC)) dsRNA activation profiles and binding affinities for adenoviral virus-associated RNA I (VA RNAI) and HIV-1 trans-activation response (TAR) RNA. From these results we conclude that the IDL of PKR is not required for RNA binding or mediating changes in protein conformation or domain interactions necessary for PKR regulation by RNA. In contrast, inhibition of rat PKR by VA RNAI and TAR RNA was found to be weaker than for hPKR by 7- and >300-fold, respectively, and each human-rat chimeric domain-swapped protein showed intermediate levels of inhibition. These findings indicate that PKR sequence or structural elements in the kinase domain, present in hPKR but absent in rat PKR, are exploited by viral non-coding RNAs to accomplish efficient inhibition of PKR.

  12. Substrate recognition and specificity of double-stranded RNA binding proteins.

    PubMed

    Vuković, Lela; Koh, Hye Ran; Myong, Sua; Schulten, Klaus

    2014-06-01

    Recognition of double-stranded (ds) RNA is an important part of many cellular pathways, including RNA silencing, viral recognition, RNA editing, processing, and transport. dsRNA recognition is often achieved by dsRNA binding domains (dsRBDs). We use atomistic molecular dynamics simulations to examine the binding interface of the transactivation response RNA binding protein (TRBP) dsRBDs to dsRNA substrates. Our results explain the exclusive selectivity of dsRBDs toward dsRNA and against DNA-RNA hybrid and dsDNA duplexes. We also provide corresponding experimental evidence. The dsRNA duplex is recognized by dsRBDs through the A-form of three duplex grooves and by the chemical properties of RNA bases, which have 2'-hydroxyl groups on their sugar rings. Our simulations show that TRBP dsRBD discriminates dsRNA- from DNA-containing duplexes primarily through interactions at two duplex grooves. The simulations also reveal that the conformation of the DNA-RNA duplex can be altered by dsRBD proteins, resulting in a weak binding of dsRBDs to DNA-RNA hybrids. Our study reveals the structural and molecular basis of protein-RNA interaction that gives rise to the observed substrate specificity of dsRNA binding proteins. PMID:24801449

  13. ATP-independent diffusion of double-stranded RNA binding proteins

    PubMed Central

    Koh, Hye Ran; Kidwell, Mary Anne; Ragunathan, Kaushik; Doudna, Jennifer A.; Myong, Sua

    2013-01-01

    The proteins harboring double-stranded RNA binding domains (dsRBDs) play diverse functional roles such as RNA localization, splicing, editing, export, and translation, yet mechanistic basis and functional significance of dsRBDs remain unclear. To unravel this enigma, we investigated transactivation response RNA binding protein (TRBP) consisting of three dsRBDs, which functions in HIV replication, protein kinase R(PKR)–mediated immune response, and RNA silencing. Here we report an ATP-independent diffusion activity of TRBP exclusively on dsRNA in a length-dependent manner. The first two dsRBDs of TRBP are essential for diffusion, whereas the third dsRBD is dispensable. Two homologs of TRBP, PKR activator and R3D1-L, displayed the same diffusion, implying a universality of the diffusion activity among this protein family. Furthermore, a Dicer–TRBP complex on dsRNA exhibited dynamic diffusion, which was correlated with Dicer’s catalytic activity. These results implicate the dsRNA-specific diffusion activity of TRBP that contributes to enhancing siRNA and miRNA processing by Dicer. PMID:23251028

  14. ATP-independent diffusion of double-stranded RNA binding proteins.

    PubMed

    Koh, Hye Ran; Kidwell, Mary Anne; Ragunathan, Kaushik; Doudna, Jennifer A; Myong, Sua

    2013-01-01

    The proteins harboring double-stranded RNA binding domains (dsRBDs) play diverse functional roles such as RNA localization, splicing, editing, export, and translation, yet mechanistic basis and functional significance of dsRBDs remain unclear. To unravel this enigma, we investigated transactivation response RNA binding protein (TRBP) consisting of three dsRBDs, which functions in HIV replication, protein kinase R(PKR)-mediated immune response, and RNA silencing. Here we report an ATP-independent diffusion activity of TRBP exclusively on dsRNA in a length-dependent manner. The first two dsRBDs of TRBP are essential for diffusion, whereas the third dsRBD is dispensable. Two homologs of TRBP, PKR activator and R3D1-L, displayed the same diffusion, implying a universality of the diffusion activity among this protein family. Furthermore, a Dicer-TRBP complex on dsRNA exhibited dynamic diffusion, which was correlated with Dicer's catalytic activity. These results implicate the dsRNA-specific diffusion activity of TRBP that contributes to enhancing siRNA and miRNA processing by Dicer. PMID:23251028

  15. Effects of double-strand break repair proteins on vertebrate telomere structure

    PubMed Central

    Wei, Chao; Skopp, Rose; Takata, Minoru; Takeda, Shunichi; Price, Carolyn M.

    2002-01-01

    Although telomeres are not recognized as double-strand breaks (DSBs), some DSB repair proteins are present at telomeres and are required for telomere maintenance. To learn more about the telomeric function of proteins from the homologous recombination (HR) and non-homologous end joining pathways (NHEJ), we have screened a panel of chicken DT40 knockout cell lines for changes in telomere structure. In contrast to what has been observed in Ku-deficient mice, we found that Ku70 disruption did not result in telomere–telomere fusions and had no effect on telomere length or the structure of the telomeric G-strand overhang. G-overhang length was increased by Rad51 disruption but unchanged by disruption of DNA-PKcs, Mre11, Rad52, Rad54, XRCC2 or XRCC3. The effect of Rad51 depletion was unexpected because gross alterations in telomere structure have not been detected in yeast HR mutants. Thus, our results indicate that Rad51 has a previously undiscovered function at vertebrate telomeres. They also indicate that Mre11 is not required to generate G-overhangs. Although Mre11 has been implicated in overhang generation, overhang structure had not previously been examined in Mre11-deficient cells. Overall our findings indicate that there are significant species-specific differences in the telomeric function of DSB repair proteins. PMID:12087170

  16. Sequestration and protection of double-stranded RNA by the betanodavirus b2 protein.

    PubMed

    Fenner, Beau J; Goh, Winnie; Kwang, Jimmy

    2006-07-01

    Betanodavirus B2 belongs to a group of functionally related proteins from the sense-strand RNA virus family Nodaviridae that suppress cellular RNA interference. The B2 proteins of insect alphanodaviruses block RNA interference by binding to double-stranded RNA (dsRNA), thus preventing Dicer-mediated cleavage and the subsequent generation of short interfering RNAs. We show here that the fish betanodavirus B2 protein also binds dsRNA. Binding is sequence independent, and maximal binding occurs with dsRNA substrates greater than 20 bp in length. The binding of B2 to long dsRNA is sufficient to completely block Dicer cleavage of dsRNA in vitro. Protein-protein interaction studies indicated that B2 interacts with itself and with other dsRNA binding proteins, the interaction occurring through binding to shared dsRNA substrates. Induction of the dsRNA-dependent interferon response was not antagonized by B2, as the interferon-responsive Mx gene of permissive fish cells was induced by wild-type viral RNA1 but not by a B2 mutant. The induction of Mx instead relied solely on viral RNA1 accumulation, which is impaired in the B2 mutant. Hyperediting of virus dsRNA and site-specific editing of 5-HT2C mRNA were both antagonized by B2. RNA editing was not, however, observed in transfected wild-type or B2 mutant RNA1, suggesting that this pathway does not contribute to the RNA1 accumulation defect of the B2 mutant. We thus conclude that betanodavirus B2 is a dsRNA binding protein that sequesters and protects both long and short dsRNAs to protect betanodavirus from cellular RNA interference.

  17. Cockayne syndrome group B protein regulates DNA double-strand break repair and checkpoint activation

    PubMed Central

    Batenburg, Nicole L; Thompson, Elizabeth L; Hendrickson, Eric A; Zhu, Xu-Dong

    2015-01-01

    Mutations of CSB account for the majority of Cockayne syndrome (CS), a devastating hereditary disorder characterized by physical impairment, neurological degeneration and segmental premature aging. Here we report the generation of a human CSB-knockout cell line. We find that CSB facilitates HR and represses NHEJ. Loss of CSB or a CS-associated CSB mutation abrogating its ATPase activity impairs the recruitment of BRCA1, RPA and Rad51 proteins to damaged chromatin but promotes the formation of 53BP1-Rif1 damage foci in S and G2 cells. Depletion of 53BP1 rescues the formation of BRCA1 damage foci in CSB-knockout cells. In addition, knockout of CSB impairs the ATM- and Chk2-mediated DNA damage responses, promoting a premature entry into mitosis. Furthermore, we show that CSB accumulates at sites of DNA double-strand breaks (DSBs) in a transcription-dependent manner. The kinetics of DSB-induced chromatin association of CSB is distinct from that of its UV-induced chromatin association. These results reveal novel, important functions of CSB in regulating the DNA DSB repair pathway choice as well as G2/M checkpoint activation. PMID:25820262

  18. Characterizing the Mechanism of Action of Double-Stranded RNA Activity against Western Corn Rootworm (Diabrotica virgifera virgifera LeConte)

    PubMed Central

    Bolognesi, Renata; Ramaseshadri, Parthasarathy; Anderson, Jerry; Bachman, Pamela; Clinton, William; Flannagan, Ronald; Ilagan, Oliver; Lawrence, Christina; Levine, Steven; Moar, William; Mueller, Geoffrey; Tan, Jianguo; Uffman, Joshua; Wiggins, Elizabeth; Heck, Gregory; Segers, Gerrit

    2012-01-01

    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. PMID:23071820

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

    PubMed Central

    Hermo-Parrado, X. Lois; Guardado-Calvo, Pablo; Llamas-Saiz, Antonio L.; Fox, Gavin C.; Vazquez-Iglesias, Lorena; Martínez-Costas, José; Benavente, Javier; van Raaij, Mark J.

    2007-01-01

    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. PMID:17565188

  20. Direct Activation of Ribosome-Associated Double-Stranded RNA-Dependent Protein Kinase (PKR) by Deoxynivalenol, Anisomycin and Ricin: A New Model for Ribotoxic Stress Response Induction

    PubMed Central

    Zhou, Hui-Ren; He, Kaiyu; Landgraf, Jeff; Pan, Xiao; Pestka, James J.

    2014-01-01

    Double-stranded RNA (dsRNA)-activated protein kinase (PKR) is a critical upstream mediator of the ribotoxic stress response (RSR) to the trichothecene deoxynivalenol (DON) and other translational inhibitors. Here, we employed HeLa cell lysates to: (1) characterize PKR’s interactions with the ribosome and ribosomal RNA (rRNA); (2) demonstrate cell-free activation of ribosomal-associated PKR and (3) integrate these findings in a unified model for RSR. Robust PKR-dependent RSR was initially confirmed in intact cells. PKR basally associated with 40S, 60S, 80S and polysome fractions at molar ratios of 7, 2, 23 and 3, respectively. Treatment of ATP-containing HeLa lysates with DON or the ribotoxins anisomycin and ricin concentration-dependently elicited phosphorylation of PKR and its substrate eIF2α. These phosphorylations could be blocked by PKR inhibitors. rRNA immunoprecipitation (RNA-IP) of HeLa lysates with PKR-specific antibody and sequencing revealed that in the presence of DON or not, the kinase associated with numerous discrete sites on both the 18S and 28S rRNA molecules, a number of which contained double-stranded hairpins. These findings are consistent with a sentinel model whereby multiple PKR molecules basally associate with the ribosome positioning them to respond to ribotoxin-induced alterations in rRNA structure by dimerizing, autoactivating and, ultimately, evoking RSR. PMID:25521494

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

    SciTech Connect

    Hermo-Parrado, X. Lois; Guardado-Calvo, Pablo; Llamas-Saiz, Antonio L.; Fox, Gavin C.; Vazquez-Iglesias, Lorena; Martínez-Costas, José; Benavente, Javier; Raaij, Mark J. van

    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.

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

    PubMed

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

    2014-10-01

    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 distribution, which was promoted by the addition of 1 mM Mg, Ca, or Sr ions. The dissociation constant of the PprA-supercoiled pUC19 DNA complex, calculated from the relative portions of shifted bands, was 0.6 μM with Hill coefficient of 3.3 in the presence of 1 mM Mg acetate. This indicates that at least 281 PprA molecules are required to saturate a supercoiled pUC19 DNA, which is consistent with the number (280) of bound PprA molecules estimated by the UV absorption of the PprA-pUC19 complex purified by gel filtration. This saturation also suggests linear polymerization of PprA along the dsDNA. On the other hand, the bands of linear dsDNA and nicked circular dsDNA that eventually formed PprA complexes did not saturate, but created larger molecular complexes when the PprA concentration was >1.3 μM. This result implies that DNA-bound PprA aids association of the termini of damaged DNAs, which is regulated by the concentration of PprA. These findings are important for the understanding of the mechanism underlying effective DNA repair involving PprA. PMID:25044036

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

    PubMed

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

    2014-10-01

    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 distribution, which was promoted by the addition of 1 mM Mg, Ca, or Sr ions. The dissociation constant of the PprA-supercoiled pUC19 DNA complex, calculated from the relative portions of shifted bands, was 0.6 μM with Hill coefficient of 3.3 in the presence of 1 mM Mg acetate. This indicates that at least 281 PprA molecules are required to saturate a supercoiled pUC19 DNA, which is consistent with the number (280) of bound PprA molecules estimated by the UV absorption of the PprA-pUC19 complex purified by gel filtration. This saturation also suggests linear polymerization of PprA along the dsDNA. On the other hand, the bands of linear dsDNA and nicked circular dsDNA that eventually formed PprA complexes did not saturate, but created larger molecular complexes when the PprA concentration was >1.3 μM. This result implies that DNA-bound PprA aids association of the termini of damaged DNAs, which is regulated by the concentration of PprA. These findings are important for the understanding of the mechanism underlying effective DNA repair involving PprA.

  4. Role of double-stranded RNA-binding proteins in RNA silencing and antiviral defense

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In plants as well as in animals, the intracellular presence of double-stranded RNA (dsRNA) triggers a signal transduction pathway that uses the sequence information of dsRNA to direct silencing of homologous genes. This process, designated RNA silencing or RNA interference (RNAi), relies on a family...

  5. Rotavirus RNA polymerase requires the core shell protein to synthesize the double-stranded RNA genome.

    PubMed

    Patton, J T; Jones, M T; Kalbach, A N; He, Y W; Xiaobo, J

    1997-12-01

    Rotavirus cores contain the double-stranded RNA (dsRNA) genome, RNA polymerase VP1, and guanylyltransferase VP3 and are enclosed within a lattice formed by the RNA-binding protein VP2. Analysis of baculovirus-expressed core-like particles (CLPs) has shown that VP1 and VP2 assemble into the simplest core-like structures with replicase activity and that VP1, but not VP3, is essential for replicase activity. To further define the role of VP1 and VP2 in the synthesis of dsRNA from viral mRNA, recombinant baculoviruses containing gene 1 (rBVg1) and gene 2 (rBVg2) of SA11 rotavirus were generated and used to express recombinant VP1 (rVP1) and rVP2, respectively. After purification, the proteins were assayed individually and together for the ability to catalyze the synthesis of dsRNA in a cell-free replication system. The results showed that dsRNA was synthesized only in assays containing rVP1 and rVP2, thus establishing that both proteins are essential for replicase activity. Even in assays containing a primer-linked mRNA template, neither rVP1 nor rVP2 alone directed RNA synthesis. Characterization of the cis-acting replication signals in mRNA recognized by the replicase of rVP1 and rVP2 showed that they were the same as those recognized by the replicase of virion-derived cores, thus excluding a role for VP3 in recognition of the mRNA template by the replicase. Analysis of RNA-protein interactions indicated that the mRNA template binds strongly to VP2 in replicase assays but that the majority of the dsRNA product neither is packaged nor stably associates with VP2. The results of replicase assays performed with mutant VP2 containing a deletion in its RNA-binding domain suggests that the essential role for VP2 in replication is linked to the protein's ability to bind the mRNA template for minus-strand synthesis.

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

    PubMed Central

    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 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

  7. Double stranded nucleic acid biochips

    DOEpatents

    Chernov, Boris; Golova, Julia

    2006-05-23

    This invention describes a new method of constructing double-stranded DNA (dsDNA) microarrays based on the use of pre-synthesized or natural DNA duplexes without a stem-loop structure. The complementary oligonucleotide chains are bonded together by a novel connector that includes a linker for immobilization on a matrix. A non-enzymatic method for synthesizing double-stranded nucleic acids with this novel connector enables the construction of inexpensive and robust dsDNA/dsRNA microarrays. DNA-DNA and DNA-protein interactions are investigated using the microarrays.

  8. Binding to the Minor Groove of the Double-Strand, Tau Protein Prevents DNA from Damage by Peroxidation

    PubMed Central

    Wang, Xing-Sheng; Chen, Lan; Wang, Dong-Liang; Liu, Ying; Hua, Qian; He, Rong-Qiao

    2008-01-01

    Tau, an important microtubule associated protein, has been found to bind to DNA, and to be localized in the nuclei of both neurons and some non-neuronal cells. Here, using electrophoretic mobility shifting assay (EMSA) in the presence of DNA with different chain-lengths, we observed that tau protein favored binding to a 13 bp or a longer polynucleotide. The results from atomic force microscopy also showed that tau protein preferred a 13 bp polynucleotide to a 12 bp or shorter polynucleotide. In a competitive assay, a minor groove binder distamycin A was able to replace the bound tau from the DNA double helix, indicating that tau protein binds to the minor groove. Tau protein was able to protect the double-strand from digestion in the presence of DNase I that was bound to the minor groove. On the other hand, a major groove binder methyl green as a negative competitor exhibited little effect on the retardation of tau-DNA complex in EMSA. This further indicates the DNA minor groove as the binding site for tau protein. EMSA with truncated tau proteins showed that both the proline-rich domain (PRD) and the microtubule-binding domain (MTBD) contributed to the interaction with DNA; that is to say, both PRD and MTBD bound to the minor groove of DNA and bent the double-strand, as observed by electron microscopy. To investigate whether tau protein is able to prevent DNA from the impairment by hydroxyl free radical, the chemiluminescence emitted by the phen-Cu/H2O2/ascorbate was measured. The emission intensity of the luminescence was markedly decreased when tau protein was present, suggesting a significant protection of DNA from the damage in the presence of hydroxyl free radical. PMID:18596978

  9. Double-Stranded RNA as an Inhibitor of Protein Synthesis and as a Substrate for a Nuclease in Extracts of Krebs II Ascites Cells

    PubMed Central

    Robertson, Hugh D.; Mathews, Michael B.

    1973-01-01

    Concentrations of double-stranded RNA above about 0.1 μg/ml inhibit translation of encephalo-myocarditis viral RNA and mouse globin messenger RNA in extracts of Krebs II ascites cells. Protein synthesis initially proceeds at the control rate, then abruptly shuts off in a manner similar to that observed in reticulocyte lysates [Hunt, T. & Ehrenfeld, E. (1971) Nature New Biol. 230, 91-94]. Substantially higher concentrations of double-stranded RNA are required to give this effect in ascites extracts. Subcellular fractions of Krebs II ascites cells contain a nucleolytic activity capable of digesting several natural and synthetic double-stranded RNAs. This nuclease is most active under conditions of protein synthesis, and part of the activity remains associated with ribosomes upon sedimentation. It is probably because of digestion of double-stranded RNA by this nuclease that higher concentrations of double-stranded RNA are required for inhibition of protein synthesis in Krebs cell extracts than in reticulocyte lysates. PMID:4346034

  10. Surface shapes and surrounding environment analysis of single- and double-stranded DNA-binding proteins in protein-DNA interface.

    PubMed

    Wang, Wei; Liu, Juan; Sun, Lin

    2016-07-01

    Protein-DNA bindings are critical to many biological processes. However, the structural mechanisms underlying these interactions are not fully understood. Here, we analyzed the residues shape (peak, flat, or valley) and the surrounding environment of double-stranded DNA-binding proteins (DSBs) and single-stranded DNA-binding proteins (SSBs) in protein-DNA interfaces. In the results, we found that the interface shapes, hydrogen bonds, and the surrounding environment present significant differences between the two kinds of proteins. Built on the investigation results, we constructed a random forest (RF) classifier to distinguish DSBs and SSBs with satisfying performance. In conclusion, we present a novel methodology to characterize protein interfaces, which will deepen our understanding of the specificity of proteins binding to ssDNA (single-stranded DNA) or dsDNA (double-stranded DNA). Proteins 2016; 84:979-989. © 2016 Wiley Periodicals, Inc.

  11. Nuclear localization of a double-stranded RNA-binding protein encoded by the vaccinia virus E3L gene.

    PubMed

    Yuwen, H; Cox, J H; Yewdell, J W; Bennink, J R; Moss, B

    1993-08-01

    We produced a B cell hybridoma (TW2.3) from vaccinia virus-infected mice that secreted a monoclonal antibody (MAb) reactive with a 25-kDA early viral protein that was localized by laser scanning confocal microscopy to the nucleus and cytoplasmic viral factory regions of infected cells. By cell-free translation of mRNA selected by hybridization to a complete library of vaccinia virus DNA fragments, the immunoreactive polypeptide was mapped to open reading frame E3L. The RNA start site of an early promoter was located 26 nucleotides upstream of the first methionine codon of E3L. Evidence was obtained that translation initiation occurs in vivo and in vitro at both the first and second methionine codons to produce major and minor polypeptides of 25 and 19 kDa, respectively. Both polypeptides bound double-stranded RNA, confirming the recent report of H.-W. Chang, J. C. Watson, and B. L. Jacobs (Proc. Natl. Acad. Sci. USA 89, 4825-4829, 1992). Other vaccinia virus proteins were not required for the nuclear localization of the E3L protein, since MAb TW2.3 bound to the nuclei of uninfected cells that were transfected with the E3L gene under the control of the SV40 early promoter. We also demonstrated that the E3L protein can bind to nuclei of aldehyde fixed and detergent permeabilized uninfected cells. This binding was abrogated by treatment of the cells with RNase but not DNase. The nuclear and cytoplasmic locations of the double-stranded RNA binding protein are consistent with multiple functions in the vaccinia virus infectious cycle.

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

    PubMed

    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

  13. Measles virus C protein impairs production of defective copyback double-stranded viral RNA and activation of protein kinase R.

    PubMed

    Pfaller, Christian K; Radeke, Monte J; Cattaneo, Roberto; Samuel, Charles E

    2014-01-01

    Measles virus (MV) lacking expression of C protein (C(KO)) is a potent activator of the double-stranded RNA (dsRNA)-dependent protein kinase (PKR), whereas the isogenic parental virus expressing C protein is not. Here, we demonstrate that significant amounts of dsRNA accumulate during C(KO) mutant infection but not following parental virus infection. dsRNA accumulated during late stages of infection and localized with virus replication sites containing N and P proteins. PKR autophosphorylation and stress granule formation correlated with the timing of dsRNA appearance. Phospho-PKR localized to dsRNA-containing structures as revealed by immunofluorescence. Production of dsRNA was sensitive to cycloheximide but resistant to actinomycin D, suggesting that dsRNA is a viral product. Quantitative PCR (qPCR) analyses revealed reduced viral RNA synthesis and a steepened transcription gradient in C(KO) virus-infected cells compared to those in parental virus-infected cells. The observed alterations were further reflected in lower viral protein expression levels and reduced C(KO) virus infectious yield. RNA deep sequencing confirmed the viral RNA expression profile differences seen by qPCR between C(KO) mutant and parental viruses. After one subsequent passage of the C(KO) virus, defective interfering RNA (DI-RNA) with a duplex structure was obtained that was not seen with the parental virus. We conclude that in the absence of C protein, the amount of PKR activator RNA, including DI-RNA, is increased, thereby triggering innate immune responses leading to impaired MV growth. PMID:24155404

  14. Melatonin sensitizes human breast cancer cells to ionizing radiation by downregulating proteins involved in double-strand DNA break repair.

    PubMed

    Alonso-González, Carolina; González, Alicia; Martínez-Campa, Carlos; Gómez-Arozamena, José; Cos, Samuel

    2015-03-01

    Radiation and adjuvant endocrine therapy are nowadays considered a standard treatment option after surgery in breast cancer. Melatonin exerts oncostatic actions on human breast cancer cells. In the current study, we investigated the effects of a combination of radiotherapy and melatonin on human breast cancer cells. Melatonin (1 mm, 10 μm and 1 nm) significantly inhibited the proliferation of MCF-7 cells. Radiation alone inhibited the MCF-7 cell proliferation in a dose-dependent manner. Pretreatment of breast cancer cells with melatonin 1 wk before radiation led to a significantly greater decrease of MCF-7 cell proliferation compared with radiation alone. Melatonin pretreatment before radiation also decreased G2 -M phase arrest compared with irradiation alone, with a higher percentage of cells in the G0 -G1 phase and a lower percentage of cells in S phase. Radiation alone diminished RAD51 and DNA-protein kinase (PKcs) mRNA expression, two main proteins involved in double-strand DNA break repair. Treatment with melatonin for 7 days before radiation led to a significantly greater decrease in RAD51 and DNA-PKcs mRNA expression compared with radiation alone. Our findings suggest that melatonin pretreatment before radiation sensitizes breast cancer cells to the ionizing effects of radiation by decreasing cell proliferation, inducing cell cycle arrest and downregulating proteins involved in double-strand DNA break repair. These findings may have implications for designing clinical trials using melatonin and radiotherapy. PMID:25623566

  15. The Axial Element Protein DESYNAPTIC2 Mediates Meiotic Double-Strand Break Formation and Synaptonemal Complex Assembly in Maize.

    PubMed

    Lee, Ding Hua; Kao, Yu-Hsin; Ku, Jia-Chi; Lin, Chien-Yu; Meeley, Robert; Jan, Ya-Shiun; Wang, Chung-Ju Rachel

    2015-09-01

    During meiosis, homologous chromosomes pair and recombine via repair of programmed DNA double-strand breaks (DSBs). DSBs are formed in the context of chromatin loops, which are anchored to the proteinaceous axial element (AE). The AE later serves as a framework to assemble the synaptonemal complex (SC) that provides a transient but tight connection between homologous chromosomes. Here, we showed that DESYNAPTIC2 (DSY2), a coiled-coil protein, mediates DSB formation and is directly involved in SC assembly in maize (Zea mays). The dsy2 mutant exhibits homologous pairing defects, leading to sterility. Analyses revealed that DSB formation and the number of RADIATION SENSITIVE51 (RAD51) foci are largely reduced, and synapsis is completely abolished in dsy2 meiocytes. Super-resolution structured illumination microscopy showed that DSY2 is located on the AE and forms a distinct alternating pattern with the HORMA-domain protein ASYNAPTIC1 (ASY1). In the dsy2 mutant, localization of ASY1 is affected, and loading of the central element ZIPPER1 (ZYP1) is disrupted. Yeast two-hybrid and bimolecular fluorescence complementation experiments further demonstrated that ZYP1 interacts with DSY2 but does not interact with ASY1. Therefore, DSY2, an AE protein, not only mediates DSB formation but also bridges the AE and central element of SC during meiosis. PMID:26296964

  16. Concentration-dependent exchange accelerates turnover of proteins bound to double-stranded DNA

    PubMed Central

    Graham, John S.; Johnson, Reid C.; Marko, John F.

    2011-01-01

    The multistep kinetics through which DNA-binding proteins bind their targets are heavily studied, but relatively little attention has been paid to proteins leaving the double helix. Using single-DNA stretching and fluorescence detection, we find that sequence-neutral DNA-binding proteins Fis, HU and NHP6A readily exchange with themselves and with each other. In experiments focused on the Escherichia coli nucleoid-associated protein Fis, only a small fraction of protein bound to DNA spontaneously dissociates into protein-free solution. However, if Fis is present in solution, we find that a concentration-dependent exchange reaction occurs which turns over the bound protein, with a rate of kexch = 6 × 104 M−1s−1. The bacterial DNA-binding protein HU and the yeast HMGB protein NHP6A display the same phenomenon of protein in solution accelerating dissociation of previously bound labeled proteins as exchange occurs. Thus, solvated proteins can play a key role in facilitating removal and renewal of proteins bound to the double helix, an effect that likely plays a major role in promoting the turnover of proteins bound to DNA in vivo and, therefore, in controlling the dynamics of gene regulation. PMID:21097894

  17. Crystal Structure of E. coli RecE Protein Reveals a Toroidal Tetramer for Processing Double-Stranded DNA Breaks

    SciTech Connect

    Zhang, Jinjin; Xing, Xu; Herr, Andrew B.; Bell, Charles E.

    2009-07-21

    Escherichia coli RecE protein is part of the classical RecET recombination system that has recently been used in powerful new methods for genetic engineering. RecE binds to free double-stranded DNA (dsDNA) ends and processively digests the 5{prime}-ended strand to form 5{prime}-mononucleotides and a 3{prime}-overhang that is a substrate for single strand annealing promoted by RecT. Here, we report the crystal structure of the C-terminal nuclease domain of RecE at 2.8 {angstrom} resolution. RecE forms a toroidal tetramer with a central tapered channel that is wide enough to bind dsDNA at one end, but is partially plugged at the other end by the C-terminal segment of the protein. Four narrow tunnels, one within each subunit of the tetramer, lead from the central channel to the four active sites, which lie about 15 {angstrom} from the channel. The structure, combined with mutational studies, suggests a mechanism in which dsDNA enters through the open end of the central channel, the 5{prime}-ended strand passes through a tunnel to access one of the four active sites, and the 3{prime}-ended strand passes through the plugged end of the channel at the back of the tetramer.

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

    PubMed

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

    2012-10-19

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

  19. Distinct binding sites for zinc and double-stranded RNA in the reovirus outer capsid protein sigma 3.

    PubMed Central

    Schiff, L A; Nibert, M L; Co, M S; Brown, E G; Fields, B N

    1988-01-01

    By atomic absorption analysis, we determined that the reovirus outer capsid protein sigma 3, which binds double-stranded RNA (dsRNA), is a zinc metalloprotein. Using Northwestern blots and a novel zinc blotting technique, we localized the zinc- and dsRNA-binding activities of sigma 3 to distinct V8 protease-generated fragments. Zinc-binding activity was contained within an amino-terminal fragment that contained a transcription factor IIIA-like zinc-binding sequence, and dsRNA-binding activity was associated with a carboxy-terminal fragment. By these techniques, new zinc- and dsRNA-binding activities were also detected in reovirus core proteins. A sequence similarity was observed between the catalytic site of the picornavirus proteases and the transcription factor IIIA-like zinc-binding site within sigma 3. We suggest that the zinc- and dsRNA-binding activities of sigma 3 may be important for its proposed regulatory effects on viral and host cell transcription and translation. Images PMID:3275869

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

    PubMed Central

    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

  1. Straightening of bulged RNA by the double-stranded RNA-binding domain from the protein kinase PKR

    PubMed Central

    Zheng, Xiaofeng; Bevilacqua, Philip C.

    2000-01-01

    The human interferon-induced protein kinase, PKR, is an antiviral agent that is activated by long stretches of double-stranded (ds)RNA. PKR has an N-terminal dsRNA-binding domain that contains two tandem copies of the dsRNA-binding motif and interacts with dsRNA in a nonsequence-specific fashion. Surprisingly, PKR can be regulated by certain viral and cellular RNAs containing non-Watson–Crick features. We found that RNAs containing bulges in the middle of a helix can bind to p20, a C-terminal truncated PKR containing the dsRNA-binding domain. Bulges are known to change the global geometry of RNA by bending the helical axis; therefore, we investigated the conformational changes of bulged RNA caused by PKR binding. A 66-mer DNA-RNA(+/− A3 bulge)-DNA chimera was constructed and annealed to a complementary RNA strand. This duplex forces the protein to bind in the middle. A 66-mer duplex with a top strand composed of DNA-DNA(+/−A3 bulge)-RNA was used as a control. Gel mobility-shift changes among the RNA-protein complexes are consistent with straightening of bulged RNA on protein binding. In addition, a van't Hoff analysis of p20 binding to bulged RNA reveals a favorable ΔΔH° and an unfavorable ΔΔS° relative to binding to straight dsRNA. These thermodynamic parameters are in good agreement with predictions from a nearest-neighbor analysis for RNA straightening and support a model in which the helical junction flanking the bulge stacks on protein binding. The ability of dsRNA-binding motif proteins to recognize and straighten bent RNA has implications for modulating the topology of RNAs in vivo. PMID:11114159

  2. SIRT6 stabilizes DNA-dependent Protein Kinase at chromatin for DNA double-strand break repair

    PubMed Central

    McCord, Ronald A.; Michishita, Eriko; Hong, Tao; Berber, Elisabeth; Boxer, Lisa D.; Kusumoto, Rika; Guan, Shenheng; Shi, Xiaobing; Gozani, Or; Burlingame, Alma L.; Bohr, Vilhelm A.; Chua, Katrin F.

    2009-01-01

    The Sir2 chromatin regulatory factor links maintenance of genomic stability to life span extension in yeast. The mammalian Sir2 family member SIRT6 has been proposed to have analogous functions, because SIRT6-deficiency leads to shortened life span and an aging-like degenerative phenotype in mice, and SIRT6 knockout cells exhibit genomic instability and DNA damage hypersensitivity. However, the molecular mechanisms underlying these defects are not fully understood. Here, we show that SIRT6 forms a macromolecular complex with the DNA double-strand break (DSB) repair factor DNA-PK (DNA-dependent protein kinase) and promotes DNA DSB repair. In response to DSBs, SIRT6 associates dynamically with chromatin and is necessary for an acute decrease in global cellular acetylation levels on histone H3 Lysine 9. Moreover, SIRT6 is required for mobilization of the DNA-PK catalytic subunit (DNA-PKcs) to chromatin in response to DNA damage and stabilizes DNA-PKcs at chromatin adjacent to an induced site-specific DSB. Abrogation of these SIRT6 activities leads to impaired resolution of DSBs. Together, these findings elucidate a mechanism whereby regulation of dynamic interaction of a DNA repair factor with chromatin impacts on the efficiency of repair, and establish a link between chromatin regulation, DNA repair, and a mammalian Sir2 factor. PMID:20157594

  3. siRNA targeting vaccinia virus double-stranded RNA binding protein [E3L] exerts potent antiviral effects.

    PubMed

    Dave, Rajnish S; McGettigan, James P; Qureshi, Tazeen; Schnell, Matthias J; Nunnari, Giuseppe; Pomerantz, Roger J

    2006-05-10

    The Vaccinia virus gene, E3L, encodes a double-stranded RNA [dsRNA]-binding protein. We hypothesized that, owing to the critical nature of dsRNA in triggering host innate antiviral responses, E3L-specific small-interfering RNAs [siRNAs] should be effective antiviral agents against pox viruses, for which Vaccinia virus is an appropriate surrogate. In this study, we have utilized two human cell types, namely, HeLa and 293T, one which responds to interferon [IFN]-beta and the other produces and responds to IFN-beta, respectively. The antiviral effects were equally robust in HeLa and 293T cells. However, in the case of 293T cells, several distinct features were observed, when IFN-beta is activated in these cells. Vaccinia virus replication was inhibited by 97% and 98% as compared to control infection in HeLa and 293T cells transfected with E3L-specific siRNAs, respectively. These studies demonstrate the utility of E3L-specific siRNAs as potent antiviral agents for small pox and related pox viruses.

  4. p53 binding protein 1 foci as a biomarker of DNA double strand breaks induced by ionizing radiation

    NASA Astrophysics Data System (ADS)

    Ng, C. K. M.; Wong, M. Y. P.; Lam, R. K. K.; Ho, J. P. Y.; Chiu, S. K.; Yu, K. N.

    2011-12-01

    Foci of p53 binding protein 1 (53 BP1) have been used as a biomarker of DNA double-strand breaks (DSBs) in cells induced by ionizing radiations. 53 BP1 was shown to relocalize into foci shortly after irradiation, with the number of foci closely paralleling the number of DNA DSBs. However, consensus on criteria in terms of the numbers of 53 BP1 foci to define cells damaged by direct irradiation or by bystander signals has not been reached, which is partly due to the presence of 53 BP1 also in normal cells. The objective of the present work was to study the changes in the distribution of cells with different numbers of 53 BP1 foci in a cell population after low-dose ionizing irradiation (<0.1 Gy) provided by alpha particles, with a view to propose feasible criteria for defining cells damaged by direct irradiation or by bystander signals. It was proposed that the change in the percentage of cells with 1-3 foci should be used for such purposes. The underlying reasons were discussed.

  5. A moonlighting metabolic protein influences repair at DNA double-stranded breaks

    PubMed Central

    Torres-Machorro, Ana Lilia; Aris, John P.; Pillus, Lorraine

    2015-01-01

    Catalytically active proteins with divergent dual functions are often described as ‘moonlighting’. In this work we characterize a new, chromatin-based function of Lys20, a moonlighting protein that is well known for its role in metabolism. Lys20 was initially described as homocitrate synthase (HCS), the first enzyme in the lysine biosynthetic pathway in yeast. Its nuclear localization led to the discovery of a key role for Lys20 in DNA damage repair through its interaction with the MYST family histone acetyltransferase Esa1. Overexpression of Lys20 promotes suppression of DNA damage sensitivity of esa1 mutants. In this work, by taking advantage of LYS20 mutants that are active in repair but not in lysine biosynthesis, the mechanism of suppression of esa1 was characterized. First we analyzed the chromatin landscape of esa1 cells, finding impaired histone acetylation and eviction. Lys20 was recruited to sites of DNA damage, and its overexpression promoted enhanced recruitment of the INO80 remodeling complex to restore normal histone eviction at the damage sites. This study improves understanding of the evolutionary, structural and biological relevance of independent activities in a moonlighting protein and links metabolism to DNA damage repair. PMID:25628362

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

    PubMed

    Nagarajan, G; Swami, Shelesh Kumar; Dahiya, Shyam Singh; Sivakumar, G; Tuteja, F C; Narnaware, S D; Mehta, S C; Singh, Raghvendar; Patil, N V

    2014-03-01

    The dsRNA binding protein (RBP) encoding gene of parapoxviruses (PPVs) from the Dromedary camels, inhabitating different geographical region of Rajasthan, India were amplified by polymerase chain reaction using the primers of pseudocowpoxvirus (PCPV) from Finnish reindeer and cloned into pGEM-T for sequence analysis. Analysis of RBP encoding gene revealed that PPV DNA from Bikaner shared 98.3% and 76.6% sequence identity at the amino acid level, with Pali and Udaipur PPV DNA, respectively. Reference strains of Bovine papular stomatitis virus (BPSV) and PCPV (reindeer PCPV and human PCPV) shared 52.8% and 86.9% amino acid identity with RBP gene of camel PPVs from Bikaner, respectively. But different strains of orf virus (ORFV) from different geographical areas of the world shared 69.5-71.7% amino acid identity with RBP gene of camel PPVs from Bikaner. These findings indicate that the camel PPVs described are closely related to bovine PPV (PCPV) in comparison to caprine and ovine PPV (ORFV). PMID:25685494

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

    SciTech Connect

    Chen, Shan-Shan; Jiang, Teng; Wang, Yi; Gu, Li-Ze; Wu, Hui-Wen; Tan, Lan; Guo, Jun

    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. Double stranded RNA-dependent protein kinase is involved in osteoclast differentiation of RAW264.7 cells in vitro

    SciTech Connect

    Teramachi, Junpei; Morimoto, Hiroyuki; Baba, Ryoko; Doi, Yoshiaki; Hirashima, Kanji; Haneji, Tatsuji

    2010-11-15

    Double-stranded RNA-dependent protein kinase (PKR) plays a critical role in antiviral defence of the host cells. PKR is also involved in cell cycle progression, cell proliferation, cell differentiation, tumorigenesis, and apoptosis. We previously reported that PKR is required for differentiation and calcification of osteoblasts. However, it is unknown about the role of PKR in osteoclast differentiation. A dominant-negative PKR mutant cDNA, in which the amino acid lysine at 296 was replaced with arginine, was transfected into RAW264.7 cells. We have established the cell line that stably expresses the PKR mutant gene (PKR-K/R). Phosphorylation of PKR and {alpha}-subunit of eukaryotic initiation factor 2 was not stimulated by polyinosic-polycytidylic acid in the PKR-K/R cells. RANKL stimulated the formation of TRAP-positive multinuclear cells in RAW264.7 cells. However, TRAP-positive multinuclear cells were not formed in the PKR-K/R cells even when the cells were stimulated with higher doses of RANKL. A specific inhibitor of PKR, 2-aminopurine, also suppressed the RANKL-induced osteoclast differentiation in RAW264.7 cells. The expression of macrophage fusion receptor and dendritic cell-specific transmembrane protein significantly decreased in the PKR-K/R cells by real time PCR analysis. The results of RT-PCR revealed that the mRNA expression of osteoclast markers (cathepsin K and calcitonin receptor) was suppressed in the PKR-K/R cells and RAW264.7 cells treated with 2-aminopurine. Expression of NF-{kappa}B protein was suppressed in the PKR-K/R cells and 2-aminopurine-treated RAW264.7 cells. The level of STAT1 protein expression was elevated in the PKR-K/R cells compared with that of the wild-type cells. Immunohistochemical study showed that PKR was localized in osteoclasts of metatarsal bone of newborn mouse. The finding that the PKR-positive multinuclear cells should be osteoclasts was confirmed by TRAP-staining. Our present study indicates that PKR plays important

  9. ATM protein is indispensable to repair complex-type DNA double strand breaks induced by high LET heavy ion irradiation.

    NASA Astrophysics Data System (ADS)

    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.

  10. Overexpression of the scaffold WD40 protein WRAP53β enhances the repair of and cell survival from DNA double-strand breaks.

    PubMed

    Rassoolzadeh, H; Böhm, S; Hedström, E; Gad, H; Helleday, T; Henriksson, S; Farnebo, M

    2016-01-01

    Altered expression of the multifunctional protein WRAP53β (WD40 encoding RNA Antisense to p53), which targets repair factors to DNA double-strand breaks and factors involved in telomere elongation to Cajal bodies, is linked to carcinogenesis. While loss of WRAP53β function has been shown to disrupt processes regulated by this protein, the consequences of its overexpression remain unclear. Here we demonstrate that overexpression of WRAP53β disrupts the formation of and impairs the localization of coilin to Cajal bodies. At the same time, the function of this protein in the repair of DNA double-strand breaks is enhanced. Following irradiation, cells overexpressing WRAP53β exhibit more rapid clearance of phospho-histone H2AX (γH2AX), and more efficient homologous recombination and non-homologous end-joining, in association with fewer DNA breaks. Moreover, in these cells the ubiquitylation of damaged chromatin, which is known to facilitate the recruitment of repair factors and subsequent repair, is elevated. Knockdown of the ubiquitin ligase involved, ring-finger protein 8 (RNF8), which is recruited to DNA breaks by WRAP53β, attenuated this effect, suggesting that overexpression of WRAP53β leads to more rapid repair, as well as improved cell survival, by enhancing RNF8-mediated ubiquitylation at DNA breaks. Our present findings indicate that WRAP53β and RNF8 are rate-limiting factors in the repair of DNA double-strand breaks and raise the possibility that upregulation of WRAP53β may contribute to genomic stability in and survival of cancer cells. PMID:27310875

  11. Overexpression of the scaffold WD40 protein WRAP53β enhances the repair of and cell survival from DNA double-strand breaks.

    PubMed

    Rassoolzadeh, H; Böhm, S; Hedström, E; Gad, H; Helleday, T; Henriksson, S; Farnebo, M

    2016-01-01

    Altered expression of the multifunctional protein WRAP53β (WD40 encoding RNA Antisense to p53), which targets repair factors to DNA double-strand breaks and factors involved in telomere elongation to Cajal bodies, is linked to carcinogenesis. While loss of WRAP53β function has been shown to disrupt processes regulated by this protein, the consequences of its overexpression remain unclear. Here we demonstrate that overexpression of WRAP53β disrupts the formation of and impairs the localization of coilin to Cajal bodies. At the same time, the function of this protein in the repair of DNA double-strand breaks is enhanced. Following irradiation, cells overexpressing WRAP53β exhibit more rapid clearance of phospho-histone H2AX (γH2AX), and more efficient homologous recombination and non-homologous end-joining, in association with fewer DNA breaks. Moreover, in these cells the ubiquitylation of damaged chromatin, which is known to facilitate the recruitment of repair factors and subsequent repair, is elevated. Knockdown of the ubiquitin ligase involved, ring-finger protein 8 (RNF8), which is recruited to DNA breaks by WRAP53β, attenuated this effect, suggesting that overexpression of WRAP53β leads to more rapid repair, as well as improved cell survival, by enhancing RNF8-mediated ubiquitylation at DNA breaks. Our present findings indicate that WRAP53β and RNF8 are rate-limiting factors in the repair of DNA double-strand breaks and raise the possibility that upregulation of WRAP53β may contribute to genomic stability in and survival of cancer cells.

  12. Massive parallel analysis of the binding specificity of histone-like protein HU to single- and double-stranded DNA with generic oligodeoxyribonucleotide microchips.

    SciTech Connect

    Krylov, A. S.; Zasedateleva, O. A.; Prokopenko, D. V.; Rouviere-Yaniv, J.; Mirzabekov, A. D.; Biochip Technology Center; Engelhardt Inst. of Molecular Biology; Inst. de Biologie Physico-Chimique

    2001-06-15

    A generic hexadeoxyribonucleotide microchip has been applied to test the DNA-binding properties of HU histone-like bacterial protein, which is known to have a low sequence specificity. All 4096 hexamers flanked within 8mers by degenerate bases at both the 3'- and 5'-ends were immobilized within the 100 x 100 x 20 mm polyacrylamide gel pads of the microchip. Single-stranded immobilized oligonucleotides were converted in some experiments to the double-stranded form by hybridization with a specified mixture of 8mers. The DNA interaction with HU was characterized by three type of measurements: (i) binding of FITC-labeled HU to microchip oligonucleotides; (ii) melting curves of complexes of labeled HU with single-stranded microchip oligonucleotides; (iii) the effect of HU binding on melting curves of microchip double-stranded DNA labeled with another fluorescent dye, Texas Red. Large numbers of measurements of these parameters were carried out in parallel for all or many generic microchip elements in real time with a multi-wavelength fluorescence microscope. Statistical analysis of these data suggests some preference for HU binding to G/C-rich single-stranded oligonucleotides. HU complexes with double-stranded microchip 8mers can be divided into two groups in which HU binding either increased the melting temperature (T{sub m}) of duplexes or decreased it. The stabilized duplexes showed some preference for presence of the sequence motifs AAG, AGA and AAGA. In the second type of complex, enriched with A/T base pairs, the destabilization effect was higher for longer stretches of A/T duplexes. Binding of HU to labeled duplexes in the second type of complex caused some decrease in fluorescence. This decrease also correlates with the higher A/T content and lower T{sub m}. The results demonstrate that generic microchips could be an efficient approach in analysis of sequence specificity of proteins.

  13. Creating Directed Double-strand Breaks with the Ref Protein: A Novel Rec A-Dependent Nuclease from Bacteriophage P1

    SciTech Connect

    Gruenig, Marielle C.; Lu, Duo; Won, Sang Joon; Dulberger, Charles L.; Manlick, Angela J.; Keck, James L.; Cox, Michael M.

    2012-03-16

    The bacteriophage P1-encoded Ref protein enhances RecA-dependent recombination in vivo by an unknown mechanism. We demonstrate that Ref is a new type of enzyme; that is, a RecA-dependent nuclease. Ref binds to ss- and dsDNA but does not cleave any DNA substrate until RecA protein and ATP are added to form RecA nucleoprotein filaments. Ref cleaves only where RecA protein is bound. RecA functions as a co-nuclease in the Ref/RecA system. Ref nuclease activity can be limited to the targeted strands of short RecA-containing D-loops. The result is a uniquely programmable endonuclease activity, producing targeted double-strand breaks at any chosen DNA sequence in an oligonucleotide-directed fashion. We present evidence indicating that cleavage occurs in the RecA filament groove. The structure of the Ref protein has been determined to 1.4 {angstrom} resolution. The core structure, consisting of residues 77-186, consists of a central 2-stranded {beta}-hairpin that is sandwiched between several {alpha}-helical and extended loop elements. The N-terminal 76 amino acid residues are disordered; this flexible region is required for optimal activity. The overall structure of Ref, including several putative active site histidine residues, defines a new subclass of HNH-family nucleases. We propose that enhancement of recombination by Ref reflects the introduction of directed, recombinogenic double-strand breaks.

  14. Creating directed double-strand breaks with the Ref protein: a novel RecA-dependent nuclease from bacteriophage P1.

    PubMed

    Gruenig, Marielle C; Lu, Duo; Won, Sang Joon; Dulberger, Charles L; Manlick, Angela J; Keck, James L; Cox, Michael M

    2011-03-11

    The bacteriophage P1-encoded Ref protein enhances RecA-dependent recombination in vivo by an unknown mechanism. We demonstrate that Ref is a new type of enzyme; that is, a RecA-dependent nuclease. Ref binds to ss- and dsDNA but does not cleave any DNA substrate until RecA protein and ATP are added to form RecA nucleoprotein filaments. Ref cleaves only where RecA protein is bound. RecA functions as a co-nuclease in the Ref/RecA system. Ref nuclease activity can be limited to the targeted strands of short RecA-containing D-loops. The result is a uniquely programmable endonuclease activity, producing targeted double-strand breaks at any chosen DNA sequence in an oligonucleotide-directed fashion. We present evidence indicating that cleavage occurs in the RecA filament groove. The structure of the Ref protein has been determined to 1.4 Å resolution. The core structure, consisting of residues 77-186, consists of a central 2-stranded β-hairpin that is sandwiched between several α-helical and extended loop elements. The N-terminal 76 amino acid residues are disordered; this flexible region is required for optimal activity. The overall structure of Ref, including several putative active site histidine residues, defines a new subclass of HNH-family nucleases. We propose that enhancement of recombination by Ref reflects the introduction of directed, recombinogenic double-strand breaks.

  15. Stress Granules Regulate Double-Stranded RNA-Dependent Protein Kinase Activation through a Complex Containing G3BP1 and Caprin1

    PubMed Central

    Reineke, Lucas C.; Kedersha, Nancy; Langereis, Martijn A.; van Kuppeveld, Frank J. M.

    2015-01-01

    ABSTRACT Stress granules (SGs) are dynamic cytoplasmic repositories containing translationally silenced mRNAs that assemble upon cellular stress. We recently reported that the SG nucleating protein G3BP1 promotes antiviral activity and is essential in double-stranded RNA-dependent protein kinase (PKR) recruitment to stress granules, thereby driving phosphorylation of the α subunit of eukaryotic initiation factor 2 (eIF2α). Here, we delineate the mechanism for SG-dependent PKR activation. We show that G3BP1 and inactive PKR directly interact with each other, dependent on both the NTF2-like and PXXP domains of G3BP1. The G3BP1-interacting protein Caprin1 also directly interacts with PKR, regulates efficient PKR activation at the stress granule, and is also integral for the release of active PKR into the cytoplasm to engage in substrate recognition. The G3BP1-Caprin1-PKR complex represents a new mode of PKR activation and is important for antiviral activity of G3BP1 and PKR during infection with mengovirus. Our data links stress responses and their resultant SGs with innate immune activation through PKR without a requirement for foreign double-stranded RNA (dsRNA) pattern recognition. PMID:25784705

  16. Distribution of reversing factor in reticulocyte lysates during active protein synthesis and on inhibition by heme deprivation or double-stranded RNA.

    PubMed Central

    Thomas, N S; Matts, R L; Petryshyn, R; London, I M

    1984-01-01

    We have recently shown a direct correlation between protein synthetic activity and the function of reversing factor (RF) as a catalyst of GDP-GTP exchange in whole reticulocyte lysates under normal conditions and on inhibition of protein synthesis by heme deficiency, double-stranded RNA, or oxidized glutathione. In this paper we report that RF is detectable as a nonribosomal complex with eukaryotic initiation factor 2 phosphorylated in its alpha subunit [eIF-2(alpha P)] in whole lysates inhibited by heme deprivation or by double-stranded RNA. The complex contains no unphosphorylated eIF-2 alpha, and the GDP present is freely dissociable. All nonribosomal eIF-2(alpha P) is complexed with RF in fully inhibited lysates; we have not detected free eIF-2(alpha P). RF in this [RF X eIF-2(alpha P)] complex is unavailable to catalyze the release of GDP from eIF-2-GDP. Dephosphorylation of eIF-2(alpha P) present in nonribosomal fractions releases active RF, which is able to carry out its normal guanine nucleotide exchange function. Images PMID:6594676

  17. Complex formation in yeast double-strand break repair: participation of Rad51, Rad52, Rad55, and Rad57 proteins.

    PubMed Central

    Hays, S L; Firmenich, A A; Berg, P

    1995-01-01

    The repair of DNA double-strand breaks in Saccharomyces cerevisiae requires genes of the RAD52 epistasis group, of which RAD55 and RAD57 are members. Here, we show that the x-ray sensitivity of rad55 and rad57 mutant strains is suppressible by overexpression of RAD51 or RAD52. Virtually complete suppression is provided by the simultaneous overexpression of RAD51 and RAD52. This suppression occurs at 23 degrees C, where these mutants are more sensitive to x-rays, as well as at 30 degrees C and 36 degrees C. In addition, a recombination defect of rad55 and rad57 mutants is similarly suppressed. Direct in vivo interactions between the Rad51 and Rad55 proteins, and between Rad55 and Rad57, have also been identified by using the two-hybrid system. These results indicate that these four proteins constitute part of a complex, a "recombinosome," to effect the recombinational repair of double-strand breaks. PMID:7624345

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

    PubMed

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

    2016-05-01

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

  19. Dynamics and Cell-Type Specificity of the DNA Double-Strand Break Repair Protein RecN in the Developmental Cyanobacterium Anabaena sp. Strain PCC 7120.

    PubMed

    Hu, Sheng; Wang, Jinglan; Wang, Li; Zhang, Cheng-Cai; Chen, Wen-Li

    2015-01-01

    DNA replication and repair are two fundamental processes required in life proliferation and cellular defense and some common proteins are involved in both processes. The filamentous cyanobacterium Anabaena sp. strain PCC 7120 is capable of forming heterocysts for N2 fixation in the absence of a combined-nitrogen source. This developmental process is intimately linked to cell cycle control. In this study, we investigated the localization of the DNA double-strand break repair protein RecN during key cellular events, such as chromosome damaging, cell division, and heterocyst differentiation. Treatment by a drug causing DNA double-strand breaks (DSBs) induced reorganization of the RecN focus preferentially towards the mid-cell position. RecN-GFP was absent in most mature heterocysts. Furthermore, our results showed that HetR, a central player in heterocyst development, was involved in the proper positioning and distribution of RecN-GFP. These results showed the dynamics of RecN in DSB repair and suggested a differential regulation of DNA DSB repair in vegetative cell and heterocysts. The absence of RecN in mature heterocysts is compatible with the terminal nature of these cells.

  20. Dynamics and Cell-Type Specificity of the DNA Double-Strand Break Repair Protein RecN in the Developmental Cyanobacterium Anabaena sp. Strain PCC 7120

    PubMed Central

    Hu, Sheng; Wang, Jinglan; Wang, Li; Zhang, Cheng-Cai; Chen, Wen-Li

    2015-01-01

    DNA replication and repair are two fundamental processes required in life proliferation and cellular defense and some common proteins are involved in both processes. The filamentous cyanobacterium Anabaena sp. strain PCC 7120 is capable of forming heterocysts for N2 fixation in the absence of a combined-nitrogen source. This developmental process is intimately linked to cell cycle control. In this study, we investigated the localization of the DNA double-strand break repair protein RecN during key cellular events, such as chromosome damaging, cell division, and heterocyst differentiation. Treatment by a drug causing DNA double-strand breaks (DSBs) induced reorganization of the RecN focus preferentially towards the mid-cell position. RecN-GFP was absent in most mature heterocysts. Furthermore, our results showed that HetR, a central player in heterocyst development, was involved in the proper positioning and distribution of RecN-GFP. These results showed the dynamics of RecN in DSB repair and suggested a differential regulation of DNA DSB repair in vegetative cell and heterocysts. The absence of RecN in mature heterocysts is compatible with the terminal nature of these cells. PMID:26431054

  1. Dynamics and Cell-Type Specificity of the DNA Double-Strand Break Repair Protein RecN in the Developmental Cyanobacterium Anabaena sp. Strain PCC 7120.

    PubMed

    Hu, Sheng; Wang, Jinglan; Wang, Li; Zhang, Cheng-Cai; Chen, Wen-Li

    2015-01-01

    DNA replication and repair are two fundamental processes required in life proliferation and cellular defense and some common proteins are involved in both processes. The filamentous cyanobacterium Anabaena sp. strain PCC 7120 is capable of forming heterocysts for N2 fixation in the absence of a combined-nitrogen source. This developmental process is intimately linked to cell cycle control. In this study, we investigated the localization of the DNA double-strand break repair protein RecN during key cellular events, such as chromosome damaging, cell division, and heterocyst differentiation. Treatment by a drug causing DNA double-strand breaks (DSBs) induced reorganization of the RecN focus preferentially towards the mid-cell position. RecN-GFP was absent in most mature heterocysts. Furthermore, our results showed that HetR, a central player in heterocyst development, was involved in the proper positioning and distribution of RecN-GFP. These results showed the dynamics of RecN in DSB repair and suggested a differential regulation of DNA DSB repair in vegetative cell and heterocysts. The absence of RecN in mature heterocysts is compatible with the terminal nature of these cells. PMID:26431054

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

    PubMed

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

    2016-05-01

    In this work, we retrace the evolutionary history of plant double-stranded RNA binding proteins (DRBs), a group of non-catalytic factors containing one or more double-stranded RNA binding motif (dsRBM) that play important roles in small RNA biogenesis and functions. Using a phylogenetic approach, we show that multiple dsRBM DRBs are systematically composed of two different types of dsRBMs evolving under different constraints and likely fulfilling complementary functions. In vascular plants, four distinct clades of multiple dsRBM DRBs are always present with the exception of Brassicaceae species, that do not possess member of the newly identified clade we named DRB6. We also identified a second new and highly conserved DRB family (we named DRB7) whose members possess a single dsRBM that shows concerted evolution with the most C-terminal dsRBM domain of the Dicer-like 4 (DCL4) proteins. Using a BiFC approach, we observed that Arabidopsis thaliana DRB7.2 (AtDRB7.2) can directly interact with AtDRB4 but not with AtDCL4 and we provide evidence that both AtDRB7.2 and AtDRB4 participate in the epigenetically activated siRNAs pathway. PMID:26858002

  3. Double-Stranded RNA-Induced Activation of Activating Protein-1 Promoter Is Differentially Regulated by the Non-structural Protein 1 of Avian Influenza A Viruses

    PubMed Central

    Zohari, Siamak; Belák, Sándor; Berg, Mikael

    2012-01-01

    Abstract Non-structural protein 1 (NS1) of influenza A viruses is a multifunctional protein that antagonizes the host immune response by interfering with several host signaling pathways. Based on putative amino acid sequences, NS1 proteins are categorized into two gene pools, allele A and allele B. Here we identified that allele A NS1 proteins of H6N8 and H4N6 are able to inhibit double-stranded RNA (dsRNA)-induced activating protein-1 (AP-1) promoter in cultured cell lines (human A549 and mink lung cells). Allele B NS1 proteins from corresponding subtypes of influenza A viruses are weak in this inhibition, despite significant levels of expression of each NS1 protein in human A549 cells. Furthermore, the capability to inhibit AP-1 promoter was mapped in the effector domain, since RNA binding domain alone lost its ability to inhibit this promoter activation. Chimeric forms of NS1 protein, composed of either RNA binding domain of allele A or B and effector domain of allele A or B, showed comparable inhibition to that of their wild-type NS1 proteins, or to the effector domain of corresponding NS1 proteins. Both alleles A and B NS1 proteins of H6N8 and H4N6 were expressed to significant levels, and were localized predominantly in the nucleus of human A549 cells. These results underscore the importance of the effector domain in inhibiting AP-1 promoter activation, and the biological function of the effector domain in stabilizing the RNA binding domain. Further, we revealed the versatile nature of NS1 in inhibiting the AP-1 transcription factor, in a manner dependent on allele type. Comprehensive studies, focusing on the molecular mechanisms behind this differential inhibition, may facilitate exploration of the zoonotic and pathogenic potential of influenza A viruses. PMID:22239235

  4. Mouse HORMAD1 is a meiosis i checkpoint protein that modulates DNA double- strand break repair during female meiosis.

    PubMed

    Shin, Yong-Hyun; McGuire, Megan M; Rajkovic, Aleksandar

    2013-08-01

    Oocytes in embryonic ovaries enter meiosis I and arrest in the diplonema stage. Perturbations in meiosis I, such as abnormal double-strand break (DSB) formation and repair, adversely affect oocyte survival. We previously discovered that HORMAD1 is a critical component of the synaptonemal complex but not essential for oocyte survival. No significant differences were observed in the number of primordial, primary, secondary, and developing follicles between wild-type and Hormad1(−/−)newborn, 8-day, and 80-day ovaries. Meiosis I progression in Hormad1(−/−) embryonic ovaries was normal through the zygotene stage and in oocytes arrested in diplonema; however, we did not visualize oocytes with completely synapsed chromosomes. We investigated effects of HORMAD1 deficiency on the kinetics of DNA DSB formation and repair in the mouse ovary. We irradiated Embryonic Day 16.5 wild-type and Hormad1(−/−) ovaries and monitored DSB repair using gammaH2AX, RAD51, and DMC1 immunofluorescence. Our results showed a significant drop in unrepaired DSBs in the irradiated Hormad1(−/−) zygotene oocytes as compared to the wild-type oocytes. Moreover, Hormad1 deficiency rescued Dmc1(−/−) oocytes. These results indicate that Hormad1 deficiency promotes DMC1-independent DSB repairs, which in turn helps asynaptic Hormad1(−/−) oocytes resist perinatal loss. PMID:23759310

  5. Protein kinase CK2 is required for the recruitment of 53BP1 to sites of DNA double-strand break induced by radiomimetic drugs.

    PubMed

    Guerra, Barbara; Iwabuchi, Kuniyoshi; Issinger, Olaf-Georg

    2014-04-01

    The ataxia telangiectasia mutated (ATM) signaling pathway responds rapidly to DNA double-strand breaks (DSBs) and it is characterized by recruitment of sensor, mediator, transducer and repair proteins to sites of DNA damage. Data suggest that CK2 is implicated in the early cellular response to DSBs. We demonstrate that CK2 binds constitutively the adaptor protein 53BP1 through the tandem Tudor domains and that the interaction is disrupted upon induction of DNA damage. Down-regulation of CK2 results in significant reduction of (i) 53BP1 foci formation, (ii) binding to dimethylated histone H4 and (iii) ATM autophosphorylation. Our data suggest that CK2 is required for 53BP1 accumulation at sites of DSBs which is a prerequisite for efficient activation of the ATM-mediated signaling pathway.

  6. Alignment of Homologous Chromosomes and Effective Repair of Programmed DNA Double-Strand Breaks during Mouse Meiosis Require the Minichromosome Maintenance Domain Containing 2 (MCMDC2) Protein

    PubMed Central

    Ravindranathan, Ramya; Dereli, Ihsan; Stanzione, Marcello; Tóth, Attila

    2016-01-01

    Orderly chromosome segregation during the first meiotic division requires meiotic recombination to form crossovers between homologous chromosomes (homologues). Members of the minichromosome maintenance (MCM) helicase family have been implicated in meiotic recombination. In addition, they have roles in initiation of DNA replication, DNA mismatch repair and mitotic DNA double-strand break repair. Here, we addressed the function of MCMDC2, an atypical yet conserved MCM protein, whose function in vertebrates has not been reported. While we did not find an important role for MCMDC2 in mitotically dividing cells, our work revealed that MCMDC2 is essential for fertility in both sexes due to a crucial function in meiotic recombination. Meiotic recombination begins with the introduction of DNA double-strand breaks into the genome. DNA ends at break sites are resected. The resultant 3-prime single-stranded DNA overhangs recruit RAD51 and DMC1 recombinases that promote the invasion of homologous duplex DNAs by the resected DNA ends. Multiple strand invasions on each chromosome promote the alignment of homologous chromosomes, which is a prerequisite for inter-homologue crossover formation during meiosis. We found that although DNA ends at break sites were evidently resected, and they recruited RAD51 and DMC1 recombinases, these recombinases were ineffective in promoting alignment of homologous chromosomes in the absence of MCMDC2. Consequently, RAD51 and DMC1 foci, which are thought to mark early recombination intermediates, were abnormally persistent in Mcmdc2-/- meiocytes. Importantly, the strand invasion stabilizing MSH4 protein, which marks more advanced recombination intermediates, did not efficiently form foci in Mcmdc2-/- meiocytes. Thus, our work suggests that MCMDC2 plays an important role in either the formation, or the stabilization, of DNA strand invasion events that promote homologue alignment and provide the basis for inter-homologue crossover formation during

  7. The telomeric protein TRF2 is critical for the protection of A549 cells from both telomere erosion and DNA double-strand breaks driven by salvicine.

    PubMed

    Zhang, Yong-Wei; Zhang, Zhi-Xiang; Miao, Ze-Hong; Ding, Jian

    2008-03-01

    Telomere repeat binding factor 2 (TRF2) has been increasingly recognized to be involved in DNA damage response and telomere maintenance. Our previous report found that salvicine (SAL), a novel topoisomerase II poison, elicited DNA double-strand breaks and telomere erosion in separate experimental systems. However, it remains to be clarified whether they share a common response to these two events and in particular whether TRF2 is involved in this process. In this study, we found that SAL concurrently induced DNA double-strand breaks, telomeric DNA damage, and telomere erosion in lung carcinoma A549 cells. It was unexpected to find that SAL led to disruption of TRF2, independently of either its transcription or proteasome-mediated degradation. By overexpressing the full-length trf2 gene and transfecting TRF2 small interfering RNAs, we showed that TRF2 protein protected both telomeric and genomic DNA from the SAL-elicited events. It is noteworthy that although both the Ataxia-telangiectasia-mutated (ATM) and the ATM- and Rad3-related (ATR) kinases responded to the SAL-induced DNA damages, only ATR was essential for the telomere erosion. The study also showed that the activated ATR augmented the SAL-triggered TRF2 disruption, whereas TRF2 reduction in turn enhanced ATR function. All of these findings suggest the emerging significance of TRF2 protecting both telomeric DNA and genomic DNA on the one hand and reveal the mutual modulation between ATR and TRF2 in sensing DNA damage signaling during cancer development on the other hand.

  8. Crystal structure of clustered regularly interspaced short palindromic repeats (CRISPR)-associated Csn2 protein revealed Ca2+-dependent double-stranded DNA binding activity.

    PubMed

    Nam, Ki Hyun; Kurinov, Igor; Ke, Ailong

    2011-09-01

    Clustered regularly interspaced short palindromic repeats (CRISPR) and their associated protein genes (cas genes) are widespread in bacteria and archaea. They form a line of RNA-based immunity to eradicate invading bacteriophages and malicious plasmids. A key molecular event during this process is the acquisition of new spacers into the CRISPR loci to guide the selective degradation of the matching foreign genetic elements. Csn2 is a Nmeni subtype-specific cas gene required for new spacer acquisition. Here we characterize the Enterococcus faecalis Csn2 protein as a double-stranded (ds-) DNA-binding protein and report its 2.7 Å tetrameric ring structure. The inner circle of the Csn2 tetrameric ring is ∼26 Å wide and populated with conserved lysine residues poised for nonspecific interactions with ds-DNA. Each Csn2 protomer contains an α/β domain and an α-helical domain; significant hinge motion was observed between these two domains. Ca(2+) was located at strategic positions in the oligomerization interface. We further showed that removal of Ca(2+) ions altered the oligomerization state of Csn2, which in turn severely decreased its affinity for ds-DNA. In summary, our results provided the first insight into the function of the Csn2 protein in CRISPR adaptation by revealing that it is a ds-DNA-binding protein functioning at the quaternary structure level and regulated by Ca(2+) ions.

  9. Crystal Structure of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated Csn2 Protein Revealed Ca[superscript 2+]-dependent Double-stranded DNA Binding Activity

    SciTech Connect

    Nam, Ki Hyun; Kurinov, Igor; Ke, Ailong

    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.

  10. Possible Involvement of the Double-Stranded RNA-Binding Core Protein ςA in the Resistance of Avian Reovirus to Interferon

    PubMed Central

    Martínez-Costas, José; González-López, Claudia; Vakharia, Vikram N.; Benavente, Javier

    2000-01-01

    Treatment of primary cultures of chicken embryo fibroblasts with a recombinant chicken alpha/beta interferon (rcIFN) induces an antiviral state that causes a strong inhibition of vaccinia virus and vesicular stomatitis virus replication but has no effect on avian reovirus S1133 replication. The fact that avian reovirus polypeptides are synthesized normally in rcIFN-treated cells prompted us to investigate whether this virus expresses factors that interfere with the activation and/or the activity of the IFN-induced, double-stranded RNA (dsRNA)-dependent enzymes. Our results demonstrate that extracts of avian-reovirus-infected cells, but not those of uninfected cells, are able to relieve the translation-inhibitory activity of dsRNA in reticulocyte lysates, by blocking the activation of the dsRNA-dependent enzymes. In addition, our results show that protein ςA, an S1133 core polypeptide, binds to dsRNA in an irreversible manner and that clearing this protein from extracts of infected cells abolishes their protranslational capacity. Taken together, our results raise the interesting possibility that protein ςA antagonizes the IFN-induced cellular response against avian reovirus by blocking the intracellular activation of enzyme pathways dependent on dsRNA, as has been suggested for several other viral dsRNA-binding proteins. PMID:10627522

  11. The Axial Element Protein DESYNAPTIC2 Mediates Meiotic Double-Strand Break Formation and Synaptonemal Complex Assembly in Maize[OPEN

    PubMed Central

    Kao, Yu-Hsin; Ku, Jia-Chi; Lin, Chien-Yu; Meeley, Robert

    2015-01-01

    During meiosis, homologous chromosomes pair and recombine via repair of programmed DNA double-strand breaks (DSBs). DSBs are formed in the context of chromatin loops, which are anchored to the proteinaceous axial element (AE). The AE later serves as a framework to assemble the synaptonemal complex (SC) that provides a transient but tight connection between homologous chromosomes. Here, we showed that DESYNAPTIC2 (DSY2), a coiled-coil protein, mediates DSB formation and is directly involved in SC assembly in maize (Zea mays). The dsy2 mutant exhibits homologous pairing defects, leading to sterility. Analyses revealed that DSB formation and the number of RADIATION SENSITIVE51 (RAD51) foci are largely reduced, and synapsis is completely abolished in dsy2 meiocytes. Super-resolution structured illumination microscopy showed that DSY2 is located on the AE and forms a distinct alternating pattern with the HORMA-domain protein ASYNAPTIC1 (ASY1). In the dsy2 mutant, localization of ASY1 is affected, and loading of the central element ZIPPER1 (ZYP1) is disrupted. Yeast two-hybrid and bimolecular fluorescence complementation experiments further demonstrated that ZYP1 interacts with DSY2 but does not interact with ASY1. Therefore, DSY2, an AE protein, not only mediates DSB formation but also bridges the AE and central element of SC during meiosis. PMID:26296964

  12. The RSF1 Histone-Remodelling Factor Facilitates DNA Double-Strand Break Repair by Recruiting Centromeric and Fanconi Anaemia Proteins

    PubMed Central

    Pessina, Fabio; Lowndes, Noel F.

    2014-01-01

    ATM is a central regulator of the cellular responses to DNA double-strand breaks (DSBs). Here we identify a biochemical interaction between ATM and RSF1 and we characterise the role of RSF1 in this response. The ATM–RSF1 interaction is dependent upon both DSBs and ATM kinase activity. Together with SNF2H/SMARCA5, RSF1 forms the RSF chromatin-remodelling complex. Although RSF1 is specific to the RSF complex, SNF2H/SMARCA5 is a catalytic subunit of several other chromatin-remodelling complexes. Although not required for checkpoint signalling, RSF1 is required for efficient repair of DSBs via both end-joining and homology-directed repair. Specifically, the ATM-dependent recruitment to sites of DSBs of the histone fold proteins CENPS/MHF1 and CENPX/MHF2, previously identified at centromeres, is RSF1-dependent. In turn these proteins recruit and regulate the mono-ubiquitination of the Fanconi Anaemia proteins FANCD2 and FANCI. We propose that by depositing CENPS/MHF1 and CENPX/MHF2, the RSF complex either directly or indirectly contributes to the reorganisation of chromatin around DSBs that is required for efficient DNA repair. PMID:24800743

  13. P53 Binding Protein 1 (53bp1) Is an Early Participant in the Cellular Response to DNA Double-Strand Breaks

    PubMed Central

    Schultz, Linda B.; Chehab, Nabil H.; Malikzay, Asra; Halazonetis, Thanos D.

    2000-01-01

    p53 binding protein 1 (53BP1), a protein proposed to function as a transcriptional coactivator of the p53 tumor suppressor, has BRCT domains with high homology to the Saccharomyces cerevisiae Rad9p DNA damage checkpoint protein. To examine whether 53BP1 has a role in the cellular response to DNA damage, we probed its intracellular localization by immunofluorescence. In untreated primary cells and U2OS osteosarcoma cells, 53BP1 exhibited diffuse nuclear staining; whereas, within 5–15 min after exposure to ionizing radiation (IR), 53BP1 localized at discreet nuclear foci. We propose that these foci represent sites of processing of DNA double-strand breaks (DSBs), because they were induced by IR and chemicals that cause DSBs, but not by ultraviolet light; their peak number approximated the number of DSBs induced by IR and decreased over time with kinetics that parallel the rate of DNA repair; and they colocalized with IR-induced Mre11/NBS and γ-H2AX foci, which have been previously shown to localize at sites of DSBs. Formation of 53BP1 foci after irradiation was not dependent on ataxia-telangiectasia mutated (ATM), Nijmegen breakage syndrome (NBS1), or wild-type p53. Thus, the fast kinetics of 53BP1 focus formation after irradiation and the lack of dependency on ATM and NBS1 suggest that 53BP1 functions early in the cellular response to DNA DSBs. PMID:11134068

  14. Loss of the catalytic subunit of the DNA-dependent protein kinase in DNA double-strand-break-repair mutant mammalian cells.

    PubMed

    Peterson, S R; Kurimasa, A; Oshimura, M; Dynan, W S; Bradbury, E M; Chen, D J

    1995-04-11

    The DNA-dependent protein kinase (DNA-PK) consists of three polypeptide components: Ku-70, Ku-80, and an approximately 350-kDa catalytic subunit (p350). The gene encoding the Ku-80 subunit is identical to the x-ray-sensitive group 5 complementing gene XRCC5. Expression of the Ku-80 cDNA rescues both DNA double-strand break (DSB) repair and V(D)J recombination in group 5 mutant cells. The involvement of Ku-80 in these processes suggests that the underlying defect in these mutant cells may be disruption of the DNA-PK holoenzyme. In this report we show that the p350 kinase subunit is deleted in cells derived from the severe combined immunodeficiency mouse and in the Chinese hamster ovary cell line V-3, both of which are defective in DSB repair and V(D)J recombination. A centromeric fragment of human chromosome 8 that complements the scid defect also restores p350 protein expression and rescues in vitro DNA-PK activity. These data suggest the scid gene may encode the p350 protein or regulate its expression and are consistent with a model whereby DNA-PK is a critical component of the DSB-repair pathway.

  15. Protein phosphatases pph3, ptc2, and ptc3 play redundant roles in DNA double-strand break repair by homologous recombination.

    PubMed

    Kim, Jung-Ae; Hicks, Wade M; Li, Jin; Tay, Sue Yen; Haber, James E

    2011-02-01

    In response to a DNA double-strand break (DSB), cells undergo a transient cell cycle arrest prior to mitosis until the break is repaired. In budding yeast (Saccharomyces cerevisiae), the DNA damage checkpoint is regulated by a signaling cascade of protein kinases, including Mec1 and Rad53. When DSB repair is complete, cells resume cell cycle progression (a process called "recovery") by turning off the checkpoint. Recovery involves two members of the protein phosphatase 2C (PP2C) family, Ptc2 and Ptc3, as well as the protein phosphatase 4 (PP4) enzyme, Pph3. Here, we demonstrate a new function of these three phosphatases in DSB repair. Cells lacking all three phosphatases Pph3, Ptc2, and Ptc3 exhibit synergistic sensitivities to the DNA-damaging agents camptothecin and methyl methanesulfonate, as well as hydroxyurea but not to UV light. Moreover, the simultaneous absence of Pph3, Ptc2, and Ptc3 results in defects in completing DSB repair, whereas neither single nor double deletion of the phosphatases causes a repair defect. Specifically, cells lacking all three phosphatases are defective in the repair-mediated DNA synthesis. Interestingly, the repair defect caused by the triple deletion of Pph3, Ptc2, and Ptc3 is most prominent when a DSB is slowly repaired and the DNA damage checkpoint is fully activated.

  16. Loss of the catalytic subunit of the DNA-dependent protein kinase in DNA double-strand-break-repair mutant mammalian cells

    SciTech Connect

    Peterson, S.R. |; Kurimasa, Akihiro; Oshimura, Mitsuo; Dynan, W.S.; Bradbury, E.M. |; Chen, D.J.

    1995-04-11

    The DNA-dependent protein kinase (DNA-PK) consists of three polypeptide components: Ku-70, Ku-80, and an {approx}350-kDa catalytic subunit (p350). The gene encoding the Ku-80 subunit is identical to the x-ray-sensitive group 5 complementing gene XRCC5. Expression of the Ku-80 cDNA rescues both DNA double-strand break (DSB) repair and V(D)J recombination in group 5 mutant cells. The involvement of Ku-80 in these processes suggests that the underlying defect in these mutant cells may be disruption of the DNA-PK holoenzyme. In this report we show that the p350 kinase subunit is deleted in cells derived from the severe combined immunodeficiency mouse and in the Chinese hamster ovary cell line V-3, both of which are defective in DSB repair and V(D)J recombination. A centromeric fragment of human chromosome 8 that complements the scid defect also restores p350 protein expression and rescues in vitro DNA-PK activity. These data suggest the scid gene may encode the p350 protein or regulate its expression and are consistent with a model whereby DNA-PK is a critical component of the DSB-repair pathway. 38 refs., 3 figs.

  17. XRCC4 and XLF form long helical protein filaments suitable for DNA end protection and alignment to facilitate DNA double strand break repair

    PubMed Central

    Mahaney, Brandi L.; Hammel, Michal; Meek, Katheryn; Tainer, John A.; Lees-Miller, Susan P.

    2013-01-01

    DNA double strand breaks (DSBs), induced by ionizing radiation (IR) and endogenous stress including replication failure, are the most cytotoxic form of DNA damage. In human cells, most IR-induced DSBs are repaired by the non-homologous end joining (NHEJ) pathway. One of the most critical steps in NHEJ is ligation of DNA ends by DNA ligase IV (LIG4), which interacts with, and is stabilized by, the scaffolding protein X-ray cross-complementing gene 4 (XRCC4). XRCC4 also interacts with XRCC4-like factor (XLF, also called Cernunnos); yet, XLF has been one of the least mechanistically understood proteins and precisely how XLF functions in NHEJ has been enigmatic. Here, we examine current combined structural and mutational findings that uncover integrated functions of XRCC4 and XLF and reveal their interactions to form long, helical protein filaments suitable to protect and align DSB ends. XLF-XRCC4 provides a global structural scaffold for ligating DSBs without requiring long complementary DNA ends, thus ensuring accurate and efficient ligation and repair. The assembly of these XRCC4-XLF filaments, providing both DNA end protection and alignment, may commit cells to NHEJ with general biological implications for NHEJ and DSB repair processes and their links to cancer predispositions and interventions. PMID:23442139

  18. Molecular architecture of tailed double-stranded DNA phages

    PubMed Central

    Fokine, Andrei; Rossmann, Michael G

    2014-01-01

    The tailed double-stranded DNA bacteriophages, or Caudovirales, constitute ~96% of all the known phages. Although these phages come in a great variety of sizes and morphology, their virions are mainly constructed of similar molecular building blocks via similar assembly pathways. Here we review the structure of tailed double-stranded DNA bacteriophages at a molecular level, emphasizing the structural similarity and common evolutionary origin of proteins that constitute these virions. PMID:24616838

  19. Mixed ligand copper(II) dicarboxylate complexes: the role of co-ligand hydrophobicity in DNA binding, double-strand DNA cleavage, protein binding and cytotoxicity.

    PubMed

    Loganathan, Rangasamy; Ramakrishnan, Sethu; Ganeshpandian, Mani; Bhuvanesh, Nattamai S P; Palaniandavar, Mallayan; Riyasdeen, Anvarbatcha; Akbarsha, Mohamad Abdulkadhar

    2015-06-14

    A few water soluble mixed ligand copper(ii) complexes of the type [Cu(bimda)(diimine)] , where bimda is N-benzyliminodiacetic acid and diimine is 2,2'-bipyridine (bpy, ) or 1,10-phenanthroline (phen, ) or 5,6-dimethyl-1,10-phenanthroline (5,6-dmp, ) or 3,4,7,8-tetramethyl-1,10-phenanthroline (3,4,7,8-tmp, ) and dipyrido[3,2-d: 2',3'-f]quinoxaline (dpq, ), have been successfully isolated and characterized by elemental analysis and other spectral techniques. The coordination geometry around copper(ii) in is described as distorted square based pyramidal while that in is described as square pyramidal. Absorption spectral titrations and competitive DNA binding studies reveal that the intrinsic DNA binding affinity of the complexes depends upon the diimine co-ligand, dpq () > 3,4,7,8-tmp () > 5,6-dmp () > phen () > bpy (). The phen and dpq co-ligands are involved in the π-stacking interaction with DNA base pairs while the 3,4,7,8-tmp/5,6-dmp and bpy co-ligands are involved in respectively hydrophobic and surface mode of binding with DNA. The small enhancement in the relative viscosity of DNA upon binding to supports the DNA binding modes proposed. Interestingly, and are selective in exhibiting a positive induced CD band (ICD) upon binding to DNA suggesting that they induce B to A conformational change. In contrast, and show CD responses which reveal their involvement in strong DNA binding. The complexes are unique in displaying prominent double-strand DNA cleavage while effects only single-strand DNA cleavage, and their ability to cleave DNA in the absence of an activator varies as > > > > . Also, all the complexes exhibit oxidative double-strand DNA cleavage activity in the presence of ascorbic acid, which varies as > > > > . The ability of the complexes to bind and cleave the protein BSA varies in the order > > > > . Interestingly, and cleave the protein non-specifically in the presence of H2O2 as an activator suggesting that they can act also as chemical proteases

  20. Mixed ligand copper(II) dicarboxylate complexes: the role of co-ligand hydrophobicity in DNA binding, double-strand DNA cleavage, protein binding and cytotoxicity.

    PubMed

    Loganathan, Rangasamy; Ramakrishnan, Sethu; Ganeshpandian, Mani; Bhuvanesh, Nattamai S P; Palaniandavar, Mallayan; Riyasdeen, Anvarbatcha; Akbarsha, Mohamad Abdulkadhar

    2015-06-14

    A few water soluble mixed ligand copper(ii) complexes of the type [Cu(bimda)(diimine)] , where bimda is N-benzyliminodiacetic acid and diimine is 2,2'-bipyridine (bpy, ) or 1,10-phenanthroline (phen, ) or 5,6-dimethyl-1,10-phenanthroline (5,6-dmp, ) or 3,4,7,8-tetramethyl-1,10-phenanthroline (3,4,7,8-tmp, ) and dipyrido[3,2-d: 2',3'-f]quinoxaline (dpq, ), have been successfully isolated and characterized by elemental analysis and other spectral techniques. The coordination geometry around copper(ii) in is described as distorted square based pyramidal while that in is described as square pyramidal. Absorption spectral titrations and competitive DNA binding studies reveal that the intrinsic DNA binding affinity of the complexes depends upon the diimine co-ligand, dpq () > 3,4,7,8-tmp () > 5,6-dmp () > phen () > bpy (). The phen and dpq co-ligands are involved in the π-stacking interaction with DNA base pairs while the 3,4,7,8-tmp/5,6-dmp and bpy co-ligands are involved in respectively hydrophobic and surface mode of binding with DNA. The small enhancement in the relative viscosity of DNA upon binding to supports the DNA binding modes proposed. Interestingly, and are selective in exhibiting a positive induced CD band (ICD) upon binding to DNA suggesting that they induce B to A conformational change. In contrast, and show CD responses which reveal their involvement in strong DNA binding. The complexes are unique in displaying prominent double-strand DNA cleavage while effects only single-strand DNA cleavage, and their ability to cleave DNA in the absence of an activator varies as > > > > . Also, all the complexes exhibit oxidative double-strand DNA cleavage activity in the presence of ascorbic acid, which varies as > > > > . The ability of the complexes to bind and cleave the protein BSA varies in the order > > > > . Interestingly, and cleave the protein non-specifically in the presence of H2O2 as an activator suggesting that they can act also as chemical proteases

  1. A novel protein, Rsf1/Pxd1, is critical for the single-strand annealing pathway of double-strand break repair in Schizosaccharomyces pombe.

    PubMed

    Wang, Hanqian; Zhang, Zhanlu; Zhang, Lan; Zhang, Qiuxue; Zhang, Liang; Zhao, Yangmin; Wang, Weibu; Fan, Yunliu; Wang, Lei

    2015-06-01

    The process of single-strand annealing (SSA) repairs DNA double-strand breaks that are flanked by direct repeat sequences through the coordinated actions of a series of proteins implicated in recombination, mismatch repair and nucleotide excision repair (NER). Many of the molecular and mechanistic insights gained in SSA repair have principally come from studies in the budding yeast Saccharomyces cerevisiae. However, there is little molecular understanding of the SSA pathway in the fission yeast Schizosaccharomyces pombe. To further our understanding of this important process, we established a new chromosome-based SSA assay in fission yeast. Our genetic analyses showed that, although many homologous components participate in SSA repair in these species indicating that some evolutionary conservation, Saw1 and Slx4 are not principal agents in the SSA repair pathway in fission yeast. This is in marked contrast to the function of Saw1 and Slx4 in budding yeast. Additionally, a novel genus-specific protein, Rsf1/Pxd1, physically interacts with Rad16, Swi10 and Saw1 in vitro and in vivo. We find that Rsf1/Pxd1 is not required for NER and demonstrate that, in fission yeast, Rsf1/Pxd1, but not Saw1, plays a critical role in SSA recombination.

  2. Ebola virus VP35 protein binds double-stranded RNA and inhibits alpha/beta interferon production induced by RIG-I signaling.

    PubMed

    Cárdenas, Washington B; Loo, Yueh-Ming; Gale, Michael; Hartman, Amy L; Kimberlin, Christopher R; Martínez-Sobrido, Luis; Saphire, Erica Ollmann; Basler, Christopher F

    2006-06-01

    The Ebola virus (EBOV) VP35 protein blocks the virus-induced phosphorylation and activation of interferon regulatory factor 3 (IRF-3), a transcription factor critical for the induction of alpha/beta interferon (IFN-alpha/beta) expression. However, the mechanism(s) by which this blockage occurs remains incompletely defined. We now provide evidence that VP35 possesses double-stranded RNA (dsRNA)-binding activity. Specifically, VP35 bound to poly(rI) . poly(rC)-coated Sepharose beads but not control beads. In contrast, two VP35 point mutants, R312A and K309A, were found to be greatly impaired in their dsRNA-binding activity. Competition assays showed that VP35 interacted specifically with poly(rI) . poly(rC), poly(rA) . poly(rU), or in vitro-transcribed dsRNAs derived from EBOV sequences, and not with single-stranded RNAs (ssRNAs) or double-stranded DNA. We then screened wild-type and mutant VP35s for their ability to target different components of the signaling pathways that activate IRF-3. These experiments indicate that VP35 blocks activation of IRF-3 induced by overexpression of RIG-I, a cellular helicase recently implicated in the activation of IRF-3 by either virus or dsRNA. Interestingly, the VP35 mutants impaired for dsRNA binding have a decreased but measurable IFN antagonist activity in these assays. Additionally, wild-type and dsRNA-binding-mutant VP35s were found to have equivalent abilities to inhibit activation of the IFN-beta promoter induced by overexpression of IPS-1, a recently identified signaling molecule downstream of RIG-I, or by overexpression of the IRF-3 kinases IKKepsilon and TBK-1. These data support the hypothesis that dsRNA binding may contribute to VP35 IFN antagonist function. However, additional mechanisms of inhibition, at a point proximal to the IRF-3 kinases, most likely also exist.

  3. Kinetics and thermodynamics of salt-dependent T7 gene 2.5 protein binding to single- and double-stranded DNA

    PubMed Central

    Shokri, Leila; Marintcheva, Boriana; Eldib, Mootaz; Hanke, Andreas; Williams, Mark C.

    2008-01-01

    Bacteriophage T7 gene 2.5 protein (gp2.5) is a single-stranded DNA (ssDNA)-binding protein that has essential roles in DNA replication, recombination and repair. However, it differs from other ssDNA-binding proteins by its weaker binding to ssDNA and lack of cooperative ssDNA binding. By studying the rate-dependent DNA melting force in the presence of gp2.5 and its deletion mutant lacking 26 C-terminal residues, we probe the kinetics and thermodynamics of gp2.5 binding to ssDNA and double-stranded DNA (dsDNA). These force measurements allow us to determine the binding rate of both proteins to ssDNA, as well as their equilibrium association constants to dsDNA. The salt dependence of dsDNA binding parallels that of ssDNA binding. We attribute the four orders of magnitude salt-independent differences between ssDNA and dsDNA binding to nonelectrostatic interactions involved only in ssDNA binding, in contrast to T4 gene 32 protein, which achieves preferential ssDNA binding primarily through cooperative interactions. The results support a model in which dimerization interactions must be broken for DNA binding, and gp2.5 monomers search dsDNA by 1D diffusion to bind ssDNA. We also quantitatively compare the salt-dependent ssDNA- and dsDNA-binding properties of the T4 and T7 ssDNA-binding proteins for the first time. PMID:18772224

  4. Serines 440 and 467 in the Werner syndrome protein are phosphorylated by DNA-PK and affects its dynamics in response to DNA double strand breaks.

    PubMed

    Kusumoto-Matsuo, Rika; Ghosh, Deblina; Karmakar, Parimal; May, Alfred; Ramsden, Dale; Bohr, Vilhelm A

    2014-01-01

    WRN protein, defective in Werner syndrome (WS), a human segmental progeria, is a target of serine/threonine kinases involved in sensing DNA damage. DNA-PK phosphorylates WRN in response to DNA double strand breaks (DSBs). However, the main phosphorylation sites and functional importance of the phosphorylation of WRN has remained unclear. Here, we identify Ser-440 and -467 in WRN as major phosphorylation sites mediated by DNA-PK.In vitro, DNA-PK fails to phosphorylate a GST-WRN fragment with S440A and/or S467A substitution. In addition, full length WRN with the mutation expressed in 293T cells was not phosphorylated in response to DSBs produced by bleomycin. Accumulation of the mutant WRN at the site of laser-induced DSBs occurred with the same kinetics as wild type WRN in live HeLa cells. While the wild type WRN relocalized to the nucleoli after 24 hours recovery from etoposide-induced DSBs, the mutant WRN remained mostly in the nucleoplasm. Consistent with this, WS cells expressing the mutants exhibited less DNA repair efficiency and more sensitivity to etoposide, compared to those expressing wild type. Our findings indicate that phosphorylation of Ser-440 and -467 in WRN are important for relocalization of WRN to nucleoli, and that it is required for efficient DSB repair.

  5. Role of the yeast DNA repair protein Nej1 in end processing during the repair of DNA double strand breaks by non-homologous end joining

    PubMed Central

    Yang, Hui; Matsumoto, Yoshihiro; Trujillo, Kelly M.; Lees-Miller, Susan P.; Osley, Mary Ann; Tomkinson, Alan E.

    2016-01-01

    DNA double strand breaks (DSB)s often require end processing prior to joining during their repair by non-homologous end joining (NHEJ). Although the yeast proteins, Pol4, a Pol X family DNA polymerase, and Rad27, a nuclease, participate in the end processing reactions of NHEJ, the mechanisms underlying the recruitment of these factors to DSBs are not known. Here we demonstrate that Nej1, a NHEJ factor that interacts with and modulates the activity of the NHEJ DNA ligase complex (Dnl4/Lif1), physically and functionally interacts with both Pol4 and Rad27. Notably, Nej1 and Dnl4/Lif1, which also interacts with both Pol4 and Rad27, independently recruit the end processing factors to in vivo DSBs via mechanisms that are additive rather than redundant. As was observed with Dnl4/Lif1, the activities of both Pol4 and Rad27 were enhanced by the interaction with Nej1. Furthermore, Nej1 increased the joining of incompatible DNA ends in reconstituted reactions containing Pol4, Rad27 and Dnl4/Lif1, indicating that the stimulatory activities of Nej1 and Dnl4/Lif1 are also additive. Together our results reveal novel roles for Nej1 in the recruitment of Pol4 and Rad27 to in vivo DSBs and the coordination of the end processing and ligation reactions of NHEJ. PMID:25942368

  6. The proteomic investigation reveals interaction of mdig protein with the machinery of DNA double-strand break repair.

    PubMed

    Wang, Wei; Lu, Yongju; Stemmer, Paul M; Zhang, Xiangmin; Bi, Yongyi; Yi, Zhengping; Chen, Fei

    2015-09-29

    To investigate how mineral dust-induced gene (mdig, also named as mina53, MINA, or NO52) promotes carcinogenesis through inducing active chromatin, we performed proteomics analyses for the interacting proteins that were co-immunoprecipitated by anti-mdig antibody from either the lung cancer cell line A549 cells or the human bronchial epithelial cell line BEAS-2B cells. On SDS-PAGE gels, three to five unique protein bands were consistently observed in the complexes pulled-down by mdig antibody, but not the control IgG. In addition to the mdig protein, several DNA repair or chromatin binding proteins, including XRCC5, XRCC6, RBBP4, CBX8, PRMT5, and TDRD, were identified in the complexes by the proteomics analyses using both Orbitrap Fusion and Orbitrap XL nanoESI-MS/MS in four independent experiments. The interaction of mdig with some of these proteins was further validated by co-immunoprecipitation using antibodies against mdig and its partner proteins, respectively. These data, thus, provide evidence suggesting that mdig accomplishes its functions on chromatin, DNA repair and cell growth through interacting with the partner proteins. PMID:26293673

  7. The proteomic investigation reveals interaction of mdig protein with the machinery of DNA double-strand break repair.

    PubMed

    Wang, Wei; Lu, Yongju; Stemmer, Paul M; Zhang, Xiangmin; Bi, Yongyi; Yi, Zhengping; Chen, Fei

    2015-09-29

    To investigate how mineral dust-induced gene (mdig, also named as mina53, MINA, or NO52) promotes carcinogenesis through inducing active chromatin, we performed proteomics analyses for the interacting proteins that were co-immunoprecipitated by anti-mdig antibody from either the lung cancer cell line A549 cells or the human bronchial epithelial cell line BEAS-2B cells. On SDS-PAGE gels, three to five unique protein bands were consistently observed in the complexes pulled-down by mdig antibody, but not the control IgG. In addition to the mdig protein, several DNA repair or chromatin binding proteins, including XRCC5, XRCC6, RBBP4, CBX8, PRMT5, and TDRD, were identified in the complexes by the proteomics analyses using both Orbitrap Fusion and Orbitrap XL nanoESI-MS/MS in four independent experiments. The interaction of mdig with some of these proteins was further validated by co-immunoprecipitation using antibodies against mdig and its partner proteins, respectively. These data, thus, provide evidence suggesting that mdig accomplishes its functions on chromatin, DNA repair and cell growth through interacting with the partner proteins.

  8. Mutational Analysis of Vaccinia Virus E3 Protein: the Biological Functions Do Not Correlate with Its Biochemical Capacity To Bind Double-Stranded RNA

    PubMed Central

    Dueck, Kevin J.; Hu, YuanShen (Sandy); Chen, Peter; Deschambault, Yvon; Lee, Jocelyn; Varga, Jessie

    2015-01-01

    ABSTRACT Vaccinia E3 protein has the biochemical capacity of binding to double-stranded RNA (dsRNA). The best characterized biological functions of the E3 protein include its host range function, suppression of cytokine expression, and inhibition of interferon (IFN)-induced antiviral activity. Currently, the role of the dsRNA binding capacity in the biological functions of the E3 protein is not clear. To further understand the mechanism of the E3 protein biological functions, we performed alanine scanning of the entire dsRNA binding domain of the E3 protein to examine the link between its biochemical capacity of dsRNA binding and biological functions. Of the 115 mutants examined, 20 were defective in dsRNA binding. Although the majority of the mutants defective in dsRNA binding also showed defective replication in HeLa cells, nine mutants (I105A, Y125A, E138A, F148A, F159A, K171A, L182A, L183A, and I187/188A) retained the host range function to various degrees. Further examination of a set of representative E3L mutants showed that residues essential for dsRNA binding are not essential for the biological functions of E3 protein, such as inhibition of protein kinase R (PKR) activation, suppression of cytokine expression, and apoptosis. Thus, data described in this communication strongly indicate the E3 protein performs its biological functions via a novel mechanism which does not correlate with its dsRNA binding activity. IMPORTANCE dsRNAs produced during virus replication are important pathogen-associated molecular patterns (PAMPs) for inducing antiviral immune responses. One of the strategies used by many viruses to counteract such antiviral immune responses is achieved by producing dsRNA binding proteins, such as poxvirus E3 family proteins, influenza virus NS1, and Ebola virus V35 proteins. The most widely accepted model for the biological functions of this class of viral dsRNA binding proteins is that they bind to and sequester viral dsRNA PAMPs; thus, they

  9. Label-Free and Separation-Free Atomic Fluorescence Spectrometry-Based Bioassay: Sensitive Determination of Single-Strand DNA, Protein, and Double-Strand DNA.

    PubMed

    Chen, Piaopiao; Wu, Peng; Chen, Junbo; Yang, Peng; Zhang, Xinfeng; Zheng, Chengbin; Hou, Xiandeng

    2016-02-16

    Based on selective and sensitive determination of Hg(2+) released from mercury complex by cold vapor generation (CVG) atomic fluorescence spectrometry (AFS) using SnCl2 as a reductant, a novel label-free and separation-free strategy was proposed for DNA and protein bioassay. To construct the DNA bioassay platform, an Hg(2+)-mediated molecular beacon (hairpin) without labeling but possessing several thymine (T) bases at both ends was employed as the probe. It is well-known that Hg(2+) could trigger the formation of the hairpin structure through T-Hg(2+)-T connection. In the presence of a specific target, the hairpin structure could be broken and the captured Hg(2+) was released. Interestingly, it was found that SnCl2 could selectively reduce only free Hg(2+) to Hg(0) vapor in the presence of T-Hg(2+)-T complex, which could be separated from sample matrices for sensitive AFS detection. Three different types of analyte, namely, single-strand DNA (ssDNA), protein, and double-strand DNA (dsDNA), were investigated as the target analytes. Under the optimized conditions, this bioassay provided high sensitivity for ssDNA, protein, and dsDNA determination with the limits of detection as low as 0.2, 0.08, and 0.3 nM and the linear dynamic ranges of 10-150, 5-175, and 1-250 nM, respectively. The analytical performance for these analytes compares favorably with those by previously reported methods, demonstrating the potential usefulness and versatility of this new AFS-based bioassay. Moreover, the bioassay retains advantages of simplicity, cost-effectiveness, and sensitivity compared to most of the conventional methods. PMID:26781421

  10. Label-Free and Separation-Free Atomic Fluorescence Spectrometry-Based Bioassay: Sensitive Determination of Single-Strand DNA, Protein, and Double-Strand DNA.

    PubMed

    Chen, Piaopiao; Wu, Peng; Chen, Junbo; Yang, Peng; Zhang, Xinfeng; Zheng, Chengbin; Hou, Xiandeng

    2016-02-16

    Based on selective and sensitive determination of Hg(2+) released from mercury complex by cold vapor generation (CVG) atomic fluorescence spectrometry (AFS) using SnCl2 as a reductant, a novel label-free and separation-free strategy was proposed for DNA and protein bioassay. To construct the DNA bioassay platform, an Hg(2+)-mediated molecular beacon (hairpin) without labeling but possessing several thymine (T) bases at both ends was employed as the probe. It is well-known that Hg(2+) could trigger the formation of the hairpin structure through T-Hg(2+)-T connection. In the presence of a specific target, the hairpin structure could be broken and the captured Hg(2+) was released. Interestingly, it was found that SnCl2 could selectively reduce only free Hg(2+) to Hg(0) vapor in the presence of T-Hg(2+)-T complex, which could be separated from sample matrices for sensitive AFS detection. Three different types of analyte, namely, single-strand DNA (ssDNA), protein, and double-strand DNA (dsDNA), were investigated as the target analytes. Under the optimized conditions, this bioassay provided high sensitivity for ssDNA, protein, and dsDNA determination with the limits of detection as low as 0.2, 0.08, and 0.3 nM and the linear dynamic ranges of 10-150, 5-175, and 1-250 nM, respectively. The analytical performance for these analytes compares favorably with those by previously reported methods, demonstrating the potential usefulness and versatility of this new AFS-based bioassay. Moreover, the bioassay retains advantages of simplicity, cost-effectiveness, and sensitivity compared to most of the conventional methods.

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

    SciTech Connect

    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.

  12. Novel Bat Influenza Virus NS1 Proteins Bind Double-Stranded RNA and Antagonize Host Innate Immunity.

    PubMed

    Turkington, Hannah L; Juozapaitis, Mindaugas; Kerry, Philip S; Aydillo, Teresa; Ayllon, Juan; García-Sastre, Adolfo; Schwemmle, Martin; Hale, Benjamin G

    2015-10-01

    We demonstrate that novel bat HL17NL10 and HL18NL11 influenza virus NS1 proteins are effective interferon antagonists but do not block general host gene expression. Solving the RNA-binding domain structures revealed the canonical NS1 symmetrical homodimer, and RNA binding required conserved basic residues in this domain. Interferon antagonism was strictly dependent on RNA binding, and chimeric bat influenza viruses expressing NS1s defective in this activity were highly attenuated in interferon-competent cells but not in cells unable to establish antiviral immunity.

  13. Novel Bat Influenza Virus NS1 Proteins Bind Double-Stranded RNA and Antagonize Host Innate Immunity

    PubMed Central

    Turkington, Hannah L.; Juozapaitis, Mindaugas; Kerry, Philip S.; Aydillo, Teresa; Ayllon, Juan; García-Sastre, Adolfo

    2015-01-01

    We demonstrate that novel bat HL17NL10 and HL18NL11 influenza virus NS1 proteins are effective interferon antagonists but do not block general host gene expression. Solving the RNA-binding domain structures revealed the canonical NS1 symmetrical homodimer, and RNA binding required conserved basic residues in this domain. Interferon antagonism was strictly dependent on RNA binding, and chimeric bat influenza viruses expressing NS1s defective in this activity were highly attenuated in interferon-competent cells but not in cells unable to establish antiviral immunity. PMID:26246567

  14. Electrochemically modified carbon and chromium surfaces for AFM imaging of double-strand DNA interaction with transposase protein.

    PubMed

    Esnault, Charles; Chénais, Benoît; Casse, Nathalie; Delorme, Nicolas; Louarn, Guy; Pilard, Jean-François

    2013-02-01

    Carbon and chromium surfaces were modified by electrochemical reduction of a diazonium salt formed in situ from the sulfanilic acid. The organic layer formed was activated by phosphorus pentachloride (PCl(5)) to form a benzene sulfonil chloride (Ar-SO(2)Cl). An electrochemical study of the blocking effect and the activity of this surface was carried out on a carbon electrode. The chromium surface study was completed by X-ray photoelectron spectroscopy and atomic force microscopy to characterize the formation of a compact monolayer (0.8 nm height and roughness 0.2-0.3 nm). The compactness and the activity of this organic monolayer allowed us to affix a length dsDNA with the aim of analyzing the formation of a complex between dsDNA and a protein. The interaction of a transposase protein with its target dsDNA was investigated. The direct imaging of the nucleoproteic complex considered herein gives new insights in the comprehension of transposase-DNA interaction in agreement with biochemical data.

  15. Structural basis of nucleic-acid recognition and double-strand unwinding by the essential neuronal protein Pur-alpha.

    PubMed

    Weber, Janine; Bao, Han; Hartlmüller, Christoph; Wang, Zhiqin; Windhager, Almut; Janowski, Robert; Madl, Tobias; Jin, Peng; Niessing, Dierk

    2016-01-01

    The neuronal DNA-/RNA-binding protein Pur-alpha is a transcription regulator and core factor for mRNA localization. Pur-alpha-deficient mice die after birth with pleiotropic neuronal defects. Here, we report the crystal structure of the DNA-/RNA-binding domain of Pur-alpha in complex with ssDNA. It reveals base-specific recognition and offers a molecular explanation for the effect of point mutations in the 5q31.3 microdeletion syndrome. Consistent with the crystal structure, biochemical and NMR data indicate that Pur-alpha binds DNA and RNA in the same way, suggesting binding modes for tri- and hexanucleotide-repeat RNAs in two neurodegenerative RNAopathies. Additionally, structure-based in vitro experiments resolved the molecular mechanism of Pur-alpha's unwindase activity. Complementing in vivo analyses in Drosophila demonstrated the importance of a highly conserved phenylalanine for Pur-alpha's unwinding and neuroprotective function. By uncovering the molecular mechanisms of nucleic-acid binding, this study contributes to understanding the cellular role of Pur-alpha and its implications in neurodegenerative diseases. PMID:26744780

  16. Structural basis of nucleic-acid recognition and double-strand unwinding by the essential neuronal protein Pur-alpha

    PubMed Central

    Weber, Janine; Bao, Han; Hartlmüller, Christoph; Wang, Zhiqin; Windhager, Almut; Janowski, Robert; Madl, Tobias; Jin, Peng; Niessing, Dierk

    2016-01-01

    The neuronal DNA-/RNA-binding protein Pur-alpha is a transcription regulator and core factor for mRNA localization. Pur-alpha-deficient mice die after birth with pleiotropic neuronal defects. Here, we report the crystal structure of the DNA-/RNA-binding domain of Pur-alpha in complex with ssDNA. It reveals base-specific recognition and offers a molecular explanation for the effect of point mutations in the 5q31.3 microdeletion syndrome. Consistent with the crystal structure, biochemical and NMR data indicate that Pur-alpha binds DNA and RNA in the same way, suggesting binding modes for tri- and hexanucleotide-repeat RNAs in two neurodegenerative RNAopathies. Additionally, structure-based in vitro experiments resolved the molecular mechanism of Pur-alpha's unwindase activity. Complementing in vivo analyses in Drosophila demonstrated the importance of a highly conserved phenylalanine for Pur-alpha's unwinding and neuroprotective function. By uncovering the molecular mechanisms of nucleic-acid binding, this study contributes to understanding the cellular role of Pur-alpha and its implications in neurodegenerative diseases. DOI: http://dx.doi.org/10.7554/eLife.11297.001 PMID:26744780

  17. DNA Methyltransferase 1-associated Protein (DMAP1) Is a Co-repressor That Stimulates DNA Methylation Globally and Locally at Sites of Double Strand Break Repair*

    PubMed Central

    Lee, Gun E.; Kim, Joo Hee; Taylor, Michael; Muller, Mark T.

    2010-01-01

    Correction of double strand DNA breaks proceeds in an error-free pathway of homologous recombination (HR), which can result in gene silencing of half of the DNA molecules caused by action by DNA methyltransferase 1 (DNMT1) (Cuozzo, C., Porcellini, A., Angrisano, T., Morano, A., Lee, B., Di Pardo, A., Messina, S., Iuliano, R., Fusco, A., Santillo, M. R., Muller, M. T., Chiariotti, L., Gottesman, M. E., and Avvedimento, E. V. (2007) PLoS Genet. 3, e110). To explore the mechanism that leads to HR-induced silencing, a genetic screen was carried out based on the silencing of a GFP reporter to identify potential partners. DMAP1, a DNMT1 interacting protein, was identified as a mediator of this process. DMAP1 is a potent activator of DNMT1 methylation in vitro, suggesting that DMAP1 is a co-repressor that supports the maintenance and de novo action of DNMT1. To examine critical roles for DMAP1 in vivo, lentiviral shRNA was used to conditionally reduce cellular DMAP1 levels. The shRNA transduced cells grew poorly and eventually ceased their growth. Analysis of the tumor suppressor gene p16 methylation status revealed a clear reduction in methylated CpGs in the shRNA cells, suggesting that reactivation of a tumor suppressor gene pathway caused the slow growth phenotype. Analysis of HR, using a fluorescence-based reporter, revealed that knocking down DMAP1 also caused hypomethylation of the DNA repair products following gene conversion. DMAP1 was selectively enriched in recombinant GFP chromatin based on chromatin immunoprecipitation analysis. The picture that emerges is that DMAP1 activates DNMT1 preferentially at sites of HR repair. Because DMAP1 depleted cells display enhanced HR, we conclude that it has additional roles in genomic stability. PMID:20864525

  18. DNA methyltransferase 1-associated protein (DMAP1) is a co-repressor that stimulates DNA methylation globally and locally at sites of double strand break repair.

    PubMed

    Lee, Gun E; Kim, Joo Hee; Taylor, Michael; Muller, Mark T

    2010-11-26

    Correction of double strand DNA breaks proceeds in an error-free pathway of homologous recombination (HR), which can result in gene silencing of half of the DNA molecules caused by action by DNA methyltransferase 1 (DNMT1) (Cuozzo, C., Porcellini, A., Angrisano, T., Morano, A., Lee, B., Di Pardo, A., Messina, S., Iuliano, R., Fusco, A., Santillo, M. R., Muller, M. T., Chiariotti, L., Gottesman, M. E., and Avvedimento, E. V. (2007) PLoS Genet. 3, e110). To explore the mechanism that leads to HR-induced silencing, a genetic screen was carried out based on the silencing of a GFP reporter to identify potential partners. DMAP1, a DNMT1 interacting protein, was identified as a mediator of this process. DMAP1 is a potent activator of DNMT1 methylation in vitro, suggesting that DMAP1 is a co-repressor that supports the maintenance and de novo action of DNMT1. To examine critical roles for DMAP1 in vivo, lentiviral shRNA was used to conditionally reduce cellular DMAP1 levels. The shRNA transduced cells grew poorly and eventually ceased their growth. Analysis of the tumor suppressor gene p16 methylation status revealed a clear reduction in methylated CpGs in the shRNA cells, suggesting that reactivation of a tumor suppressor gene pathway caused the slow growth phenotype. Analysis of HR, using a fluorescence-based reporter, revealed that knocking down DMAP1 also caused hypomethylation of the DNA repair products following gene conversion. DMAP1 was selectively enriched in recombinant GFP chromatin based on chromatin immunoprecipitation analysis. The picture that emerges is that DMAP1 activates DNMT1 preferentially at sites of HR repair. Because DMAP1 depleted cells display enhanced HR, we conclude that it has additional roles in genomic stability. PMID:20864525

  19. Double Stranded RNA-Dependent Protein Kinase is Necessary for TNF-α-Induced Osteoclast Formation In Vitro and In Vivo.

    PubMed

    Shinohara, Hiroki; Teramachi, Jumpei; Okamura, Hirohiko; Yang, Di; Nagata, Toshihiko; Haneji, Tatsuji

    2015-09-01

    Double-stranded RNA-dependent protein kinase (PKR) is involved in cell cycle progression, cell proliferation, cell differentiation, tumorgenesis, and apoptosis. We previously reported that PKR is required for differentiation and calcification in osteoblasts. TNF-α plays a key role in osteoclast differentiation. However, it is unknown about the roles of PKR in the TNF-α-induced osteoclast differentiation. The expression of PKR in osteoclast precursor RAW264.7 cells increased during TNF-α-induced osteoclastogenesis. The TNF-α-induced osteoclast differentiation in bone marrow-derived macrophages and RAW264.7 cells was markedly suppressed by the pretreatment of PKR inhibitor, 2-aminopurine (2AP), as well as gene silencing of PKR. The expression of gene markers in the differentiated osteoclasts including TRAP, Calcitonin receptor, cathepsin K, and ATP6V0d2 was also suppressed by the 2AP treatment. Bone resorption activity of TNF-α-induced osteoclasts was also supressed by 2AP treatment. Inhibition of PKR supressed the TNF-α-induced activation of NF-κB and MAPK in RAW264.7 cells. 2AP inhibited both the nuclear translocation of NF-κB and its transcriptional activity in RAW264.7 cells. 2AP inhibited the TNF-α-induced expression of NFATc1 and c-fos, master transcription factors in osteoclastogenesis. TNF-α-induced nuclear translocation of NFATc1 in mature osteoclasts was clearly inhibited by the 2AP treatment. The PKR inhibitor C16 decreased the TNF-α-induced osteoclast formation and bone resorption in mouse calvaria. The present study indicates that PKR is necessary for the TNF-α-induced osteoclast differentiation in vitro and in vivo.

  20. Poly(ADP-Ribose) Polymerase-1 and DNA-Dependent Protein Kinase Have Equivalent Roles in Double Strand Break Repair Following Ionizing Radiation

    SciTech Connect

    Mitchell, Jody; Smith, Graeme; Curtin, Nicola J.

    2009-12-01

    Purpose: Radiation-induced DNA double strand breaks (DSBs) are predominantly repaired by nonhomologous end joining (NHEJ), involving DNA-dependent protein kinase (DNA-PK). Poly(ADP-ribose) polymerase-1 (PARP-1), well characterized for its role in single strand break repair, may also facilitate DSB repair. We investigated the activation of these enzymes by differing DNA ends and their interaction in the cellular response to ionizing radiation (IR). Methods and Materials: The effect of PARP and DNA-PK inhibitors (KU-0058684 and NU7441) on repair of IR-induced DSBs was investigated in DNA-PK and PARP-1 proficient and deficient cells by measuring gammaH2AX foci and neutral comets. Complementary in vitro enzyme kinetics assays demonstrated the affinities of DNA-PK and PARP-1 for DSBs with varying DNA termini. Results: DNA-PK and PARP-1 both promoted the fast phase of resolution of IR-induced DSBs in cells. Inactivation of both enzymes was not additive, suggesting that PARP-1 and DNA-PK cooperate within the same pathway to promote DSB repair. The affinities of the two enzymes for oligonucleotides with blunt, 3' GGG or 5' GGG overhanging termini were similar and overlapping (K{sub dapp} = 2.6-6.4nM for DNA-PK; 1.7-4.5nM for PARP-1). DNA-PK showed a slightly greater affinity for overhanging DNA and was significantly more efficient when activated by a 5' GGG overhang. PARP-1 had a preference for blunt-ended DNA and required a separate factor for efficient stimulation by a 5' GGG overhang. Conclusion: DNA-PK and PARP-1 are both required in a pathway facilitating the fast phase of DNA DSB repair.

  1. Coordinateendonucleolytic 5' and 3' trimming of terminally blocked blunt DNA double-strand break ends by Artemis nuclease and DNA-dependent protein kinase

    SciTech Connect

    Povirk, Lawrence; Yannone, Steven M.; Khan, Imran S.; Zhou, Rui-Zhe; Zhou, Tong; Valerie, Kristoffer; F., Lawrence

    2008-02-18

    Previous work showed that, in the presence of DNA-PK, Artemis slowly trims 3'-phosphoglycolate-terminated blunt ends. To examine the trimming reaction in more detail, long internally labeled DNA substrates were treated with Artemis. In the absence of DNA-PK, Artemis catalyzed extensive 5' {yields} 3' exonucleolytic resection of double-stranded DNA. This resection required a 5'-phosphate but did not require ATP, and was accompanied by endonucleolytic cleavage of the resulting 3' overhang. In the presence of DNA-PK, Artemis-mediated trimming was more limited, was ATP-dependent, and did not require a 5'-phosphate. For a blunt end with either a 3'-phosphoglycolate or 3'-hydroxyl terminus, endonucleolytic trimming of 2-4 nucleotides from the 3'-terminal strand was accompanied by trimming of 6 nucleotides from the 5'-terminal strand. The results suggest that autophosphorylated DNA-PK suppresses the exonuclease activity of Artemis toward blunt-ended DNA, and promotes slow and limited endonucleolytic trimming of the 5'-terminal strand, resulting in short 3' overhangs that are trimmed endonucleolytically. Thus, Artemis and DNA-PK can convert terminally blocked DNA ends of diverse geometry and chemical structure to a form suitable for polymerase mediated patching and ligation, with minimal loss of terminal sequence. Such processing could account for the very small deletions often found at DNA double-strand break repair sites.

  2. hnRNP-U is a specific DNA-dependent protein kinase substrate phosphorylated in response to DNA double-strand breaks

    SciTech Connect

    Berglund, Fredrik M.; Clarke, Paul R.

    2009-03-27

    Cellular responses to DNA damage are orchestrated by the large phosphoinositol-3-kinase related kinases ATM, ATR and DNA-PK. We have developed a cell-free system to dissect the biochemical mechanisms of these kinases. Using this system, we identify heterogeneous nuclear ribonucleoprotein U (hnRNP-U), also termed scaffold attachment factor A (SAF-A), as a specific substrate for DNA-PK. We show that hnRNP-U is phosphorylated at Ser59 by DNA-PK in vitro and in cells in response to DNA double-strand breaks. Phosphorylation of hnRNP-U suggests novel functions for DNA-PK in the response to DNA damage.

  3. The mitotic DNA damage checkpoint proteins Rad17 and Rad24 are required for repair of double-strand breaks during meiosis in yeast.

    PubMed Central

    Shinohara, Miki; Sakai, Kazuko; Ogawa, Tomoko; Shinohara, Akira

    2003-01-01

    We show here that deletion of the DNA damage checkpoint genes RAD17 and RAD24 in Saccharomyces cerevisiae delays repair of meiotic double-strand breaks (DSBs) and results in an altered ratio of crossover-to-noncrossover products. These mutations also decrease the colocalization of immunostaining foci of the RecA homologs Rad51 and Dmc1 and cause a delay in the disappearance of Rad51 foci, but not of Dmc1. These observations imply that RAD17 and RAD24 promote efficient repair of meiotic DSBs by facilitating proper assembly of the meiotic recombination complex containing Rad51. Consistent with this proposal, extra copies of RAD51 and RAD54 substantially suppress not only the spore inviability of the rad24 mutant, but also the gamma-ray sensitivity of the mutant. Unexpectedly, the entry into meiosis I (metaphase I) is delayed in the checkpoint single mutants compared to wild type. The control of the cell cycle in response to meiotic DSBs is also discussed. PMID:12871899

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

    PubMed

    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

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

    PubMed

    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.

  6. The protein ORF80 from the acidophilic and thermophilic archaeon Sulfolobus islandicus binds highly site-specifically to double-stranded DNA and represents a novel type of basic leucine zipper protein

    PubMed Central

    Lipps, Georg; Ibanez, Pablo; Stroessenreuther, Thomas; Hekimian, Katya; Krauss, Gerhard

    2001-01-01

    The cryptic high copy number plasmid pRN1 from the thermophilic and acidophilic crenarchaeote Sulfolobus islandicus shares three conserved open reading frames with other S.islandicus plasmids. One of the open reading frames, namely orf80, encodes a 9.5 kDa protein that has no homology to any characterised protein. Recombinant ORF80 purified from Escherichia coli binds to double-stranded DNA in a sequence-specific manner as suggested by EMSA experiments and DNase I footprints. Two highly symmetrical binding sites separated by ∼60 bp were found upstream of the orf80 gene. Both binding sites contain two TTAA motifs as well as other conserved bases. Fluorescence measurements show that short duplex DNAs derived from a single binding site sequence are bound with submicromolar affinity and moderate cooperativity by ORF80. On DNA fragments carrying both binding sites, a rather large protein–DNA complex is formed in a highly cooperative manner. ORF80 contains an N-terminal leucine zipper motif and a highly basic domain at its C-terminus. Compared to all known basic leucine zipper proteins the order of the domains is reversed in ORF80. ORF80 may therefore constitute a new subclass of basic leucine zipper DNA-binding proteins. PMID:11812827

  7. Double-strand break-induced targeted mutagenesis in plants.

    PubMed

    Lyznik, L Alexander; Djukanovic, Vesna; Yang, Meizhu; Jones, Spencer

    2012-01-01

    Double-strand breaks are very potent inducers of DNA recombination. There is no recombination between DNA molecules unless one or two DNA strands are broken. It has become feasible to introduce double-strand breaks at specific chromosomal loci by using dedicated, redesigned endonucleases with altered DNA-binding specificities. Such breaks are mainly repaired by error-prone nonhomologous recombination pathways in somatic cells, thus frequently producing mutations at the preselected chromosomal sites. Although the art and science of reengineering protein properties have been advancing quickly, an empirical validation of new endonucleases in a particular experimental environment is essential for successful targeted mutagenesis experiments. This chapter presents methods that were developed for a comprehensive evaluation of the DNA-binding and DNA-cutting activities of homing endonucleases in maize cells; however, they can be adopted for similar evaluation studies of other endonucleases and other plant species that are amenable for Agrobacterium-mediated transformation. PMID:22351025

  8. In vitro binding kinetics of DNA double strand break repair proteins Ku70/80 and DNA-PKcs quantified by fluorescence correlation spectroscopy and fluorescence cross-correlation spectroscopy

    NASA Astrophysics Data System (ADS)

    Abdisalaam, Salim; Chen, David J.; Alexandrakis, George

    2012-02-01

    DNA double-strand breaks (DSBs) are one of the most lethal types of DNA damage that occurs in eukaryotic cells. There are two distinct pathways of repairing DSBs, homologous recombination (HR) and non-homologous end joining (NHEJ). In the NHEJ repairing pathway, DSB recognition and repair initiation is directed by the interaction of DNAbinding subunit Ku70/80 heterodimer with the DNA-PK protein catalytic subunit (DNA-PKcs). Mutations in these proteins result in repair stalling and eventual DNA misrepair that may lead to genomic instability. Studying the binding kinetics of these repair proteins is therefore important for understanding the conditions under which DSB repair stalls. Currently open questions are, what is the minimum DNA length that this complex needs to get a foothold onto a DSB and how tightly does DNA-PKcs bind onto the DNA-Ku70/80 complex. Fluorescence Correlation Spectroscopy (FCS) and Fluorescence Cross-Correlation Spectroscopy (FCCS) techniques have the potential to give information about the binding kinetics of DNA-protein and protein-protein interactions at the single-molecule level. In this work, FCS/FCCS measurements were performed to explore the minimum DNA base-pair (bp) length that Ku70/80 needed as a foothold to bind effectively onto the tips of different lengths of double-stranded DNA (dsDNA) fragments that mimic DSBs. 25 bp, 33 bp and 50 bp of dsDNA were used for these experiments and binding was studied as a function of salt concentration in solution. It was found that the 25 bp binding was weak even at physiological salt concentrations while the dissociation constant (Kd) remained constant for 33 and 50 bp dsDNA strand lengths. These studies indicated that the minimum binding length for the Ku70/8 is in the vicinity of 25 bp. The specificity of binding of Ku70/80 was proven by competitive binding FCCS experiments between Cy5-labeled DNA, GFP-Ku70/80 and titrations of unlabeled Ku70/80. Finally, using FCCS it was possible to estimate

  9. Phosphorylation of H2AX histone as indirect evidence for double-stranded DNA breaks related to the exchange of nuclear proteins and chromatin remodeling in Chara vulgaris spermiogenesis.

    PubMed

    Wojtczak, A; Popłońska, K; Kwiatkowska, M

    2008-11-01

    Phosphorylation of H2AX histone results not only from DNA damage (caused by ionizing radiation, UV or chemical substances, e.g. hydroxyurea), but also regularly takes place during spermiogenesis, enabling correct chromatin remodeling. Immunocytochemical analysis using antibodies against H2AX histone phosphorylated at serine 139 indirectly revealed endogenous double-stranded DNA breaks in Chara vulgaris spermatids in mid-spermiogenesis (stages V, VI and VII), when protamine-type proteins appear in the nucleus. Fluorescent foci were not observed in early (stages I-IV) and late (VIII-X) spermiogenesis, after replacement of histones by protamine-type proteins was finished. A similar phenomenon exists in animals. Determination of the localization of fluorescent foci and the ultrastructure of nuclei led to the hypothesis that DNA breaks at stage V, when condensed chromatin adheres to the nuclear envelope. This is transformed into a net-like structure during stage VI, probably allowing chromosome repositioning to specific regions in the mature spermatozoid. However, at stages VI and VII, DNA breaks are necessary for transformation of the nucleosomal structure into a fibrillar and finally the extremely condensed status of sleeping genes at stage X.

  10. Mutations of the Huntington's disease protein impact on the ATM-dependent signaling and repair pathways of the radiation-induced DNA double-strand breaks: corrective effect of statins and bisphosphonates.

    PubMed

    Ferlazzo, Mélanie L; Sonzogni, Laurène; Granzotto, Adeline; Bodgi, Larry; Lartin, Océane; Devic, Clément; Vogin, Guillaume; Pereira, Sandrine; Foray, Nicolas

    2014-06-01

    Huntington's disease (HD) is a neurodegenerative syndrome caused by mutations of the IT15 gene encoding for the huntingtin protein. Some research groups have previously shown that HD is associated with cellular radiosensitivity in quiescent cells. However, there is still no mechanistic model explaining such specific clinical feature. Here, we examined the ATM-dependent signaling and repair pathways of the DNA double-strand breaks (DSB), the key damage induced by ionizing radiation, in human HD skin fibroblasts. Early after irradiation, quiescent HD fibroblasts showed an abnormally low rate of recognized DSB managed by non-homologous end-joining reflected by a low yield of nuclear foci formed by phosphorylated H2AX histones and by 53BP1 protein. Furthermore, HD cells elicited a significant but moderate yield of unrepaired DSB 24 h after irradiation. Irradiated HD cells also presented a delayed nucleo-shuttling of phosphorylated forms of the ATM kinase, potentially due to a specific binding of ATM to mutated huntingtin in the cytoplasm. Our results suggest that HD belongs to the group of syndromes associated with a low but significant defect of DSB signaling and repair defect associated with radiosensitivity. A combination of biphosphonates and statins complements these impairments by facilitating the nucleo-shuttling of ATM, increasing the yield of recognized and repaired DSB. PMID:24277524

  11. The deinococcal DdrB protein is involved in an early step of DNA double strand break repair and in plasmid transformation through its single-strand annealing activity

    PubMed Central

    de la Tour, Claire Bouthier; Boisnard, Stéphanie; Norais, Cédric; Toueille, Magali; Bentchikou, Esma; Vannier, Françoise; Cox, Michael M.; Sommer, Suzanne; Servant, Pascale

    2012-01-01

    The Deinococcus radiodurans bacterium exhibits an extreme resistance to ionizing radiation. Here, we investigated the in vivo role of DdrB, a radiation-induced Deinococcus specific protein that was previously shown to exhibit some in vitro properties akin to those of SSB protein from E. coli but also to promote annealing of single stranded DNA. First we report that the deletion of the C-terminal motif of the DdrB protein, which is similar to the SSB C-terminal motif involved in recruitment to DNA of repair proteins, did neither affect cell radioresistance nor DNA binding properties of purified DdrB protein. We show that, in spite of their different quaternary structure, DdrB and SSB occlude the same amount of ssDNA in vitro. We also showed that DdrB is recruited early and transiently after irradiation into the nucleoid to form discrete foci. Absence of DdrB increased the lag phase of the extended synthesis-dependent strand annealing (ESDSA) process, affecting neither the rate of DNA synthesis nor the efficiency of fragment reassembly, as indicated by monitoring DNA synthesis and genome reconstitution in cells exposed to a sub-lethal ionizing radiation dose. Moreover, cells devoid of DdrB were affected in the establishment of plasmid DNA during natural transformation, a process that requires pairing of internalized plasmid single stranded DNA fragments, whereas they were proficient in transformation by a chromosomal DNA marker that integrates into the host chromosome through homologous recombination. Our data are consistent with a model in which DdrB participates in an early step of DNA double strand break repair in cells exposed to very high radiation doses. DdrB might facilitate the accurate assembly of the myriad of small fragments generated by extreme radiation exposure through a single strand annealing (SSA) process to generate suitable substrates for subsequent ESDSA-promoted genome reconstitution. PMID:21968057

  12. Visualization of DNA double-strand break repair in live bacteria reveals dynamic recruitment of Bacillus subtilis RecF, RecO and RecN proteins to distinct sites on the nucleoids.

    PubMed

    Kidane, Dawit; Sanchez, Humberto; Alonso, Juan C; Graumann, Peter L

    2004-06-01

    We have found that SMC-like RecN protein, RecF and RecO proteins that are involved in DNA recombination play an important role in DNA double-strand break (DSB) repair in Bacillus subtilis. Upon induction of DNA DSBs, RecN, RecO and RecF localized as a discrete focus on the nucleoids in a majority of cells, whereas two or three foci were rarely observed. RecN, RecO and RecF co-localized to the induced foci, with RecN localizing first, while RecO localized later, followed by RecF. Thus, three repair proteins were differentially recruited to distinct sites on the nucleoids, potentially constituting active DSB repair centres (RCs). RecF did not form regular foci in the absence of RecN and failed to form any foci in recO cells, demonstrating a central role for RecN and RecO in initializing the formation of RCs. RecN/O/F foci were detected in recA, recG or recU mutant cells, indicating that the proteins act upstream of proteins involved in synapsis or post-synapsis. In the absence of exogenous DNA damage, RCs were rare, but they accumulated in recA and recU cells, suggesting that DSBs occur frequently in the absence of RecA or RecU. The results suggest a model in which RecN that forms multimers in solution and high-molecular-weight complexes in cells containing DSBs initiates the formation of RCs that mediate DSB repair with the homologous sister chromosome, which presents a novel concept for DSB repair in prokaryotes. PMID:15186413

  13. Binding Study of T7 Gene 2.5 Protein to Single- and Double--Stranded DNA from Single Molecule Stretching

    NASA Astrophysics Data System (ADS)

    Shokri, Leila; Marintcheva, Boriana; Richardson, Charles C.; Williams, Mark C.

    2006-03-01

    Bacteriophage T7 gene 2.5 protein binds preferentially to single-stranded DNA. This property is essential for its role in DNA replication, recombination, and repair. We present the first quantitative study of the thermodynamics and kinetics of equilibrium and non-equilibrium DNA helix destabilization in the presence of gp2.5 and a deletion mutant lacking 26 C-terminal amino acids that binds with higher affinity to ssDNA (gp2.5-delta26C). Our measured force-extension curves of lambda-DNA in the presence of these proteins suggest strong cooperative binding. By measuring the DNA melting force as a function of time and pulling rate, we obtained binding site size and the association constants of these proteins to ssDNA and dsDNA, over a range of salt and protein concentrations. The results are used to characterize the electrostatic interactions that determine the DNA-protein binding in each case.

  14. Production of HIV Particles Is Regulated by Altering Sub-Cellular Localization and Dynamics of Rev Induced by Double-Strand RNA Binding Protein

    PubMed Central

    Urcuqui-Inchima, Silvio; Patiño, Claudia; Zapata, Ximena; García, María Patricia; Arteaga, José; Chamot, Christophe; Kumar, Ajit; Hernandez-Verdun, Danièle

    2011-01-01

    Human immunodeficiency virus (HIV)-1 encoded Rev is essential for export from the nucleus to the cytoplasm, of unspliced and singly spliced transcripts coding for structural and nonstructural viral proteins. This process is spatially and temporally coordinated resulting from the interactions between cellular and viral proteins. Here we examined the effects of the sub-cellular localization and dynamics of Rev on the efficiency of nucleocytoplasmic transport of HIV-1 Gag transcripts and virus particle production. Using confocal microscopy and fluorescence recovery after bleaching (FRAP), we report that NF90ctv, a cellular protein involved in Rev function, alters both the sub-cellular localization and dynamics of Rev in vivo, which drastically affects the accumulation of the viral protein p24. The CRM1–dependent nuclear export of Gag mRNA linked to the Rev Response Element (RRE) is dependent on specific domains of the NF90ctv protein. Taken together, our results demonstrate that the appropriate intracellular localization and dynamics of Rev could regulate Gag assembly and HIV-1 replication. PMID:21364984

  15. Entropy in DNA Double-Strand Break, Detection and Signaling

    NASA Astrophysics Data System (ADS)

    Zhang, Yang; Schindler, Christina; Heermann, Dieter

    2014-03-01

    In biology, the term entropy is often understood as a measure of disorder - a restrictive interpretation that can even be misleading. Recently it has become clearer and clearer that entropy, contrary to conventional wisdom, can help to order and guide biological processes in living cells. DNA double-strand breaks (DSBs) are among the most dangerous lesions and efficient damage detection and repair is essential for organism viability. However, what remains unknown is the precise mechanism of targeting the site of damage within billions of intact nucleotides and a crowded nuclear environment, a process which is often referred to as recruitment or signaling. Here we show that the change in entropy associated with inflicting a DSB facilitates the recruitment of damage sensor proteins. By means of computational modeling we found that higher mobility and local chromatin structure accelerate protein association at DSB ends. We compared the effect of different chromatin architectures on protein dynamics and concentrations in the vicinity of DSBs, and related these results to experiments on repair in heterochromatin. Our results demonstrate how entropy contributes to a more efficient damage detection. We identify entropy as the physical basis for DNA double-strand break signaling.

  16. Structural polymorphism of the major capsid protein of a double-stranded RNA virus: an amphipathic alpha helix as a molecular switch.

    PubMed

    Saugar, Irene; Luque, Daniel; Oña, Ana; Rodríguez, José F; Carrascosa, José L; Trus, Benes L; Castón, José R

    2005-07-01

    The infectious bursal disease virus T=13 viral particle is composed of two major proteins, VP2 and VP3. Here, we show that the molecular basis of the conformational flexibility of the major capsid protein precursor, pVP2, is an amphipatic alpha helix formed by the sequence GFKDIIRAIR. VP2 containing this alpha helix is able to assemble into the T=13 capsid only when expressed as a chimeric protein with an N-terminal His tag. An amphiphilic alpha helix, which acts as a conformational switch, is thus responsible for the inherent structural polymorphism of VP2. The His tag mimics the VP3 C-terminal region closely and acts as a molecular triggering factor. Using cryo-electron microscopy difference imaging, both polypeptide elements were detected on the capsid inner surface. We propose that electrostatic interactions between these two morphogenic elements are transmitted to VP2 to acquire the competent conformations for capsid assembly.

  17. Interaction of Ddc1 and RPA with single-stranded/double-stranded DNA junctions in yeast whole cell extracts: Proteolytic degradation of the large subunit of replication protein A in ddc1Δ strains.

    PubMed

    Sukhanova, Maria V; D'Herin, Claudine; Boiteux, Serge; Lavrik, Olga I

    2014-10-01

    To characterize proteins that interact with single-stranded/double-stranded (ss/ds) DNA junctions in whole cell free extracts of Saccharomyces cerevisiae, we used [(32)P]-labeled photoreactive partial DNA duplexes containing a 3'-ss/ds-junction (3'-junction) or a 5'-ss/ds-junction (5'-junction). Identification of labeled proteins was achieved by MALDI-TOF mass spectrometry peptide mass fingerprinting and genetic analysis. In wild-type extract, one of the components of the Ddc1-Rad17-Mec3 complex, Ddc1, was found to be preferentially photocrosslinked at a 3'-junction. On the other hand, RPAp70, the large subunit of the replication protein A (RPA), was the predominant crosslinking product at a 5'-junction. Interestingly, ddc1Δ extracts did not display photocrosslinking of RPAp70 at a 5'-junction. The results show that RPAp70 crosslinked to DNA with a 5'-junction is subject to limited proteolysis in ddc1Δ extracts, whereas it is stable in WT, rad17Δ, mec3Δ and mec1Δ extracts. The degradation of the RPAp70-DNA adduct in ddc1Δ extract is strongly reduced in the presence of the proteasome inhibitor MG 132. We also addressed the question of the stability of free RPA, using anti-RPA antibodies. The results show that RPAp70 is also subject to proteolysis without photocrosslinking to DNA upon incubation in ddc1Δ extract. The data point to a novel property of Ddc1, modulating the turnover of DNA binding proteins such as RPAp70 by the proteasome.

  18. Regulation of the double-stranded RNA-dependent protein kinase PKR by RNAs encoded by a repeated sequence in the Epstein-Barr virus genome.

    PubMed Central

    Elia, A; Laing, K G; Schofield, A; Tilleray, V J; Clemens, M J

    1996-01-01

    During the initial infection of B lymphocytes by Epstein-Barr virus (EBV) only a few viral genes are expressed, six of which encode the EBV nuclear antigens, EBNAs 1-6. The majority of EBNA mRNAs share common 5'-ends containing a variable number of two alternating and repeated exons transcribed from the BamHI W major internal repeats of the viral DNA. These sequences can also exist as independent small RNA species in some EBV-infected cell types. We present evidence that transcripts from these W repeat regions can exert a trans-acting effect on protein synthesis, through their ability to activate the dsRNA-dependent protein kinase PKR. UV cross-linking and filter binding assays have demonstrated that the W transcripts bind specifically to PKR and can compete with another EBV-encoded small RNA, EBER-1, which was shown previously to bind this kinase. In the reticulocyte lysate system the W RNAs shut off protein synthesis through an ability to activate PKR. In contrast to EBER-1, the W RNAs are unable to block the dsRNA-dependent activation of PKR. Using a purified preparation of the protein kinase we have shown that the W transcripts directly activate PKR in vitro. The results suggest that EBV has the ability both to activate and to inhibit PKR through the actions of different products of viral transcription. PMID:8948637

  19. Why double-stranded RNA resists condensation.

    PubMed

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

    2014-01-01

    The addition of small amounts of multivalent cations to solutions containing double-stranded DNA leads to inter-DNA attraction and eventual condensation. Surprisingly, the condensation is suppressed in double-stranded RNA, which carries the same negative charge as DNA, but assumes a different double helical form. Here, we combine experiment and atomistic simulations to propose a mechanism that explains the variations in condensation of short (25 base-pairs) nucleic acid (NA) duplexes, from B-like form of homopolymeric DNA, to mixed sequence DNA, to DNA:RNA hybrid, to A-like RNA. Circular dichroism measurements suggest that duplex helical geometry is not the fundamental property that ultimately determines the observed differences in condensation. Instead, these differences are governed by the spatial variation of cobalt hexammine (CoHex) binding to NA. There are two major NA-CoHex binding modes--internal and external--distinguished by the proximity of bound CoHex to the helical axis. We find a significant difference, up to 5-fold, in the fraction of ions bound to the external surfaces of the different NA constructs studied. NA condensation propensity is determined by the fraction of CoHex ions in the external binding mode.

  20. Translocation of double strand DNA into a biological nanopore

    NASA Astrophysics Data System (ADS)

    Chatkaew, Sunita; Mlayeh, Lamia; Leonetti, Marc; Homble, Fabrice

    2009-03-01

    Translocation of double strand DNA across a unique mitochondrial biological nanopore (VDAC) is observed by an electrophysiological method. Characteristics of opened and sub-conductance states of VDAC are studied. When the applied electric potential is beyond ± 20 mV, VDAC transits to a sub-conductance state. Plasmids (circular double strand DNA) with a diameter greater than that of the channel shows the current reduction into the channel during the interaction but the state with zero-current is not observed. On the contrary, the interaction of linear double strand DNA with the channel shows the current reduction along with the zero-current state. These show the passages of linear double strand DNA across the channel and the electrostatic effect due to the surface charges of double strand DNA and channel for circular and linear double strand DNA.

  1. A double-stranded RNA element from a hypovirulent strain of Rhizoctonia solani occurs in DNA form and is genetically related to the pentafunctional AROM protein of the shikimate pathway.

    PubMed

    Lakshman, D K; Jian, J; Tavantzis, S M

    1998-05-26

    M2 is a double-stranded RNA (dsRNA) element occurring in the hypovirulent isolate Rhs 1A1 of the plant pathogenic basidiomycete Rhizoctonia solani. Rhs 1A1 originated as a sector of the virulent field isolate Rhs 1AP, which contains no detectable amount of the M2 dsRNA. The complete sequence (3,570 bp) of the M2 dsRNA has been determined. A 6.9-kbp segment of total DNA from either Rhs 1A1 or Rhs 1AP hybridizes with an M2-specific cDNA probe. The sequences of M2 dsRNA and of PCR products generated from Rhs 1A1 total DNA were found to be identical. Thus this report describes a fungal host containing full-length DNA copies of a dsRNA element. A major portion of the M2 dsRNA is located in the cytoplasm, whereas a smaller amount is found in mitochondria. Based on either the universal or the mitochondrial genetic code of filamentous fungi, one strand of M2 encodes a putative protein of 754 amino acids. The resulting polypeptide has all four motifs of a dsRNA viral RNA-dependent RNA polymerase (RDRP) and is phylogenetically related to the RDRP of a mitochondrial dsRNA associated with hypovirulence in strain NB631 of Cryphonectria parasitica, incitant of chestnut blight. This polypeptide also has significant sequence similarity with two domains of a pentafunctional polypeptide, which catalyzes the five central steps of the shikimate pathway in yeast and filamentous fungi.

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

    PubMed Central

    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 plays myriad roles in the transmission and regulation of genetic information that are fundamentally constrained by its mechanical properties, including the elasticity and conformational transitions of the double-stranded (dsRNA) form. Although double-stranded DNA (dsDNA) mechanics have been dissected with exquisite precision, much less is known about dsRNA. Here we present a comprehensive characterization of dsRNA under external forces and torques using magnetic tweezers. We find that dsRNA has a force–torque phase diagram similar to that of dsDNA, including plectoneme formation, melting of the double helix induced by torque, a highly overwound state termed “P-RNA,” and a highly underwound, left-handed state denoted “L-RNA.” Beyond these similarities, our experiments reveal two unexpected behaviors of dsRNA: Unlike dsDNA, dsRNA shortens upon overwinding, and its characteristic transition rate at the plectonemic buckling transition is two orders of magnitude slower than for dsDNA. Our results challenge current models of nucleic acid mechanics, provide a baseline for modeling RNAs in biological contexts, and pave the way for new classes of magnetic tweezers experiments to dissect the role of twist and torque for RNA–protein interactions at the single-molecule level. PMID:25313077

  3. Why double-stranded RNA resists condensation

    SciTech Connect

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

    2014-09-15

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

  4. Saliva of Lygus lineolaris digests double stranded ribonucleic acids

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The prospects for development of highly specific pesticides based on double stranded ribonucleic acid have been a recent focus of scientific research. Creative applications have been proposed and demonstrated. However, not all insects are sensitive to double stranded RNA (dsRNA) gene knockdown effec...

  5. Repair of DNA Double-Strand Breaks

    NASA Astrophysics Data System (ADS)

    Falk, Martin; Lukasova, Emilie; Kozubek, Stanislav

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

  6. A double-stranded DNA rotaxane

    NASA Astrophysics Data System (ADS)

    Ackermann, Damian; Schmidt, Thorsten L.; Hannam, Jeffrey S.; Purohit, Chandra S.; Heckel, Alexander; Famulok, Michael

    2010-06-01

    Mechanically interlocked molecules such as rotaxanes and catenanes have potential as components of molecular machinery. Rotaxanes consist of a dumb-bell-shaped molecule encircled by a macrocycle that can move unhindered along the axle, trapped by bulky stoppers. Previously, rotaxanes have been made from a variety of molecules, but not from DNA. Here, we report the design, assembly and characterization of rotaxanes in which both the dumb-bell-shaped molecule and the macrocycle are made of double-stranded DNA, and in which the axle of the dumb-bell is threaded through the macrocycle by base pairing. The assembly involves the formation of pseudorotaxanes, in which the macrocycle and the axle are locked together by hybridization. Ligation of stopper modules to the axle leads to the characteristic dumb-bell topology. When an oligonucleotide is added to release the macrocycle from the axle, the pseudorotaxanes are either converted to mechanically stable rotaxanes, or they disassemble by means of a slippage mechanism to yield a dumb-bell and a free macrocycle. Our DNA rotaxanes allow the fields of mechanically interlocked molecules and DNA nanotechnology to be combined, thus opening new possibilities for research into molecular machines and synthetic biology.

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

    PubMed Central

    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

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

    SciTech Connect

    Budd, M.E.

    1982-07-01

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

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

    PubMed

    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.

  10. [Double-stranded DNA microarray: principal, techniques and applications].

    PubMed

    Pan, Yan; Wang, Jin-Ke

    2013-03-01

    Double-stranded DNA (dsDNA) microarray, also known as protein binding microarray (PBM), is an important technique that can be used to assay the interaction of DNA-binding protein (such as transcription factor, TF) with vast amount of DNA molecules in high-throughput format. This technique immobilizes large amount of various dsDNA molecules on the surface of a solid support (such as glass slide) for detecting the binding interaction of a DNA-binding protein with all of the immobilized dsDNA molecules, and thus determining the DNA-binding affinity, specificity and preference of TFs. In recent years, this technique has demonstrated its valuable applications in several aspects, including rapidly characterizing DNA-binding specificity of large number of TFs, building DNA-binding profiles of TFs, identifying DNA-binding sites and target genes of TFs, discriminating the subtle DNA-binding preferences of members and their dimmers of a TF family, and examining the effects of a cofactor on the DNA-binding specificity of TFs. This paper reviews the principal, techniques, and applications of dsDNA microarray.

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

    NASA Astrophysics Data System (ADS)

    Fields, Alexander; Axelrod, Kevin; Cohen, Adam

    2012-02-01

    Bare double-stranded DNA is a stiff biopolymer with a persistence length of roughly 53 nm under physiological conditions. Cells and viruses employ extensive protein machinery to overcome this stiffness and bend, twist, and loop DNA to accomplish tasks such as packaging, recombination, gene regulation, and repair. The mechanical properties of DNA are of fundamental importance to the mechanism and thermodynamics of these processes, but physiologically relevant curvature has been difficult to access experimentally. We designed and synthesized a DNA hairpin construct in which base-pairing interactions generated a compressive force on a short segment of duplex DNA, inducing Euler buckling followed by bending to thermally inaccessible radii of curvature. The efficiency of F"orster resonance energy transfer (FRET) between two fluorophores covalently linked to the hairpin indicated the degree of buckling. Bulk and single-molecule measurements yielded distinctly different force-compression curves for intact DNA and for strands with single nicks, base pair mismatches, and damage sites. These results suggest that changes in local mechanical properties may play a significant role in the recognition of these features by DNA-binding proteins.

  12. ATM controls meiotic double-strand break formation

    PubMed Central

    Lange, Julian; Pan, Jing; Cole, Francesca; Thelen, Michael P.; Jasin, Maria; Keeney, Scott

    2011-01-01

    In many organisms, developmentally programmed double-strand breaks (DSBs) formed by the SPO11 transesterase initiate meiotic recombination, which promotes pairing and segregation of homologous chromosomes1. Because every chromosome must receive a minimum number of DSBs, attention has focused on factors that support DSB formation2. However, improperly repaired DSBs can cause meiotic arrest or mutation3,4, thus having too many DSBs is likely as deleterious as having too few. Only a small fraction of SPO11 protein ever makes a DSB in yeast or mouse5, and SPO11 and its accessory factors remain abundant long after most DSB formation ceases1, implying the existence of mechanisms that restrain SPO11 activity to limit DSB numbers. Here we report that the number of meiotic DSBs in mouse is controlled by ATM, a kinase activated by DNA damage to trigger checkpoint signaling and promote DSB repair. Levels of SPO11-oligonucleotide complexes, by-products of meiotic DSB formation, are elevated at least ten-fold in spermatocytes lacking ATM. Moreover, Atm mutation renders SPO11-oligonucleotide levels sensitive to genetic manipulations that modulate SPO11 protein levels. We propose that ATM restrains SPO11 via a negative feedback loop in which kinase activation by DSBs suppresses further DSB formation. Our findings explain previously puzzling phenotypes of Atm-null mice and provide a molecular basis for the gonadal dysgenesis observed in ataxia telangiectasia, the human syndrome caused by ATM deficiency. PMID:22002603

  13. Error-Prone Repair of DNA Double-Strand Breaks.

    PubMed

    Rodgers, Kasey; McVey, Mitch

    2016-01-01

    Preserving the integrity of the DNA double helix is crucial for the maintenance of genomic stability. Therefore, DNA double-strand breaks represent a serious threat to cells. In this review, we describe the two major strategies used to repair double strand breaks: non-homologous end joining and homologous recombination, emphasizing the mutagenic aspects of each. We focus on emerging evidence that homologous recombination, long thought to be an error-free repair process, can in fact be highly mutagenic, particularly in contexts requiring large amounts of DNA synthesis. Recent investigations have begun to illuminate the molecular mechanisms by which error-prone double-strand break repair can create major genomic changes, such as translocations and complex chromosome rearrangements. We highlight these studies and discuss proposed models that may explain some of the more extreme genetic changes observed in human cancers and congenital disorders.

  14. Repair of DNA double strand breaks: in vivo biochemistry.

    PubMed

    Sugawara, Neal; Haber, James E

    2006-01-01

    Double strand breaks (DSBs) can cause damage to the genomic integrity of a cell as well as initiate genetic recombination processes. The HO and I-SceI endonucleases from budding yeast have provided a way to study these events by inducing a unique DSB in vivo under the control of a galactose-inducible promoter. The GAL::HO construct has been used extensively to study processes such as nonhomologous end joining, intra- and interchromosomal gene conversion, single strand annealing and break-induced recombination. Synchronously induced DSBs have also been important in the study of the DNA damage checkpoint, adaptation, and recovery pathways of yeast. This chapter describes methods of using GAL::HO to physically monitor the progression of events following a DSB, specifically the events leading to the switching of mating type by gene conversion of MAT using the silent donors at HML and HMR. Southern blot analysis can be used to follow the overall events in this process such as the formation of the DSB and product. Denaturing alkaline gels and slot blot techniques can be employed to follow the 5' to 3' resection of DNA starting at the DSB. After resection, the 3' tail initiates a homology search and then strand invades its homologous sequence at the donor cassette. Polymerase chain reaction is an important means to assay strand invasion and the priming of new DNA synthesis as well as the completion of gene conversion. Methods such as chromatin immunoprecipitation have provided a means to study many proteins that associate with a DSB, including not only recombination proteins, but also proteins involved in nonhomologous end joining, cell cycle arrest, chromatin remodeling, cohesin function, and mismatch repair.

  15. Blocking single-stranded transferred DNA conversion to double-stranded intermediates by overexpression of yeast DNA REPLICATION FACTOR A.

    PubMed

    Dafny-Yelin, Mery; Levy, Avner; Dafny, Raz; Tzfira, Tzvi

    2015-01-01

    Agrobacterium tumefaciens delivers its single-stranded transferred DNA (T-strand) into the host cell nucleus, where it can be converted into double-stranded molecules. Various studies have revealed that double-stranded transfer DNA (T-DNA) intermediates can serve as substrates by as yet uncharacterized integration machinery. Nevertheless, the possibility that T-strands are themselves substrates for integration cannot be ruled out. We attempted to block the conversion of T-strands into double-stranded intermediates prior to integration in order to further investigate the route taken by T-DNA molecules on their way to integration. Transgenic tobacco (Nicotiana benthamiana) plants that overexpress three yeast (Saccharomyces cerevisiae) protein subunits of DNA REPLICATION FACTOR A (RFA) were produced. In yeast, these subunits (RFA1-RFA3) function as a complex that can bind single-stranded DNA molecules, promoting the repair of genomic double strand breaks. Overexpression of the RFA complex in tobacco resulted in decreased T-DNA expression, as determined by infection with A. tumefaciens cells carrying the β-glucuronidase intron reporter gene. Gene expression was not blocked when the reporter gene was delivered by microbombardment. Enhanced green fluorescent protein-assisted localization studies indicated that the three-protein complex was predominantly nuclear, thus indicating its function within the plant cell nucleus, possibly by binding naked T-strands and blocking their conversion into double-stranded intermediates. This notion was further supported by the inhibitory effect of RFA expression on the cell-to-cell movement of Bean dwarf mosaic virus, a single-stranded DNA virus. The observation that RFA complex plants dramatically inhibited the transient expression level of T-DNA and only reduced T-DNA integration by 50% suggests that double-stranded T-DNA intermediates, as well as single-stranded T-DNA, play significant roles in the integration process. PMID:25424309

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

    NASA Astrophysics Data System (ADS)

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

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

  17. Theory for the force-stretched double-stranded chain molecule

    NASA Astrophysics Data System (ADS)

    Liu, Fei; Dai, Luru; Ou-Yang, Zhong-can

    2003-10-01

    We modify and extend the recently developed statistical mechanical theory of chain molecules having noncovalent double-stranded conformations, as in RNA or single-stranded DNA, and β sheets in protein, to the force-stretched case as in a typical single-molecule experiment. The conformations of double-stranded regions of the molecules are calculated based on polymer graph-theoretic approach [S.-J. Chen and K. A. Dill, J. Chem. Phys. 109, 4602 (1998)], while the unpaired single-stranded regions are treated as self-avoiding walks. Two classes of conformations—the hairpin conformations and RNA secondary structures—are explored. For the hairpin conformations, all possible end-to-end distances corresponding to the different types of double-stranded regions are enumerated exactly. For the RNA secondary structures, a recursive formula incorporating the secondary structure and end-to-end distribution has been derived. The sequence dependence and excluded volume interaction are taken into account explicitly. Using the extended theory, we investigate the extension-force and force-extension curves, distribution of the extensions at fixed forces, and reentering phenomenon, respectively. We find that the mechanical behaviors of homogeneous chains of hairpin conformations and secondary structures are quite different: the unfolding of the hairpin case is two state, while the unfolding of the latter is one state. In addition, a reentering transition is observed in hairpin conformations.

  18. A general solution for opening double-stranded DNA for isothermal amplification

    PubMed Central

    Chen, Gangyi; Dong, Juan; Yuan, Yi; Li, Na; Huang, Xin; Cui, Xin; Tang, Zhuo

    2016-01-01

    Nucleic acid amplification is the core technology of molecular biology and genetic engineering. Various isothermal amplification techniques have been developed as alternatives to polymerase chain reaction (PCR). However, most of these methods can only detect single stranded nucleic acid. Herein, we put forward a simple solution for opening double-stranded DNA for isothermal detection methods. The strategy employs recombination protein from E. coli (RecA) to form nucleoprotein complex with single-stranded DNA, which could scan double-stranded template for homologous sites. Then, the nucleoprotein can invade the double-stranded template to form heteroduplex in the presence of ATP, resulting in the strand exchange. The ATP regeneration system could be eliminated by using high concentration of ATP, and the 3′-OH terminal of the invasion strand can be recognized by other DNA modifying enzymes such as DNA polymerase or DNA ligase. Moreover, dATP was found to be a better cofactor for RecA, which make the system more compatible to DNA polymerase. The method described here is a general solution to open dsDNA, serving as a platform to develop more isothermal nucleic acids detection methods for real DNA samples based on it. PMID:27687498

  19. Staufen Negatively Modulates MicroRNA Activity in Caenorhabditis elegans

    PubMed Central

    Ren, Zhiji; Veksler-Lublinsky, Isana; Morrissey, David; Ambros, Victor

    2016-01-01

    The double-stranded RNA-binding protein Staufen has been implicated in various posttranscriptional gene regulatory processes. Here, we demonstrate that the Caenorhabditis elegans homolog of Staufen, STAU-1, functionally interacts with microRNAs. Loss-of-function mutations of stau-1 significantly suppress phenotypes of let-7 family microRNA mutants, a hypomorphic allele of dicer, and a lsy-6 microRNA partial loss-of-function mutant. Furthermore, STAU-1 modulates the activity of lin-14, a target of lin-4 and let-7 family microRNAs, and this modulation is abolished when the 3′ untranslated region of lin-14 is removed. Deep sequencing of small RNA cDNA libraries reveals no dramatic change in the levels of microRNAs or other small RNA populations between wild-type and stau-1 mutants, with the exception of certain endogenous siRNAs in the WAGO pathway. The modulation of microRNA activity by STAU-1 does not seem to be associated with the previously reported enhanced exogenous RNAi (Eri) phenotype of stau-1 mutants, since eri-1 exhibits the opposite effect on microRNA activity. Altogether, our results suggest that STAU-1 negatively modulates microRNA activity downstream of microRNA biogenesis, possibly by competing with microRNAs for binding on the 3′ untranslated region of target mRNAs. PMID:26921297

  20. Staufen Negatively Modulates MicroRNA Activity in Caenorhabditis elegans.

    PubMed

    Ren, Zhiji; Veksler-Lublinsky, Isana; Morrissey, David; Ambros, Victor

    2016-01-01

    The double-stranded RNA-binding protein Staufen has been implicated in various posttranscriptional gene regulatory processes. Here, we demonstrate that the Caenorhabditis elegans homolog of Staufen, STAU-1, functionally interacts with microRNAs. Loss-of-function mutations of stau-1 significantly suppress phenotypes of let-7 family microRNA mutants, a hypomorphic allele of dicer, and a lsy-6 microRNA partial loss-of-function mutant. Furthermore, STAU-1 modulates the activity of lin-14, a target of lin-4 and let-7 family microRNAs, and this modulation is abolished when the 3' untranslated region of lin-14 is removed. Deep sequencing of small RNA cDNA libraries reveals no dramatic change in the levels of microRNAs or other small RNA populations between wild-type and stau-1 mutants, with the exception of certain endogenous siRNAs in the WAGO pathway. The modulation of microRNA activity by STAU-1 does not seem to be associated with the previously reported enhanced exogenous RNAi (Eri) phenotype of stau-1 mutants, since eri-1 exhibits the opposite effect on microRNA activity. Altogether, our results suggest that STAU-1 negatively modulates microRNA activity downstream of microRNA biogenesis, possibly by competing with microRNAs for binding on the 3' untranslated region of target mRNAs. PMID:26921297

  1. Double-Stranded Water on Stepped Platinum Surfaces

    NASA Astrophysics Data System (ADS)

    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.

  2. Human Immunodeficiency Virus Type 1 Tat Protein Activates Transcription Factor NF-κB through the Cellular Interferon-Inducible, Double-Stranded RNA-Dependent Protein Kinase, PKR

    PubMed Central

    Demarchi, Francesca; Gutierrez, Maria Ines; Giacca, Mauro

    1999-01-01

    The transactivator protein of human immunodeficiency virus type 1 (HIV-1) (Tat) is a powerful activator of nuclear factor-κB (NF-κB), acting through degradation of the inhibitor IκB-α (F. Demarchi, F. d’Adda di Fagagna, A. Falaschi, and M. Giacca, J. Virol. 70:4427–4437, 1996). Here, we show that this activity of Tat requires the function of the cellular interferon-inducible protein kinase PKR. Tat-mediated NF-κB activation and transcriptional induction of the HIV-1 long terminal repeat were impaired in murine cells in which the PKR gene was knocked out. Both functions were restored by cotransfection of Tat with the cDNA for PKR. Expression of a dominant-negative mutant of PKR specifically reduced the levels of Tat transactivation in different human cell types. Activation of NF-κB by Tat required integrity of the basic domain of Tat; previous studies have indicated that this domain is necessary for specific Tat-PKR interaction. PMID:10400814

  3. Cloning of the cDNA of the heme-regulated eukaryotic initiation factor 2 alpha (eIF-2 alpha) kinase of rabbit reticulocytes: homology to yeast GCN2 protein kinase and human double-stranded-RNA-dependent eIF-2 alpha kinase.

    PubMed Central

    Chen, J J; Throop, M S; Gehrke, L; Kuo, I; Pal, J K; Brodsky, M; London, I M

    1991-01-01

    We have cloned the cDNA of the heme-regulated eIF-2 alpha kinase (HRI) of rabbit reticulocytes. In vitro translation of mRNA transcribed from the HRI cDNA yields a 90-kDa polypeptide that exhibits eIF-2 alpha kinase activity and is recognized by a monoclonal antibody directed against authentic HRI. The open reading frame sequence of the HRI cDNA contains all 11 catalytic domains of protein kinases with consensus sequences of protein-serine/threonine kinases in conserved catalytic domains VI and VIII. The HRI cDNA also contains an insert of approximately 140 amino acids between catalytic domains V and VI. The HRI cDNA coding sequence has extensive homology to GCN2 protein kinase of Saccharomyces cerevisiae and to human double-stranded-RNA-dependent eIF-2 alpha kinase. This observation suggests that GCN2 protein kinase may be an eIF-2 alpha kinase in yeast. In addition, HRI has an unusually high degree of homology to three protein kinases (NimA, Wee1, and CDC2) that are involved in the regulation of the cell cycle. Images PMID:1679235

  4. Nuclear organization in DNA end processing: Telomeres vs double-strand breaks.

    PubMed

    Marcomini, Isabella; Gasser, Susan M

    2015-08-01

    Many proteins ligands are shared between double-strand breaks and natural chromosomal ends or telomeres. The structural similarity of the 3' overhang, and the efficiency of cellular DNA end degradation machineries, highlight the need for mechanisms that resect selectively to promote or restrict recombination events. Here we examine the means used by eukaryotic cells to suppress resection at telomeres, target telomerase to short telomeres, and process broken ends for appropriate repair. Not only molecular ligands, but the spatial sequestration of telomeres and damage likely ensure that these two very similar structures have very distinct outcomes with respect to the DNA damage response and repair.

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

    SciTech Connect

    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.

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

    PubMed

    Azeroglu, Benura; Mawer, Julia S P; Cockram, Charlotte A; White, Martin A; Hasan, A M Mahedi; Filatenkova, Milana; Leach, David R F

    2016-02-01

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

  7. The double-stranded RNA binding domain of human Dicer functions as a nuclear localization signal.

    PubMed

    Doyle, Michael; Badertscher, Lukas; Jaskiewicz, Lukasz; Güttinger, Stephan; Jurado, Sabine; Hugenschmidt, Tabea; Kutay, Ulrike; Filipowicz, Witold

    2013-09-01

    Dicer is a key player in microRNA (miRNA) and RNA interference (RNAi) pathways, processing miRNA precursors and double-stranded RNA into ∼21-nt-long products ultimately triggering sequence-dependent gene silencing. Although processing of substrates in vertebrate cells occurs in the cytoplasm, there is growing evidence suggesting Dicer is also present and functional in the nucleus. To address this possibility, we searched for a nuclear localization signal (NLS) in human Dicer and identified its C-terminal double-stranded RNA binding domain (dsRBD) as harboring NLS activity. We show that the dsRBD-NLS can mediate nuclear import of a reporter protein via interaction with importins β, 7, and 8. In the context of full-length Dicer, the dsRBD-NLS is masked. However, duplication of the dsRBD localizes the full-length protein to the nucleus. Furthermore, deletion of the N-terminal helicase domain results in partial accumulation of Dicer in the nucleus upon leptomycin B treatment, indicating that CRM1 contributes to nuclear export of Dicer. Finally, we demonstrate that human Dicer has the ability to shuttle between the nucleus and the cytoplasm. We conclude that Dicer is a shuttling protein whose steady-state localization is cytoplasmic.

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

    PubMed Central

    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 polarization in ionic solution is discussed based on the frequency, viscosity, and field dependence of the observed trapping force. PMID:12324434

  9. Double strand breaks: hurdles for RNA polymerase II transcription?

    PubMed

    Pankotai, Tibor; Soutoglou, Evi

    2013-01-01

    DNA lesions pose a physical obstacle to DNA-dependent cellular transactions such as replication and transcription. A great deal is known regarding RNA polymerase II (RNAP II) transcription stalling in the presence of lesions induced by UV, but recent studies have uncovered previously uncharacterized behavior of the RNAP II machinery in the presence of double strand breaks (DSBs). These new data, although contradictory, contribute to our understanding of a vital cellular mechanism that defends against the production of aberrant transcripts and protects cell viability.

  10. Herpetic keratoconjunctivitis: Therapy with synthetic double-stranded RNA

    USGS Publications Warehouse

    Friedman, I.; Evans, C.; Meighan, C.W.; Foote, L.J.; Aiello, P.V.; Park, J.H.; Baron, S.

    1968-01-01

    A study was undertaken in rabbits to determine how late in the course of keratoconjunctivitis caused by herpes simplex recovery could be effected by an inducer of interferon. Interferon was induced by means of synthetic double-stranded RNA copolymer formed with polynosinic acid : polycytidilic acid RNA. Therapy promotes recovery from severe and fully established keratoconjunctivitis for which treatment was begun as late as 3 days after virus inoculation. No drug toxicity was observed in the therapeutic dose range. These findings further support the proposed role of the interferon mechanism in the natural recovery of already established viral infection. They also suggest the usefulness of interferon inducers in viral infections of man.

  11. Adenosine triphosphatases of thermophilic archaeal double-stranded DNA viruses

    PubMed Central

    2014-01-01

    Adenosine triphosphatases (ATPases) of double-stranded (ds) DNA archaeal viruses are structurally related to the AAA+ hexameric helicases and translocases. These ATPases have been implicated in viral life cycle functions such as DNA entry into the host, and viral genome packaging into preformed procapsids. We summarize bioinformatical analyses of a wide range of archaeal ATPases, and review the biochemical and structural properties of those archaeal ATPases that have measurable ATPase activity. We discuss their potential roles in genome delivery into the host, virus assembly and genome packaging in comparison to hexameric helicases and packaging motors from bacteriophages. PMID:25105011

  12. Long noncoding RNA LINP1 regulates double strand DNA break repair in triple negative breast cancer

    PubMed Central

    Zhang, Youyou; He, Qun; Hu, Zhongyi; Feng, Yi; Fan, Lingling; Tang, Zhaoqing; Yuan, Jiao; Shan, Weiwei; Li, Chunsheng; Hu, Xiaowen; Tanyi, Janos L; Fan, Yi; Huang, Qihong; Montone, Kathleen; Dang, Chi V; Zhang, Lin

    2016-01-01

    Long noncoding RNAs (lncRNAs), which are transcripts that are larger than 200 nucleotides but do not appear to have protein-coding potential, play critical roles during tumorigenesis by functioning as scaffolds to regulate protein-protein, protein-DNA or protein-RNA interactions. Using a clinically guided genetic screening approach, we identified (lncRNA in Non-homologous end joining [NHEJ] pathway 1) as a lncRNA that is overexpressed in human triple-negative breast cancer. We found that LINP1 enhances double-strand DNA break repair by serving as a scaffold that links Ku80 and DNA-PKcs, thereby coordinating the NHEJ pathway. Importantly, blocking LINP1, which is regulated by the p53 and epidermal growth factor receptor (EGFR) signaling, increases sensitivity of tumor cell response to radiotherapy in breast cancer. PMID:27111890

  13. Solid phase sequencing of double-stranded nucleic acids

    DOEpatents

    Fu, Dong-Jing; Cantor, Charles R.; Koster, Hubert; Smith, Cassandra L.

    2002-01-01

    This invention relates to methods for detecting and sequencing of target double-stranded nucleic acid sequences, to nucleic acid probes and arrays of probes useful in these methods, and to kits and systems which contain these probes. Useful methods involve hybridizing the nucleic acids or nucleic acids which represent complementary or homologous sequences of the target to an array of nucleic acid probes. These probe comprise a single-stranded portion, an optional double-stranded portion and a variable sequence within the single-stranded portion. The molecular weights of the hybridized nucleic acids of the set can be determined by mass spectroscopy, and the sequence of the target determined from the molecular weights of the fragments. Nucleic acids whose sequences can be determined include nucleic acids in biological samples such as patient biopsies and environmental samples. Probes may be fixed to a solid support such as a hybridization chip to facilitate automated determination of molecular weights and identification of the target sequence.

  14. Nucleolar responses to DNA double-strand breaks.

    PubMed

    Larsen, Dorthe Helena; Stucki, Manuel

    2016-01-29

    Maintenance of cellular homeostasis is key to prevent transformation and disease. The cellular response to DNA double-strand breaks, primarily orchestrated by the ATM/ATR kinases is one of many mechanisms that serve to uphold genome stability and homeostasis. Upon detection of double-strand breaks (DSBs), several signaling cascades are activated to halt cell cycle progression and initiate repair. Furthermore, the DNA damage response (DDR) controls cellular processes such as transcription, splicing and metabolism. Recent studies have uncovered aspects of how the DDR operates within nucleoli. It appears that the DDR controls transcription in the nucleoli, not only when DNA breaks occur in the rDNA repeats, but also when a nuclear DDR is activated. In addition, we have gained first insights into how repair of DSBs is organized in the nucleolus. Collectively, these recent studies provide a more comprehensive picture of how the DDR regulates basic cellular functions to maintain cellular homeostasis. In this review we will summarize recent findings and discuss their implications for our understanding of how the DDR regulates transcription and repair in the nucleolus.

  15. Nucleolar responses to DNA double-strand breaks

    PubMed Central

    Larsen, Dorthe Helena; Stucki, Manuel

    2016-01-01

    Maintenance of cellular homeostasis is key to prevent transformation and disease. The cellular response to DNA double-strand breaks, primarily orchestrated by the ATM/ATR kinases is one of many mechanisms that serve to uphold genome stability and homeostasis. Upon detection of double-strand breaks (DSBs), several signaling cascades are activated to halt cell cycle progression and initiate repair. Furthermore, the DNA damage response (DDR) controls cellular processes such as transcription, splicing and metabolism. Recent studies have uncovered aspects of how the DDR operates within nucleoli. It appears that the DDR controls transcription in the nucleoli, not only when DNA breaks occur in the rDNA repeats, but also when a nuclear DDR is activated. In addition, we have gained first insights into how repair of DSBs is organized in the nucleolus. Collectively, these recent studies provide a more comprehensive picture of how the DDR regulates basic cellular functions to maintain cellular homeostasis. In this review we will summarize recent findings and discuss their implications for our understanding of how the DDR regulates transcription and repair in the nucleolus. PMID:26615196

  16. Force-Driven Separation of Short Double-Stranded DNA

    PubMed Central

    Ho, Dominik; Zimmermann, Julia L.; Dehmelt, Florian A.; Steinbach, Uta; Erdmann, Matthias; Severin, Philip; Falter, Katja; Gaub, Hermann E.

    2009-01-01

    Abstract Short double-stranded DNA is used in a variety of nanotechnological applications, and for many of them, it is important to know for which forces and which force loading rates the DNA duplex remains stable. In this work, we develop a theoretical model that describes the force-dependent dissociation rate for DNA duplexes tens of basepairs long under tension along their axes (“shear geometry”). Explicitly, we set up a three-state equilibrium model and apply the canonical transition state theory to calculate the kinetic rates for strand unpairing and the rupture-force distribution as a function of the separation velocity of the end-to-end distance. Theory is in excellent agreement with actual single-molecule force spectroscopy results and even allows for the prediction of the rupture-force distribution for a given DNA duplex sequence and separation velocity. We further show that for describing double-stranded DNA separation kinetics, our model is a significant refinement of the conventionally used Bell-Evans model. PMID:20006953

  17. Splicing controls the ubiquitin response during DNA double-strand break repair

    PubMed Central

    Pederiva, C; Böhm, S; Julner, A; Farnebo, M

    2016-01-01

    Although evidence that splicing regulates DNA repair is accumulating, the underlying mechanism(s) remain unclear. Here, we report that short-term inhibition of pre-mRNA splicing by spliceosomal inhibitors impairs cellular repair of DNA double-strand breaks. Indeed, interference with splicing as little as 1 h prior to irradiation reduced ubiquitylation of damaged chromatin and impaired recruitment of the repair factors WRAP53β, RNF168, 53BP1, BRCA1 and RAD51 to sites of DNA damage. Consequently, splicing-deficient cells exhibited significant numbers of residual γH2AX foci, as would be expected if DNA repair is defective. Furthermore, we show that this is due to downregulation of the E3 ubiquitin ligase RNF8 and that re-introduction of this protein into splicing-deficient cells restores ubiquitylation at sites of DNA damage, accumulation of downstream factors and subsequent repair. Moreover, downregulation of RNF8 explains the defective repair associated with knockdown of various splicing factors in recent genome-wide siRNA screens and, significantly, overexpression of RNF8 counteracts this defect. These discoveries reveal a mechanism that may not only explain how splicing regulates repair of double-strand breaks, but also may underlie various diseases caused by deregulation of splicing factors, including cancer. PMID:27315300

  18. Origin of the catalytic activity of bovine seminal ribonuclease against double-stranded RNA

    NASA Technical Reports Server (NTRS)

    Opitz, J. G.; Ciglic, M. I.; Haugg, M.; Trautwein-Fritz, K.; Raillard, S. A.; Jermann, T. M.; Benner, S. A.

    1998-01-01

    Bovine seminal ribonuclease (RNase) binds, melts, and (in the case of RNA) catalyzes the hydrolysis of double-stranded nucleic acid 30-fold better under physiological conditions than its pancreatic homologue, the well-known RNase A. Reported here are site-directed mutagenesis experiments that identify the sequence determinants of this enhanced catalytic activity. These experiments have been guided in part by experimental reconstructions of ancestral RNases from extinct organisms that were intermediates in the evolution of the RNase superfamily. It is shown that the enhanced interactions between bovine seminal RNase and double-stranded nucleic acid do not arise from the increased number of basic residues carried by the seminal enzyme. Rather, a combination of a dimeric structure and the introduction of two glycine residues at positions 38 and 111 on the periphery of the active site confers the full catalytic activity of bovine seminal RNase against duplex RNA. A structural model is presented to explain these data, the use of evolutionary reconstructions to guide protein engineering experiments is discussed, and a new variant of RNase A, A(Q28L K31C S32C D38G E111G), which contains all of the elements identified in these experiments as being important for duplex activity, is prepared. This is the most powerful catalyst within this subfamily yet observed, some 46-fold more active against duplex RNA than RNase A.

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

    PubMed

    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.

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

    PubMed

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

    2016-06-15

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

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

    PubMed

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

    2016-06-15

    Here we have developed a novel method of isothermal amplification detection of double-stranded DNA (dsDNA) based on double-stranded fluorescence probe (ds-probe). Target dsDNA repeatedly generated single-stranded DNA (ssDNA) with polymerase and nicking enzyme. The ds-probe as a primer hybridized with ssDNA and extended to its 5'-end. The displaced ssDNA served as a new detection target to initiate above-described reaction. Meanwhile, the extended ds-probe could dynamically dissociate from ssDNA and self-hybridize, converting into a turn-back structure to initiate another amplification reaction. In particular, the ds-probe played a key role in the entire experimental process, which not only was as a primer but also produced the fluorescent signal by an extension and displacement reaction. Our method could detect the pBluescript II KS(+) plasmid with a detection limit of 2.3 amol, and it was also verified to exhibit a high specificity, even one-base mismatch. Overall, it was a true isothermal dsDNA detection strategy with a strongly anti-jamming capacity and one-pot, only requiring one ds-probe, which greatly reduced the cost and the probability of contamination. With its advantages, the approach of dsDNA detection will offer a promising tool in the field of point-of-care testing (POCT). PMID:26803414

  2. SMCHD1 accumulates at DNA damage sites and facilitates the repair of DNA double-strand breaks

    PubMed Central

    Coker, Heather; Brockdorff, Neil

    2014-01-01

    ABSTRACT SMCHD1 is a structural maintenance of chromosomes (SMC) family protein involved in epigenetic gene silencing and chromosome organisation on the female inactive X chromosome and at a limited number of autosomal loci. Here, we demonstrate that SMCHD1 also has a role in DNA repair of double-strand breaks; SMCHD1 is recruited to sites of laser micro-irradiated damage along with other DNA repair factors, including Ku80 (also known as XRCC5 in mammals) and RAD51. Cells deficient in SMCHD1 show evidence of decreased efficiency of repair and cell viability after DNA damage. We suggest that SMCHD1 responds to DNA double-strand breaks in a manner that is likely to involve its ability to alter chromatin states to facilitate DNA repair. PMID:24790221

  3. DNA double-strand break repair pathway choice and cancer

    PubMed Central

    Aparicio, Tomas; Baer, Richard

    2014-01-01

    Summary Since DNA double-strand breaks (DSBs) contribute to the genomic instability that drives cancer development, DSB repair pathways serve as important mechanisms for tumor suppression. Thus, genetic lesions, such as BRCA1 and BRCA2 mutations, that disrupt DSB repair are often associated with cancer susceptibility. In addition, recent evidence suggests that DSB “mis-repair”, in which DSBs are resolved by an inappropriate repair pathway, can also promote genomic instability and presumably tumorigenesis. This notion has gained currency from recent cancer genome sequencing studies which have uncovered numerous chromosomal rearrangements harboring pathological DNA repair signatures. In this perspective, we discuss the factors that regulate DSB repair pathway choice and their consequences for genome stability and cancer. PMID:24746645

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

    PubMed

    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.

  5. Do DNA Double-Strand Breaks Drive Aging?

    PubMed

    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

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

    PubMed

    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

  7. Blocking Single-Stranded Transferred DNA Conversion to Double-Stranded Intermediates by Overexpression of Yeast DNA REPLICATION FACTOR A1

    PubMed Central

    Levy, Avner; Dafny, Raz; Tzfira, Tzvi

    2015-01-01

    Agrobacterium tumefaciens delivers its single-stranded transferred DNA (T-strand) into the host cell nucleus, where it can be converted into double-stranded molecules. Various studies have revealed that double-stranded transfer DNA (T-DNA) intermediates can serve as substrates by as yet uncharacterized integration machinery. Nevertheless, the possibility that T-strands are themselves substrates for integration cannot be ruled out. We attempted to block the conversion of T-strands into double-stranded intermediates prior to integration in order to further investigate the route taken by T-DNA molecules on their way to integration. Transgenic tobacco (Nicotiana benthamiana) plants that overexpress three yeast (Saccharomyces cerevisiae) protein subunits of DNA REPLICATION FACTOR A (RFA) were produced. In yeast, these subunits (RFA1–RFA3) function as a complex that can bind single-stranded DNA molecules, promoting the repair of genomic double strand breaks. Overexpression of the RFA complex in tobacco resulted in decreased T-DNA expression, as determined by infection with A. tumefaciens cells carrying the β-glucuronidase intron reporter gene. Gene expression was not blocked when the reporter gene was delivered by microbombardment. Enhanced green fluorescent protein-assisted localization studies indicated that the three-protein complex was predominantly nuclear, thus indicating its function within the plant cell nucleus, possibly by binding naked T-strands and blocking their conversion into double-stranded intermediates. This notion was further supported by the inhibitory effect of RFA expression on the cell-to-cell movement of Bean dwarf mosaic virus, a single-stranded DNA virus. The observation that RFA complex plants dramatically inhibited the transient expression level of T-DNA and only reduced T-DNA integration by 50% suggests that double-stranded T-DNA intermediates, as well as single-stranded T-DNA, play significant roles in the integration process. PMID

  8. Coordination and processing of DNA ends during double-strand break repair: the role of the bacteriophage T4 Mre11/Rad50 (MR) complex.

    PubMed

    Almond, Joshua R; Stohr, Bradley A; Panigrahi, Anil K; Albrecht, Dustin W; Nelson, Scott W; Kreuzer, Kenneth N

    2013-11-01

    The in vivo functions of the bacteriophage T4 Mre11/Rad50 (MR) complex (gp46/47) in double-strand-end processing, double-strand break repair, and recombination-dependent replication were investigated. The complex is essential for T4 growth, but we wanted to investigate the in vivo function during productive infections. We therefore generated a suppressed triple amber mutant in the Rad50 subunit to substantially reduce the level of complex and thereby reduce phage growth. Growth-limiting amounts of the complex caused a concordant decrease in phage genomic recombination-dependent replication. However, the efficiencies of double-strand break repair and of plasmid-based recombination-dependent replication remained relatively normal. Genetic analyses of linked markers indicated that double-strand ends were less protected from nuclease erosion in the depleted infection and also that end coordination during repair was compromised. We discuss models for why phage genomic recombination-dependent replication is more dependent on Mre11/Rad50 levels when compared to plasmid recombination-dependent replication. We also tested the importance of the conserved histidine residue in nuclease motif I of the T4 Mre11 protein. Substitution with multiple different amino acids (including serine) failed to support phage growth, completely blocked plasmid recombination-dependent replication, and led to the stabilization of double-strand ends. We also constructed and expressed an Mre11 mutant protein with the conserved histidine changed to serine. The mutant protein was found to be completely defective for nuclease activities, but retained the ability to bind the Rad50 subunit and double-stranded DNA. These results indicate that the nuclease activity of Mre11 is critical for phage growth and recombination-dependent replication during T4 infections.

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

    SciTech Connect

    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.

  10. A link between double-strand break-related repair and V(D)J recombination: the scid mutation

    SciTech Connect

    Hendrickson, E.A.; Qin, X.Q.; Bump, E.A.; Schatz, D.G.; Oettinger, M.; Weaver, D.T. )

    1991-05-15

    We show here that mammalian site-specific recombination and DNA-repair pathways share a common factor. The effects of DNA-damaging agents on cell lines derived from mice homozygous for the scid (severe combined immune deficiency) mutation were studied. Surprisingly, all scid cell lines exhibited a profound hypersensitivity to DNA-damaging agents that caused double-strand breaks (x-irradiation and bleomycin) but not to other chemicals that caused single-strand breaks or cross-links. Neutral filter elution assays demonstrated that the x-irradiation hypersensitivity could be correlated with a deficiency in repairing double-strand breaks. These data suggest that the scid gene product is involved in two pathways: DNA repair of random double-strand breaks and the site-specific and lymphoid-restricted variable-(diversity)-joining (V(D)J) DNA rearrangement process. We propose that the scid gene product performs a similar function in both pathways and may be a ubiquitous protein.

  11. Tracking Cryptosporidium parvum by sequence analysis of small double-stranded RNA.

    PubMed Central

    Xiao, L.; Limor, J.; Bern, C.; Lal, A. A.

    2001-01-01

    We sequenced a 173-nucleotide fragment of the small double-stranded viruslike RNA of Cryptosporidium parvum isolates from 23 calves and 38 humans. Sequence diversity was detected at 17 sites. Isolates from the same outbreak had identical double-stranded RNA sequences, suggesting that this technique may be useful for tracking Cryptosporidium infection sources. PMID:11266306

  12. 75 FR 62820 - Screening Framework Guidance for Providers of Synthetic Double-Stranded DNA

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-10-13

    ...- Stranded DNA AGENCY: Department of Health and Human Services, Office of the Secretary. ACTION: Notice... provides a framework for screening synthetic double-stranded DNA (dsDNA). This document, the Screening Framework Guidance for Providers of Synthetic Double-Stranded DNA (the Guidance), sets forth...

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

    PubMed

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

    2014-07-01

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

  14. Subdiffusion Supports Joining Of Correct Ends During Repair Of DNA Double-Strand Breaks

    NASA Astrophysics Data System (ADS)

    Girst, S.; Hable, V.; Drexler, G. A.; Greubel, C.; Siebenwirth, C.; Haum, M.; Friedl, A. A.; Dollinger, G.

    2013-08-01

    The mobility of damaged chromatin regions in the nucleus may affect the probability of mis-repair. In this work, live-cell observation and distance tracking of GFP-tagged DNA damage response protein MDC1 was used to study the random-walk behaviour of chromatin domains containing radiation-induced DNA double-strand breaks (DSB). Our measurements indicate a subdiffusion-type random walk process with similar time dependence for isolated and clustered DSBs that were induced by 20 MeV proton or 43 MeV carbon ion micro-irradiation. As compared to normal diffusion, subdiffusion enhances the probability that both ends of a DSB meet, thus promoting high efficiency DNA repair. It also limits their probability of long-range movements and thus lowers the probability of mis-rejoining and chromosome aberrations.

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

    PubMed Central

    Lu, Lin-Yu; Yu, Xiaochun

    2015-01-01

    During meiotic prophase, DNA double-strand break (DSB) repair-mediated homologous recombination (HR) occurs for exchange of genetic information between homologous chromosomes. Unlike autosomes or female sex chromosomes, human male sex chromosomes X and Y share little homology. Although DSBs are generated throughout male sex chromosomes, homologous recombination does not occur for most regions and DSB repair process is significantly prolonged. As a result, male sex chromosomes are coated with many DNA damage response proteins and form a unique chromatin structure known as the XY body. Interestingly, associated with the prolonged DSB repair, transcription is repressed in the XY body but not in autosomes, a phenomenon known as meiotic sex chromosome inactivation (MSCI), which is critical for male meiosis. Here using mice as model organisms, we briefly summarize recent progress on DSB repair in meiotic prophase and focus on the mechanism and function of DNA damage response in the XY body. PMID:25565522

  16. Multiple Pathways of Recombination Induced by Double-Strand Breaks in Saccharomyces cerevisiae

    PubMed Central

    Pâques, Frédéric; Haber, James E.

    1999-01-01

    The budding yeast Saccharomyces cerevisiae has been the principal organism used in experiments to examine genetic recombination in eukaryotes. Studies over the past decade have shown that meiotic recombination and probably most mitotic recombination arise from the repair of double-strand breaks (DSBs). There are multiple pathways by which such DSBs can be repaired, including several homologous recombination pathways and still other nonhomologous mechanisms. Our understanding has also been greatly enriched by the characterization of many proteins involved in recombination and by insights that link aspects of DNA repair to chromosome replication. New molecular models of DSB-induced gene conversion are presented. This review encompasses these different aspects of DSB-induced recombination in Saccharomyces and attempts to relate genetic, molecular biological, and biochemical studies of the processes of DNA repair and recombination. PMID:10357855

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

    NASA Astrophysics Data System (ADS)

    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

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

    PubMed

    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.

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

    PubMed

    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

  20. How a short double-stranded DNA bends

    NASA Astrophysics Data System (ADS)

    Shin, Jaeoh; Lee, O.-Chul; Sung, Wokyung

    2015-04-01

    A recent experiment using fluorescence microscopy showed that double-stranded DNA fragments shorter than 100 base pairs loop with the probabilities higher by the factor of 102-106 than predicted by the worm-like chain (WLC) model [R. Vafabakhsh and T. Ha, Science 337, 1101(2012)]. Furthermore, the looping probabilities were found to be nearly independent of the loop size. The results signify a breakdown of the WLC model for DNA mechanics which works well on long length scales and calls for fundamental understanding for stressed DNA on shorter length scales. We develop an analytical, statistical mechanical model to investigate what emerges to the short DNA under a tight bending. A bending above a critical level initiates nucleation of a thermally induced bubble, which could be trapped for a long time, in contrast to the bubbles in both free and uniformly bent DNAs, which are either transient or unstable. The trapped bubble is none other than the previously hypothesized kink, which releases the bending energy more easily as the contour length decreases. It leads to tremendous enhancement of the cyclization probabilities, in a reasonable agreement with experiment.

  1. Fluid mechanics of DNA double-strand filter elution.

    PubMed Central

    Rudinger, George; Blazek, Ed Robert

    2002-01-01

    Measurement of infrequent DNA double-strand breaks (DSB) in mammalian cells is essential for the understanding of cell damage by ionizing radiation and many DNA-reactive drugs. One of the most important assays for measuring DSB in cellular DNA is filter elution. This study is an attempt to determine whether standard concepts of fluid mechanics can yield a self-consistent model of this process. Major assumptions of the analysis are reptation through a channel formed by surrounding strands, with only strand ends captured by filter pores. Both viscosity and entanglement with surrounding strands are considered to determine the resistance to this motion. One important result is that the average elution time of a strand depends not only on its length, but also on the size distribution of the surrounding strands. This model is consistent with experimental observations, such as the dependence of elution kinetics upon radiation dose, but independence from the size of the DNA sample up to a critical filter loading, and possible overlap of elution times for strands of different length. It indicates how the dependence of elution time on the flow rate could reveal the relative importance of viscous and entanglement resistance, and also predicts the consequences of using different filters. PMID:11751292

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

    PubMed

    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 (<41 base pairs) resisted this force and remained straight; longer strands became bent, a phenomenon called 'Euler buckling'. We monitored the buckling transition via Förster Resonance Energy Transfer (FRET) between appended fluorophores. For low-to-moderate concentrations of monovalent salt (up to ∼150 mM), our results are in quantitative agreement with the worm-like chain (WLC) model of DNA elasticity, without the need to invoke any 'kinked' states. Greater concentrations of monovalent salts or 1 mM Mg(2+) induced an apparent softening of the dsDNA, which was best accounted for by a kink in the region of highest curvature. We tested the effects of all single-nucleotide mismatches on the DNA bending. Remarkably, the propensity to kink correlated with the thermodynamic destabilization of the mismatched DNA relative the perfectly complementary strand, suggesting that the kinked state is locally melted. The molecular vise is exquisitely sensitive to the sequence-dependent linear and nonlinear elastic properties of dsDNA.

  3. Chromatin modifications and DNA repair: beyond double-strand breaks

    PubMed Central

    House, Nealia C. M.; Koch, Melissa R.; Freudenreich, Catherine H.

    2014-01-01

    DNA repair must take place in the context of chromatin, and chromatin modifications and DNA repair are intimately linked. The study of double-strand break repair has revealed numerous histone modifications that occur after induction of a DSB, and modification of the repair factors themselves can also occur. In some cases the function of the modification is at least partially understood, but in many cases it is not yet clear. Although DSB repair is a crucial activity for cell survival, DSBs account for only a small percentage of the DNA lesions that occur over the lifetime of a cell. Repair of single-strand gaps, nicks, stalled forks, alternative DNA structures, and base lesions must also occur in a chromatin context. There is increasing evidence that these repair pathways are also regulated by histone modifications and chromatin remodeling. In this review, we will summarize the current state of knowledge of chromatin modifications that occur during non-DSB repair, highlighting similarities and differences to DSB repair as well as remaining questions. PMID:25250043

  4. Drosophila ATR in double-strand break repair.

    PubMed

    LaRocque, Jeannine R; Jaklevic, Burnley; Su, Tin Tin; Sekelsky, Jeff

    2007-03-01

    The ability of a cell to sense and respond to DNA damage is essential for genome stability. An important aspect of the response is arrest of the cell cycle, presumably to allow time for repair. Ataxia telangiectasia mutated (ATM) and ATR are essential for such cell-cycle control, but some observations suggest that they also play a direct role in DNA repair. The Drosophila ortholog of ATR, MEI-41, mediates the DNA damage-dependent G2-M checkpoint. We examined the role of MEI-41 in repair of double-strand breaks (DSBs) induced by P-element excision. We found that mei-41 mutants are defective in completing the later steps of homologous recombination repair, but have no defects in end-joining repair. We hypothesized that these repair defects are the result of loss of checkpoint control. To test this, we genetically reduced mitotic cyclin levels and also examined repair in grp (DmChk1) and lok (DmChk2) mutants. Our results suggest that a significant component of the repair defects is due to loss of MEI-41-dependent cell cycle regulation. However, this does not account for all of the defects we observed. We propose a novel role for MEI-41 in DSB repair, independent of the Chk1/Chk2-mediated checkpoint response.

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

    PubMed

    Terasawa, Masahiro; Shinohara, Akira; Shinohara, Miki

    2014-12-01

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

  6. Sequence selective double strand DNA cleavage by peptide nucleic acid (PNA) targeting using nuclease S1.

    PubMed Central

    Demidov, V; Frank-Kamenetskii, M D; Egholm, M; Buchardt, O; Nielsen, P E

    1993-01-01

    A novel method for sequence specific double strand DNA cleavage using PNA (peptide nucleic acid) targeting is described. Nuclease S1 digestion of double stranded DNA gives rise to double strand cleavage at an occupied PNA strand displacement binding site, and under optimized conditions complete cleavage can be obtained. The efficiency of this cleavage is more than 10 fold enhanced when a tandem PNA site is targeted, and additionally enhanced if this site is in trans rather than in cis orientation. Thus in effect, the PNA targeting makes the single strand specific nuclease S1 behave like a pseudo restriction endonuclease. Images PMID:8502550

  7. DNA Double-Strand Break Rejoining in Complex Normal Tissues

    SciTech Connect

    Ruebe, Claudia E.; Kuehne, Martin; Fricke, Andreas

    2008-11-15

    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 {gamma}H2AX 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 {gamma}H2AX 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 {gamma}H2AX immunofluorescence allows for the accurate quantification of DSBs in complex organs. Strikingly, the various normal tissues exhibited identical kinetics for {gamma}H2AX 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.

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

    NASA Astrophysics Data System (ADS)

    Fiedler, Anja; Reinert, Tilo; Tanner, Judith; Butz, Tilman

    2007-07-01

    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 Si 3N 4 window showed a homogenous Hsp70 expression pattern.

  9. Double-strand breaks from a radical commonly produced by DNA-damaging agents.

    PubMed

    Taverna Porro, Marisa L; Greenberg, Marc M

    2015-04-20

    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 abstraction. Inferential evidence suggests that a C3'-peroxyl radical induces complementary strand damage more efficiently than does a C4'-peroxyl radical. Complementary strand hydrogen atom abstraction by the peroxyl radical is efficiently quenched by thiols. This mechanism could contribute to the higher than expected yield of double-strand breaks produced by ionizing radiation.

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

    SciTech Connect

    Wang, Chen; Lees-Miller, Susan P.

    2013-07-01

    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.

  11. BRCA1-CtIP interaction in the repair of DNA double-strand breaks.

    PubMed

    Aparicio, Tomas; Gautier, Jean

    2016-07-01

    DNA termini at double-strand breaks are often chemically heterogeneous and require processing before initiation of repair. In a recent report, we demonstrated that CtIP and the MRE11-RAD50-NBS1 (MRN) nuclease complex cooperate with BRCA1 to specifically repair topoisomerase II-DNA adducted breaks. In contrast, BRCA1 is dispensable for repair of restriction endonuclease-generated double-strand breaks. PMID:27652321

  12. BRCA1-CtIP interaction in the repair of DNA double-strand breaks.

    PubMed

    Aparicio, Tomas; Gautier, Jean

    2016-07-01

    DNA termini at double-strand breaks are often chemically heterogeneous and require processing before initiation of repair. In a recent report, we demonstrated that CtIP and the MRE11-RAD50-NBS1 (MRN) nuclease complex cooperate with BRCA1 to specifically repair topoisomerase II-DNA adducted breaks. In contrast, BRCA1 is dispensable for repair of restriction endonuclease-generated double-strand breaks.

  13. Immunofluorescent Detection of DNA Double Strand Breaks induced by High-LET Radiation

    NASA Technical Reports Server (NTRS)

    Cucinotta, Francis A.; Wu, Honglu; Desai, Nirav

    2004-01-01

    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.

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

    NASA Astrophysics Data System (ADS)

    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.

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

    PubMed Central

    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 DNAs, and 14 of these sites were found to represent coupled cleavage sites, that is, events in which one of the two strands was always cleaved first, followed by the associated site on the opposite strand. Most of these coupled sites underwent cleavage by a mechanism described previously by the Povirk laboratory and afforded cleavage patterns entirely analogous to those reported. However, at least one coupled cleavage event was noted that did not conform to the pattern of those described previously. More surprisingly, 17 double-strand cleavages were found not to result from coupled double-strand cleavage, and we posit that these cleavages resulted from a new mechanism not previously described. Enhanced double-strand cleavages at these sites appear to be a consequence of the dynamic nature of the interaction of Fe·BLM A5 with the strongly bound hairpin DNAs. PMID:24548300

  16. TrmBL2 from Pyrococcus furiosus Interacts Both with Double-Stranded and Single-Stranded DNA

    PubMed Central

    Wierer, Sebastian; Daldrop, Peter; Ud Din Ahmad, Misbha; Boos, Winfried; Drescher, Malte; Welte, Wolfram; Seidel, Ralf

    2016-01-01

    In many hyperthermophilic archaea the DNA binding protein TrmBL2 or one of its homologues is abundantly expressed. TrmBL2 is thought to play a significant role in modulating the chromatin architecture in combination with the archaeal histone proteins and Alba. However, its precise physiological role is poorly understood. It has been previously shown that upon binding TrmBL2 covers double-stranded DNA, which leads to the formation of a thick and fibrous filament. Here we investigated the filament formation process as well as the stabilization of DNA by TrmBL2 from Pyroccocus furiosus in detail. We used magnetic tweezers that allow to monitor changes of the DNA mechanical properties upon TrmBL2 binding on the single-molecule level. Extended filaments formed in a cooperative manner and were considerably stiffer than bare double-stranded DNA. Unlike Alba, TrmBL2 did not form DNA cross-bridges. The protein was found to bind double- and single-stranded DNA with similar affinities. In mechanical disruption experiments of DNA hairpins this led to stabilization of both, the double- (before disruption) and the single-stranded (after disruption) DNA forms. Combined, these findings suggest that the biological function of TrmBL2 is not limited to modulating genome architecture and acting as a global repressor but that the protein acts additionally as a stabilizer of DNA secondary structure. PMID:27214207

  17. TrmBL2 from Pyrococcus furiosus Interacts Both with Double-Stranded and Single-Stranded DNA.

    PubMed

    Wierer, Sebastian; Daldrop, Peter; Ud Din Ahmad, Misbha; Boos, Winfried; Drescher, Malte; Welte, Wolfram; Seidel, Ralf

    2016-01-01

    In many hyperthermophilic archaea the DNA binding protein TrmBL2 or one of its homologues is abundantly expressed. TrmBL2 is thought to play a significant role in modulating the chromatin architecture in combination with the archaeal histone proteins and Alba. However, its precise physiological role is poorly understood. It has been previously shown that upon binding TrmBL2 covers double-stranded DNA, which leads to the formation of a thick and fibrous filament. Here we investigated the filament formation process as well as the stabilization of DNA by TrmBL2 from Pyroccocus furiosus in detail. We used magnetic tweezers that allow to monitor changes of the DNA mechanical properties upon TrmBL2 binding on the single-molecule level. Extended filaments formed in a cooperative manner and were considerably stiffer than bare double-stranded DNA. Unlike Alba, TrmBL2 did not form DNA cross-bridges. The protein was found to bind double- and single-stranded DNA with similar affinities. In mechanical disruption experiments of DNA hairpins this led to stabilization of both, the double- (before disruption) and the single-stranded (after disruption) DNA forms. Combined, these findings suggest that the biological function of TrmBL2 is not limited to modulating genome architecture and acting as a global repressor but that the protein acts additionally as a stabilizer of DNA secondary structure.

  18. The COP9 signalosome is vital for timely repair of DNA double-strand breaks

    PubMed Central

    Meir, Michal; Galanty, Yaron; Kashani, Lior; Blank, Michael; Khosravi, Rami; Fernández-Ávila, María Jesús; Cruz-García, Andrés; Star, Ayelet; Shochot, Lea; Thomas, Yann; Garrett, Lisa J.; Chamovitz, Daniel A.; Bodine, David M.; Kurz, Thimo; Huertas, Pablo; Ziv, Yael; Shiloh, Yosef

    2015-01-01

    The DNA damage response is vigorously activated by DNA double-strand breaks (DSBs). The chief mobilizer of the DSB response is the ATM protein kinase. We discovered that the COP9 signalosome (CSN) is a crucial player in the DSB response and an ATM target. CSN is a protein complex that regulates the activity of cullin ring ubiquitin ligase (CRL) complexes by removing the ubiquitin-like protein, NEDD8, from their cullin scaffold. We find that the CSN is physically recruited to DSB sites in a neddylation-dependent manner, and is required for timely repair of DSBs, affecting the balance between the two major DSB repair pathways—nonhomologous end-joining and homologous recombination repair (HRR). The CSN is essential for the processivity of deep end-resection—the initial step in HRR. Cullin 4a (CUL4A) is recruited to DSB sites in a CSN- and neddylation-dependent manner, suggesting that CSN partners with CRL4 in this pathway. Furthermore, we found that ATM-mediated phosphorylation of CSN subunit 3 on S410 is critical for proper DSB repair, and that loss of this phosphorylation site alone is sufficient to cause a DDR deficiency phenotype in the mouse. This novel branch of the DSB response thus significantly affects genome stability. PMID:25855810

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

    PubMed Central

    De Vlaminck, Iwijn; Vidic, Iztok; van Loenhout, Marijn T. J.; Kanaar, Roland; Lebbink, Joyce H. G.; Dekker, Cees

    2010-01-01

    All cellular single-stranded (ss) DNA is rapidly bound and stabilized by single stranded DNA-binding proteins (SSBs). Replication protein A, the main eukaryotic SSB, is able to unwind double-stranded (ds) DNA by binding and stabilizing transiently forming bubbles of ssDNA. Here, we study the dynamics of human RPA (hRPA) activity on topologically constrained dsDNA with single-molecule magnetic tweezers. We find that the hRPA unwinding rate is exponentially dependent on torsion present in the DNA. The unwinding reaction is self-limiting, ultimately removing the driving torsional stress. The process can easily be reverted: release of tension or the application of a rewinding torque leads to protein dissociation and helix rewinding. Based on the force and salt dependence of the in vitro kinetics we anticipate that the unwinding reaction occurs frequently in vivo. We propose that the hRPA unwinding reaction serves to protect and stabilize the dsDNA when it is structurally destabilized by mechanical stresses. PMID:20197317

  20. Histone modifications and DNA double-strand break repair after exposure to ionizing radiations.

    PubMed

    Hunt, Clayton R; Ramnarain, Deepti; Horikoshi, Nobuo; Iyengar, Puneeth; Pandita, Raj K; Shay, Jerry W; Pandita, Tej K

    2013-04-01

    Ionizing radiation exposure induces highly lethal DNA double-strand breaks (DSBs) in all phases of the cell cycle. After DSBs are detected by the cellular machinery, these breaks are repaired by either of two mechanisms: (1) nonhomologous end joining (NHEJ), which re-ligates the broken ends of the DNA and (2) homologous recombination (HR), that makes use of an undamaged identical DNA sequence as a template to maintain the fidelity of DNA repair. DNA DSB repair must occur within the context of the natural cellular DNA structure. Among the major factors influencing DNA organization are specific histone and nonhistone proteins that form chromatin. The overall chromatin structure regulates DNA damage responses since chromatin status can impede DNA damage site access by repair proteins. During the process of DNA DSB repair, several chromatin alterations are required to sense damage and facilitate accessibility of the repair machinery. The DNA DSB response is also facilitated by hierarchical signaling networks that orchestrate chromatin structural changes that may coordinate cell-cycle checkpoints involving multiple enzymatic activities to repair broken DNA ends. During DNA damage sensing and repair, histones undergo posttranslational modifications (PTMs) including phosphorylation, acetylation, methylation and ubiquitylation. Such histone modifications represent a histone code that directs the recruitment of proteins involved in DNA damage sensing and repair processes. In this review, we summarize histone modifications that occur during DNA DSB repair processes.

  1. Triplex structures induce DNA double strand breaks via replication fork collapse in NER deficient cells

    PubMed Central

    Kaushik Tiwari, Meetu; Adaku, Nneoma; Peart, Natoya; Rogers, Faye A.

    2016-01-01

    Structural alterations in DNA can serve as natural impediments to replication fork stability and progression, resulting in DNA damage and genomic instability. Naturally occurring polypurine mirror repeat sequences in the human genome can create endogenous triplex structures evoking a robust DNA damage response. Failures to recognize or adequately process these genomic lesions can result in loss of genomic integrity. Nucleotide excision repair (NER) proteins have been found to play a prominent role in the recognition and repair of triplex structures. We demonstrate using triplex-forming oligonucleotides that chromosomal triplexes perturb DNA replication fork progression, eventually resulting in fork collapse and the induction of double strand breaks (DSBs). We find that cells deficient in the NER damage recognition proteins, XPA and XPC, accumulate more DSBs in response to chromosomal triplex formation than NER-proficient cells. Furthermore, we demonstrate that XPC-deficient cells are particularly prone to replication-associated DSBs in the presence of triplexes. In the absence of XPA or XPC, deleterious consequences of triplex-induced genomic instability may be averted by activating apoptosis via dual phosphorylation of the H2AX protein. Our results reveal that damage recognition by XPC and XPA is critical to maintaining replication fork integrity and preventing replication fork collapse in the presence of triplex structures. PMID:27298253

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

    PubMed Central

    Smeenk, Godelieve; Mailand, Niels

    2016-01-01

    DNA double-strand breaks (DSBs) are highly cytotoxic DNA lesions, whose faulty repair may alter the content and organization of cellular genomes. To counteract this threat, numerous signaling and repair proteins are recruited hierarchically to the chromatin areas surrounding DSBs to facilitate accurate lesion repair and restoration of genome integrity. In vertebrate cells, ubiquitin-dependent modifications of histones adjacent to DSBs by RNF8, RNF168, and other ubiquitin ligases have a key role in promoting the assembly of repair protein complexes, serving as direct recruitment platforms for a range of genome caretaker proteins and their associated factors. These DNA damage-induced chromatin ubiquitylation marks provide an essential component of a histone code for DSB repair that is controlled by multifaceted regulatory circuits, underscoring its importance for genome stability maintenance. In this review, we provide a comprehensive account of how DSB-induced histone ubiquitylation is sensed, decoded and modulated by an elaborate array of repair factors and regulators. We discuss how these mechanisms impact DSB repair pathway choice and functionality for optimal protection of genome integrity, as well as cell and organismal fitness. PMID:27446204

  3. Blueberry anthocyanins ameliorate radiation-induced lung injury through the protein kinase RNA-activated pathway.

    PubMed

    Liu, Yunen; Tan, Dehong; Tong, Changci; Zhang, Yubiao; Xu, Ying; Liu, Xinwei; Gao, Yan; Hou, Mingxiao

    2015-12-01

    The purpose of this study was to explore the effect of blueberry anthocyanins (BA) on radiation-induced lung injury and investigate the mechanism of action. Seven days after BA(20 and 80 mg/kg/d)administration, 6 weeks old male Sprague-Dawley rats rats were irradiated by LEKTA precise linear accelerator at a single dose of 20 Gy only once. and the rats were continuously treated with BA for 4 weeks. Moreover, human pulmonary alveolar epithelial cells (HPAEpiC) were transfected with either control-siRNA or siRNA targeting protein kinase R (PKR). Cells were then irradiated and treated with 75 μg/mL BA for 72 h. The results showed that BA significantly ameliorated radiation-induced lung inflammation, lung collagen deposition, apoptosis and PKR expression and activation. In vitro, BA significantly protected cells from radiation-induced cell death through modulating expression of Bcl-2, Bax and Caspase-3. Suppression of PKR by siRNA resulted in ablation of BA protection on radiation-induced cell death and modulation of anti-apoptotic and pro-apoptotic proteins, as well as Caspase-3 expression. These findings suggest that BA is effective in ameliorating radiation-induced lung injury, likely through the PKR signaling pathway. PMID:26551926

  4. Blueberry anthocyanins ameliorate radiation-induced lung injury through the protein kinase RNA-activated pathway.

    PubMed

    Liu, Yunen; Tan, Dehong; Tong, Changci; Zhang, Yubiao; Xu, Ying; Liu, Xinwei; Gao, Yan; Hou, Mingxiao

    2015-12-01

    The purpose of this study was to explore the effect of blueberry anthocyanins (BA) on radiation-induced lung injury and investigate the mechanism of action. Seven days after BA(20 and 80 mg/kg/d)administration, 6 weeks old male Sprague-Dawley rats rats were irradiated by LEKTA precise linear accelerator at a single dose of 20 Gy only once. and the rats were continuously treated with BA for 4 weeks. Moreover, human pulmonary alveolar epithelial cells (HPAEpiC) were transfected with either control-siRNA or siRNA targeting protein kinase R (PKR). Cells were then irradiated and treated with 75 μg/mL BA for 72 h. The results showed that BA significantly ameliorated radiation-induced lung inflammation, lung collagen deposition, apoptosis and PKR expression and activation. In vitro, BA significantly protected cells from radiation-induced cell death through modulating expression of Bcl-2, Bax and Caspase-3. Suppression of PKR by siRNA resulted in ablation of BA protection on radiation-induced cell death and modulation of anti-apoptotic and pro-apoptotic proteins, as well as Caspase-3 expression. These findings suggest that BA is effective in ameliorating radiation-induced lung injury, likely through the PKR signaling pathway.

  5. Intermediate States of Ribonuclease III in Complex with Double-Stranded RNA

    SciTech Connect

    Gan, Jianhua; Tropea, Joseph E.; Austin, Brian P.; Court, Donald L.; Waugh, David S.; Ji, Xinhua

    2010-07-19

    Bacterial ribonuclease III (RNase III) can affect RNA structure and gene expression in either of two ways: as a processing enzyme that cleaves double-stranded (ds) RNA, or as a binding protein that binds but does not cleave dsRNA. We previously proposed a model of the catalytic complex of RNase III with dsRNA based on three crystal structures, including the endonuclease domain of RNase III with and without bound metal ions and a dsRNA binding protein complexed with dsRNA. We also reported a noncatalytic assembly observed in the crystal structure of an RNase III mutant, which binds but does not cleave dsRNA, complexed with dsRNA. We hypothesize that the RNase III {center_dot} dsRNA complex can exist in two functional forms, a catalytic complex and a noncatalytic assembly, and that in between the two forms there may be intermediate states. Here, we present four crystal structures of RNase III complexed with dsRNA, representing possible intermediates.

  6. Atrazine Triggers DNA Damage Response and Induces DNA Double-Strand Breaks in MCF-10A Cells.

    PubMed

    Huang, Peixin; Yang, John; Ning, Jie; Wang, Michael; Song, Qisheng

    2015-06-24

    Atrazine, a pre-emergent herbicide in the chloro-s-triazine family, has been widely used in crop lands and often detected in agriculture watersheds, which is considered as a potential threat to human health. Although atrazine and its metabolites showed an elevated incidence of mammary tumors in female Sprague-Dawley (SD) rats, no molecular evidence was found relevant to its carcinogenesis in humans. This study aims to determine whether atrazine could induce the expression of DNA damage response-related proteins in normal human breast epithelial cells (MCF-10A) and to examine the cytotoxicity of atrazine at a molecular level. Our results indicate that a short-term exposure of MCF-10A to an environmentally-detectable concentration of atrazine (0.1 µg/mL) significantly increased the expression of tumor necrosis factor receptor-1 (TNFR1) and phosphorylated Rad17 in the cells. Atrazine treatment increased H2AX phosphorylation (γH2AX) and the formation of γH2AX foci in the nuclei of MCF-10A cells. Atrazine also sequentially elevated DNA damage checkpoint proteins of ATM- and RAD3-related (ATR), ATRIP and phospho-Chk1, suggesting that atrazine could induce DNA double-strand breaks and trigger the DNA damage response ATR-Chk1 pathway in MCF-10A cells. Further investigations are needed to determine whether atrazine-triggered DNA double-strand breaks and DNA damage response ATR-Chk1 pathway occur in vivo.

  7. Multiple pathways of DNA double-strand break processing in a mutant Indian muntjac cell line

    SciTech Connect

    Bouffler, S.D.; Jha, B.; Johnson, R.T. )

    1990-09-01

    DNA break processing is compared in the Indian muntjac cell lines, SVM and DM. The initial frequencies and resealing of X-ray generated single- and double-strand breaks are similar in the two cell lines. Inhibiting the repair of UV damage leads to greater double-strand breakage in SVM than in DM, and some of these breaks are not repaired; however, repair-associated single-strand breakage and resealing are normal. Dimethylsulfate also induces excess double-strand breakage in SVM, and these breaks are irreparable. Restricted plasmids are reconstituted correctly in SVM at approximately 30% of the frequency observed in DM. Thus SVM has a reduced capacity to repair certain types of double-strand break. This defect is not due to a DNA ligase deficiency. We conclude that DNA double-strand breaks are repaired by a variety of pathways within mammalian cells and that the structure of the break or its mode of formation determines its subsequent fate.

  8. Theory for the conformational changes of double-stranded chain molecules

    NASA Astrophysics Data System (ADS)

    Chen, Shi-Jie; Dill, Ken A.

    1998-09-01

    We develop statistical mechanical theory to predict the thermodynamic properties of chain molecules having noncovalent double-stranded conformations, as in RNA molecules and β-sheets in proteins. Sequence dependence and excluded volume interactions are explicitly taken into account. We classify conformations by their polymer graphs and enumerate all the conformations corresponding to each graph by a recently developed matrix method [S-J. Chen and K. A. Dill, J. Chem. Phys. 103, 5802 (1995)]. All such graphs are summed by a recursive method. Tests against exact computer enumeration for short chains on a 2D lattice show that the density of states and partition function are given quite accurately. So far, we have explored two classes of conformations; hairpins, which model small β-sheets, and RNA secondary structures. The main folding transition is predicted to be quite different for these two conformational classes: the hairpin transition is two-state while the RNA secondary structure transition is one-state for homopolymeric chains.

  9. A three-dimensional statistical mechanical model of folding double-stranded chain molecules

    NASA Astrophysics Data System (ADS)

    Zhang, Wenbing; Chen, Shi-Jie

    2001-05-01

    Based on a graphical representation of intrachain contacts, we have developed a new three-dimensional model for the statistical mechanics of double-stranded chain molecules. The theory has been tested and validated for the cubic lattice chain conformations. The statistical mechanical model can be applied to the equilibrium folding thermodynamics of a large class of chain molecules, including protein β-hairpin conformations and RNA secondary structures. The application of a previously developed two-dimensional model to RNA secondary structure folding thermodynamics generally overestimates the breadth of the melting curves [S-J. Chen and K. A. Dill, Proc. Natl. Acad. Sci. U.S.A. 97, 646 (2000)], suggesting an underestimation for the sharpness of the conformational transitions. In this work, we show that the new three-dimensional model gives much sharper melting curves than the two-dimensional model. We believe that the new three-dimensional model may give much improved predictions for the thermodynamic properties of RNA conformational changes than the previous two-dimensional model.

  10. RNA interference in the termite Reticulitermes flavipes through ingestion of double-stranded RNA.

    PubMed

    Zhou, Xuguo; Wheeler, Marsha M; Oi, Faith M; Scharf, Michael E

    2008-08-01

    RNA interference (RNAi) represents a breakthrough technology for conducting functional genomics research in non-model organisms and for the highly targeted control of insect pests. This study investigated RNAi via voluntary feeding in the economically important pest termite, Reticulitermes flavipes. We used a high-dose double-stranded (ds) RNA feeding approach to silence two termite genes: one encoding an endogenous digestive cellulase enzyme and the other a caste-regulatory hexamerin storage protein. Contrary to results from previous low-dose studies that examined injection-based RNAi, high-dose silencing of either gene through dsRNA feeding led to significantly reduced group fitness and mortality. Hexamerin silencing in combination with ectopic juvenile hormone treatments additionally led to lethal molting impacts and increased differentiation of presoldier caste phenotypes (a phenotype that is not capable of feeding). These results provide the first examples of insecticidal effects from dsRNA feeding in a termite. Additionally, these results validate a high-throughput bioassay approach for use in (i) termite functional genomics research, and (ii) characterizing target sites of conventional and novel RNAi-based termiticides. PMID:18625404

  11. Prolonged gene knockdown in the tsetse fly Glossina by feeding double stranded RNA.

    PubMed

    Walshe, D P; Lehane, S M; Lehane, M J; Haines, L R

    2009-02-01

    Reverse genetic studies based on RNA interference (RNAi) have revolutionized analysis of gene function in most insects. However the necessity of injecting double stranded RNA (dsRNA) inevitably compromises many investigations particularly those on immunity. Additionally, injection of tsetse flies often causes significant mortality. We demonstrate, at transcript and protein level, that delivering dsRNA in the bloodmeal to Glossina morsitans morsitans is as effective as injection in knockdown of the immunoresponsive midgut-expressed gene TsetseEP. However, feeding dsRNA fails to knockdown the fat body expressed transferrin gene, 2A192, previously shown to be silenced by dsRNA injection. Mortality rates of the dsRNA fed flies were significantly reduced compared to injected flies 14 days after treatment (Fed: 10.1%+/- 1.8%; injected: 37.9% +/- 3.6% (Mean +/- SEM)). This is the first demonstration in Diptera of gene knockdown by feeding and the first example of knockdown in a blood-sucking insect by including dsRNA in the bloodmeal. PMID:19016913

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

    PubMed

    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

  13. Widespread horizontal gene transfer from double-stranded RNA viruses to eukaryotic nuclear genomes.

    PubMed

    Liu, Huiquan; Fu, Yanping; Jiang, Daohong; Li, Guoqing; Xie, Jiatao; Cheng, Jiasen; Peng, Youliang; Ghabrial, Said A; Yi, Xianhong

    2010-11-01

    Horizontal gene transfer commonly occurs from cells to viruses but rarely occurs from viruses to their host cells, with the exception of retroviruses and some DNA viruses. However, extensive sequence similarity searches in public genome databases for various organisms showed that the capsid protein and RNA-dependent RNA polymerase genes from totiviruses and partitiviruses have widespread homologs in the nuclear genomes of eukaryotic organisms, including plants, arthropods, fungi, nematodes, and protozoa. PCR amplification and sequencing as well as comparative evidence of junction coverage between virus and host sequences support the conclusion that these viral homologs are real and occur in eukaryotic genomes. Sequence comparison and phylogenetic analysis suggest that these genes were likely transferred horizontally from viruses to eukaryotic genomes. Furthermore, we present evidence showing that some of the transferred genes are conserved and expressed in eukaryotic organisms and suggesting that these viral genes are also functional in the recipient genomes. Our findings imply that horizontal transfer of double-stranded RNA viral genes is widespread among eukaryotes and may give rise to functionally important new genes, thus entailing that RNA viruses may play significant roles in the evolution of eukaryotes.

  14. Coordinated nuclease activities counteract Ku at single-ended DNA double-strand breaks

    PubMed Central

    Chanut, Pauline; Britton, Sébastien; Coates, Julia; Jackson, Stephen P.; Calsou, Patrick

    2016-01-01

    Repair of single-ended DNA double-strand breaks (seDSBs) by homologous recombination (HR) requires the generation of a 3′ single-strand DNA overhang by exonuclease activities in a process called DNA resection. However, it is anticipated that the highly abundant DNA end-binding protein Ku sequesters seDSBs and shields them from exonuclease activities. Despite pioneering works in yeast, it is unclear how mammalian cells counteract Ku at seDSBs to allow HR to proceed. Here we show that in human cells, ATM-dependent phosphorylation of CtIP and the epistatic and coordinated actions of MRE11 and CtIP nuclease activities are required to limit the stable loading of Ku on seDSBs. We also provide evidence for a hitherto unsuspected additional mechanism that contributes to prevent Ku accumulation at seDSBs, acting downstream of MRE11 endonuclease activity and in parallel with MRE11 exonuclease activity. Finally, we show that Ku persistence at seDSBs compromises Rad51 focus assembly but not DNA resection. PMID:27641979

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

    PubMed Central

    Rao, Venigalla B.; Feiss, Michael

    2016-01-01

    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

  16. The proteasomal de-ubiquitinating enzyme POH1 promotes the double-strand DNA break response

    PubMed Central

    Butler, Laura R; Densham, Ruth M; Jia, Junying; Garvin, Alexander J; Stone, Helen R; Shah, Vandna; Weekes, Daniel; Festy, Frederic; Beesley, James; Morris, Joanna R

    2012-01-01

    The regulation of Ubiquitin (Ub) conjugates generated by the complex network of proteins that promote the mammalian DNA double-strand break (DSB) response is not fully understood. We show here that the Ub protease POH1/rpn11/PSMD14 resident in the 19S proteasome regulatory particle is required for processing poly-Ub formed in the DSB response. Proteasome activity is required to restrict tudor domain-dependent 53BP1 accumulation at sites of DNA damage. This occurs both through antagonism of RNF8/RNF168-mediated lysine 63-linked poly-Ub and through the promotion of JMJD2A retention on chromatin. Consistent with this role POH1 acts in opposition to RNF8/RNF168 to modulate end-joining DNA repair. Additionally, POH1 acts independently of 53BP1 in homologous recombination repair to promote RAD51 loading. Accordingly, POH1-deficient cells are sensitive to DNA damaging agents. These data demonstrate that proteasomal POH1 is a key de-ubiquitinating enzyme that regulates ubiquitin conjugates generated in response to damage and that several aspects of the DSB response are regulated by the proteasome. PMID:22909820

  17. Proliferation of Double-Strand Break-Resistant Polyploid Cells Requires Drosophila FANCD2.

    PubMed

    Bretscher, Heidi S; Fox, Donald T

    2016-06-01

    Conserved DNA-damage responses (DDRs) sense genome damage and prevent mitosis of broken chromosomes. How cells lacking DDRs cope with broken chromosomes during mitosis is poorly understood. DDRs are frequently inactivated in cells with extra genomes (polyploidy), suggesting that study of polyploidy can reveal how cells with impaired DDRs/genome damage continue dividing. Here, we show that continued division and normal organ development occurs in polyploid, DDR-impaired Drosophila papillar cells. As papillar cells become polyploid, they naturally accumulate broken acentric chromosomes but do not apoptose/arrest the cell cycle. To survive mitosis with acentric chromosomes, papillar cells require Fanconi anemia proteins FANCD2 and FANCI, as well as Blm helicase, but not canonical DDR signaling. FANCD2 acts independently of previous S phases to promote alignment and segregation of acentric DNA produced by double-strand breaks, thus avoiding micronuclei and organ malformation. Because polyploidy and impaired DDRs can promote cancer, our findings provide insight into disease-relevant DNA-damage tolerance mechanisms. PMID:27270041

  18. Real-time analysis of double-strand DNA break repair by homologous recombination

    PubMed Central

    Hicks, Wade M.; Yamaguchi, Miyuki; Haber, James E.

    2011-01-01

    The ability to induce synchronously a single site-specific double-strand break (DSB) in a budding yeast chromosome has made it possible to monitor the kinetics and genetic requirements of many molecular steps during DSB repair. Special attention has been paid to the switching of mating-type genes in Saccharomyces cerevisiae, a process initiated by the HO endonuclease by cleaving the MAT locus. A DSB in MATa is repaired by homologous recombination—specifically, by gene conversion—using a heterochromatic donor, HMLα. Repair results in the replacement of the a-specific sequences (Ya) by Yα and switching from MATa to MATα. We report that MAT switching requires the DNA replication factor Dpb11, although it does not require the Cdc7-Dbf4 kinase or the Mcm and Cdc45 helicase components. Using Southern blot, PCR, and ChIP analysis of samples collected every 10 min, we extend previous studies of this process to identify the times for the loading of Rad51 recombinase protein onto the DSB ends at MAT, the subsequent strand invasion by the Rad51 nucleoprotein filament into the donor sequences, the initiation of new DNA synthesis, and the removal of the nonhomologous Y sequences. In addition we report evidence for the transient displacement of well-positioned nucleosomes in the HML donor locus during strand invasion. PMID:21292986

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

    PubMed Central

    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

  20. Tel1(ATM)-mediated interference suppresses clustered meiotic double-strand-break formation.

    PubMed

    Garcia, Valerie; Gray, Stephen; Allison, Rachal M; Cooper, Tim J; Neale, Matthew J

    2015-04-01

    Meiotic recombination is a critical step in gametogenesis for many organisms, enabling the creation of genetically diverse haploid gametes. In each meiotic cell, recombination is initiated by numerous DNA double-strand breaks (DSBs) created by Spo11, the evolutionarily conserved topoisomerase-like protein, but how these DSBs are distributed relatively uniformly across the four chromatids that make up each chromosome pair is poorly understood. Here we employ Saccharomyces cerevisiae to demonstrate distance-dependent DSB interference in cis (in which the occurrence of a DSB suppresses adjacent DSB formation)--a process that is mediated by the conserved DNA damage response kinase, Tel1(ATM). The inhibitory function of Tel1 acts on a relatively local scale, while over large distances DSBs have a tendency to form independently of one another even in the presence of Tel1. Notably, over very short distances, loss of Tel1 activity causes DSBs to cluster within discrete zones of concerted DSB activity. Our observations support a hierarchical view of recombination initiation where Tel1(ATM) prevents clusters of DSBs, and further suppresses DSBs within the surrounding chromosomal region. Such collective negative regulation will help to ensure that recombination events are dispersed evenly and arranged optimally for genetic exchange and efficient chromosome segregation. PMID:25539084

  1. BRC-1 acts in the inter-sister pathway of meiotic double-strand break repair.

    PubMed

    Adamo, Adele; Montemauri, Paolo; Silva, Nicola; Ward, Jordan D; Boulton, Simon J; La Volpe, Adriana

    2008-03-01

    The breast and ovarian cancer susceptibility protein BRCA1 is evolutionarily conserved and functions in DNA double-strand break (DSB) repair through homologous recombination, but its role in meiosis is poorly understood. By using genetic analysis, we investigated the role of the Caenorhabditis elegans BRCA1 orthologue (brc-1) during meiotic prophase. The null mutant in the brc-1 gene is viable, fertile and shows the wild-type complement of six bivalents in most diakinetic nuclei, which is indicative of successful crossover recombination. However, brc-1 mutants show an abnormal increase in apoptosis and RAD-51 foci at pachytene that are abolished by loss of spo-11 function, suggesting a defect in meiosis rather than during premeiotic DNA replication. In genetic backgrounds in which chiasma formation is abrogated, such as him-14/MSH4 and syp-2, loss of brc-1 leads to chromosome fragmentation suggesting that brc-1 is dispensable for crossing over but essential for DSB repair through inter-sister recombination.

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

    PubMed Central

    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

  3. Real-time analysis of double-strand DNA break repair by homologous recombination.

    PubMed

    Hicks, Wade M; Yamaguchi, Miyuki; Haber, James E

    2011-02-22

    The ability to induce synchronously a single site-specific double-strand break (DSB) in a budding yeast chromosome has made it possible to monitor the kinetics and genetic requirements of many molecular steps during DSB repair. Special attention has been paid to the switching of mating-type genes in Saccharomyces cerevisiae, a process initiated by the HO endonuclease by cleaving the MAT locus. A DSB in MATa is repaired by homologous recombination--specifically, by gene conversion--using a heterochromatic donor, HMLα. Repair results in the replacement of the a-specific sequences (Ya) by Yα and switching from MATa to MATα. We report that MAT switching requires the DNA replication factor Dpb11, although it does not require the Cdc7-Dbf4 kinase or the Mcm and Cdc45 helicase components. Using Southern blot, PCR, and ChIP analysis of samples collected every 10 min, we extend previous studies of this process to identify the times for the loading of Rad51 recombinase protein onto the DSB ends at MAT, the subsequent strand invasion by the Rad51 nucleoprotein filament into the donor sequences, the initiation of new DNA synthesis, and the removal of the nonhomologous Y sequences. In addition we report evidence for the transient displacement of well-positioned nucleosomes in the HML donor locus during strand invasion.

  4. Coordinated nuclease activities counteract Ku at single-ended DNA double-strand breaks.

    PubMed

    Chanut, Pauline; Britton, Sébastien; Coates, Julia; Jackson, Stephen P; Calsou, Patrick

    2016-01-01

    Repair of single-ended DNA double-strand breaks (seDSBs) by homologous recombination (HR) requires the generation of a 3' single-strand DNA overhang by exonuclease activities in a process called DNA resection. However, it is anticipated that the highly abundant DNA end-binding protein Ku sequesters seDSBs and shields them from exonuclease activities. Despite pioneering works in yeast, it is unclear how mammalian cells counteract Ku at seDSBs to allow HR to proceed. Here we show that in human cells, ATM-dependent phosphorylation of CtIP and the epistatic and coordinated actions of MRE11 and CtIP nuclease activities are required to limit the stable loading of Ku on seDSBs. We also provide evidence for a hitherto unsuspected additional mechanism that contributes to prevent Ku accumulation at seDSBs, acting downstream of MRE11 endonuclease activity and in parallel with MRE11 exonuclease activity. Finally, we show that Ku persistence at seDSBs compromises Rad51 focus assembly but not DNA resection. PMID:27641979

  5. Isolation of Additional Bacteriophages with Genomes of Segmented Double-Stranded RNA

    PubMed Central

    Mindich, Leonard; Qiao, Xueying; Qiao, Jian; Onodera, Shiroh; Romantschuk, Martin; Hoogstraten, Deborah

    1999-01-01

    Eight different bacteriophages were isolated from leaves of Pisum sativum, Phaseolus vulgaris, Lycopersicon esculentum, Daucus carota sativum, Raphanus sativum, and Ocimum basilicum. All contain three segments of double-stranded RNA and have genomic-segment sizes that are similar but not identical to those of previously described bacteriophage φ6. All appear to have lipid-containing membranes. The base sequences of some of the viruses are very similar but not identical to those of φ6. Three of the viruses have little or no base sequence identity to φ6. Two of the viruses, φ8 and φ12, contain proteins with a size distribution very different from that of φ6 and do not package genomic segments of φ6. Whereas φ6 attaches to host cells by means of a pilus, several of the new isolates attach directly to the outer membrane. Although the normal hosts of these viruses seem to be pseudomonads, those viruses that attach directly to the outer membrane can establish carrier states in Escherichia coli or Salmonella typhimurium. One of the isolates, φ8, can form plaques on heptoseless strains of S. typhimurium. PMID:10419946

  6. Chronic glial activation, neurodegeneration, and APP immunoreactive deposits following acute administration of double-stranded RNA.

    PubMed

    Melton, Lisa M; Keith, Alexander B; Davis, Sue; Oakley, Arthur E; Edwardson, James A; Morris, Christopher M

    2003-10-01

    Several neurodegenerative disorders, including Alzheimer's and Parkinson's diseases, are associated with immunocompetent microglia, leading to the suggestion that chronic glial-mediated inflammation contributes to the neurodegeneration seen in these diseases. Little direct evidence supports this hypothesis, and no suitable rodent models exist that do not involve the use of blunt trauma or ischaemia, events that are infrequently encountered in the human disease state. In the present study, we report that administration of double-stranded RNA, a classical inducer of interferon-gamma (IFN-gamma), causes rapid and persistent activation of microglia and astrocytes, as well as induction of interleukin-1beta (IL-beta) and nitric oxide synthase. In close temporal succession to glial activation, there is neurodegeneration, with neuron loss involving apoptosis in selected brain regions including the septal nucleus, hippocampus, cortex and thalamus, along with hippocampal atrophy. This neuronal loss is accompanied by punctate deposits of material that are immunoreactive for amyloid precursor protein, beta-amyloid peptide (Abeta), and apolipoprotein E. The findings may have clinical relevance, since the administration of the nonsteroidal antiinflammatory agent (NSAID) ibuprofen markedly reduces the neurodegeneration observed in the absence of significant glial inhibition. These findings may be relevant to the pathogenesis of Alzheimer's disease in particular, and to other neurodegenerative diseases involving inflammation.

  7. Sulforaphane induces DNA double strand breaks predominantly repaired by homologous recombination pathway in human cancer cells

    SciTech Connect

    Sekine-Suzuki, Emiko; Yu, Dong; Kubota, Nobuo; Okayasu, Ryuichi; Anzai, Kazunori

    2008-12-12

    Cytotoxicity and DNA double strand breaks (DSBs) were studied in HeLa cells treated with sulforaphane (SFN), a well-known chemo-preventive agent. Cell survival was impaired by SFN in a concentration and treatment time-dependent manner. Both constant field gel electrophoresis (CFGE) and {gamma}-H2AX assay unambiguously indicated formation of DSBs by SFN, reflecting the cell survival data. These DSBs were predominantly processed by homologous recombination repair (HRR), judging from the SFN concentration-dependent manner of Rad51 foci formation. On the other hand, the phosphorylation of DNA-PKcs, a key non-homologous end joining (NHEJ) protein, was not observed by SFN treatment, suggesting that NHEJ may not be involved in DSBs induced by this chemical. G2/M arrest by SFN, a typical response for cells exposed to ionizing radiation was also observed. Our new data indicate the clear induction of DSBs by SFN and a useful anti-tumor aspect of SFN through the induction of DNA DSBs.

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

    NASA Astrophysics Data System (ADS)

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

    2011-12-01

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

  9. DNA Double-Strand Breaks and Telomeres Play Important Roles in Trypanosoma brucei Antigenic Variation

    PubMed Central

    2015-01-01

    Human-infecting microbial pathogens all face a serious problem of elimination by the host immune response. Antigenic variation is an effective immune evasion mechanism where the pathogen regularly switches its major surface antigen. In many cases, the major surface antigen is encoded by genes from the same gene family, and its expression is strictly monoallelic. Among pathogens that undergo antigenic variation, Trypanosoma brucei (a kinetoplastid), which causes human African trypanosomiasis, Plasmodium falciparum (an apicomplexan), which causes malaria, Pneumocystis jirovecii (a fungus), which causes pneumonia, and Borrelia burgdorferi (a bacterium), which causes Lyme disease, also express their major surface antigens from loci next to the telomere. Except for Plasmodium, DNA recombination-mediated gene conversion is a major pathway for surface antigen switching in these pathogens. In the last decade, more sophisticated molecular and genetic tools have been developed in T. brucei, and our knowledge of functions of DNA recombination in antigenic variation has been greatly advanced. VSG is the major surface antigen in T. brucei. In subtelomeric VSG expression sites (ESs), VSG genes invariably are flanked by a long stretch of upstream 70-bp repeats. Recent studies have shown that DNA double-strand breaks (DSBs), particularly those in 70-bp repeats in the active ES, are a natural potent trigger for antigenic variation in T. brucei. In addition, telomere proteins can influence VSG switching by reducing the DSB amount at subtelomeric regions. These findings will be summarized and their implications will be discussed in this review. PMID:25576484

  10. Identification of a pKa-regulating motif stabilizing imidazole-modified double-stranded DNA

    PubMed Central

    Buyst, Dieter; Gheerardijn, Vicky; Fehér, Krisztina; Van Gasse, Bjorn; Van Den Begin, Jos; Martins, José C.; Madder, Annemieke

    2015-01-01

    The predictable 3D structure of double-stranded DNA renders it ideally suited as a template for the bottom-up design of functionalized nucleic acid-based active sites. We here explore the use of a 14mer DNA duplex as a scaffold for the precise and predictable positioning of catalytic functionalities. Given the ubiquitous participation of the histidine-based imidazole group in protein recognition and catalysis events, single histidine-like modified duplexes were investigated. Tethering histamine to the C5 of the thymine base via an amide bond, allows the flexible positioning of the imidazole function in the major groove. The mutual interactions between the imidazole and the duplex and its influence on the imidazolium pKaH are investigated by placing a single modified thymine at four different positions in the center of the 14mer double helix. Using NMR and unrestrained molecular dynamics, a structural motif involving the formation of a hydrogen bond between the imidazole and the Hoogsteen side of the guanine bases of two neighboring GC base pairs is established. The motif contributes to a stabilization against thermal melting of 6°C and is key in modulating the pKaH of the imidazolium group. The general features, prerequisites and generic character of the new pKaH-regulating motif are described. PMID:25520197

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

    PubMed Central

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

    PubMed

    Lettier, Gaëlle; Feng, Qi; de Mayolo, Adriana Antúnez; Erdeniz, Naz; Reid, Robert J D; Lisby, Michael; Mortensen, Uffe H; Rothstein, Rodney

    2006-11-10

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

  13. Simultaneous characterization of sense and antisense genomic processes by the double-stranded hidden Markov model.

    PubMed

    Glas, Julia; Dümcke, Sebastian; Zacher, Benedikt; Poron, Don; Gagneur, Julien; Tresch, Achim

    2016-03-18

    Hidden Markov models (HMMs) have been extensively used to dissect the genome into functionally distinct regions using data such as RNA expression or DNA binding measurements. It is a challenge to disentangle processes occurring on complementary strands of the same genomic region. We present the double-stranded HMM (dsHMM), a model for the strand-specific analysis of genomic processes. We applied dsHMM to yeast using strand specific transcription data, nucleosome data, and protein binding data for a set of 11 factors associated with the regulation of transcription.The resulting annotation recovers the mRNA transcription cycle (initiation, elongation, termination) while correctly predicting strand-specificity and directionality of the transcription process. We find that pre-initiation complex formation is an essentially undirected process, giving rise to a large number of bidirectional promoters and to pervasive antisense transcription. Notably, 12% of all transcriptionally active positions showed simultaneous activity on both strands. Furthermore, dsHMM reveals that antisense transcription is specifically suppressed by Nrd1, a yeast termination factor. PMID:26578558

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

    PubMed

    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

  15. High levels of double-stranded transferred DNA (T-DNA) processing from an intact nopaline Ti plasmid.

    PubMed

    Steck, T R; Close, T J; Kado, C I

    1989-04-01

    To obtain bacterial-mediated oncogenic transformation of plants, the transferred DNA (T-DNA) of the tumor-inducing (Ti) plasmid of Agrobacterium tumefaciens is transferred to its plant host cells during infection. The initial phases of transformation involve the processing of the T-DNA in the bacterial cell after induction of the vir genes located on the Ti plasmid. The kinetics and conditions of this processing were examined and upon induction with acetosyringone up to 40% of the left and right borders of the T-DNA were cleaved. This cleavage was dependent upon virA, virG, and VirD and was rec-independent. Processed T-DNA was observed within 30 min after induction and was delayed by an increased concentration of phosphate in the induction medium. When DNA was isolated in the absence of protease treatment, the DNA fragment corresponding to the left side of the cut at both the left and right border region exhibited gel retardation, suggesting one or more "pilot" proteins may be involved in T-DNA transfer. Although the relative abundance of a processed product does not necessarily imply relative importance, the preponderance of double-stranded cleavage products suggests that double-stranded T-DNA should be considered as a possible intermediate in T-DNA transfer.

  16. Analysis of BRCA1 Variants in Double-Strand Break Repair by Homologous Recombination and Single-Strand Annealing

    PubMed Central

    Towler, William I.; Zhang, Jie; Ransburgh, Derek J. R.; Toland, Amanda E.; Ishioka, Chikashi; Chiba, Natsuko; Parvin, Jeffrey D.

    2014-01-01

    Missense substitutions of uncertain clinical significance in the BRCA1 gene are a vexing problem in genetic counseling for women who have a family history of breast cancer. In this study, we evaluated the functions of 29 missense substitutions of BRCA1 in two DNA repair pathways. Repair of double-strand breaks by homology-directed recombination (HDR) had been previously analyzed for 16 of these BRCA1 variants, and 13 more variants were analyzed in this study. All 29 variants were also analyzed for function in double-strand break repair by the single-strand annealing (SSA) pathway. We found that among the pathogenic mutations in BRCA1, all were defective for DNA repair by either pathway. The HDR assay was accurate because all pathogenic mutants were defective for HDR, and all nonpathogenic variants were fully functional for HDR. Repair by SSA accurately identified pathogenic mutants, but several nonpathogenic variants were scored as defective or partially defective. These results indicated that specific amino acid residues of the BRCA1 protein have different effects in the two related DNA repair pathways, and these results validate the HDR assay as highly correlative with BRCA1-associated breast cancer. PMID:23161852

  17. A Small Molecule Inhibitor of Polycomb Repressive Complex 1 Inhibits Ubiquitin Signaling at DNA Double-strand Breaks*

    PubMed Central

    Ismail, Ismail Hassan; McDonald, Darin; Strickfaden, Hilmar; Xu, Zhizhong; Hendzel, Michael J.

    2013-01-01

    Polycomb-repressive complex 1 (PRC1)-mediated histone ubiquitylation plays an important role in aberrant gene silencing in human cancers and is a potential target for cancer therapy. Here we show that 2-pyridine-3-yl-methylene-indan-1,3-dione (PRT4165) is a potent inhibitor of PRC1-mediated H2A ubiquitylation in vivo and in vitro. The drug also inhibits the accumulation of all detectable ubiquitin at sites of DNA double-strand breaks (DSBs), the retention of several DNA damage response proteins in foci that form around DSBs, and the repair of the DSBs. In vitro E3 ubiquitin ligase activity assays revealed that PRT4165 inhibits both RNF2 and RING 1A, which are partially redundant paralogues that together account for the E3 ubiquitin ligase activity found in PRC1 complexes, but not RNF8 nor RNF168. Because ubiquitylation is completely inhibited despite the efficient recruitment of RNF8 to DSBs, our results suggest that PRC1-mediated monoubiquitylation is required for subsequent RNF8- and/or RNF168-mediated polyubiquitylation. Our results demonstrate the unique feature of PRT4165 as a novel chromatin-remodeling compound and provide a new tool for the inhibition of ubiquitylation signaling at DNA double-strand breaks. PMID:23902761

  18. Selective contribution of IFN-/ signaling to the maturation of dendritic cells induced by double-stranded RNA or viral infection

    NASA Astrophysics Data System (ADS)

    Honda, Kenya; Sakaguchi, Shinya; Nakajima, Chigusa; Watanabe, Ai; Yanai, Hideyuki; Matsumoto, Misako; Ohteki, Toshiaki; Kaisho, Tsuneyasu; Takaoka, Akinori; Akira, Shizuo; Seya, Tsukasa; Taniguchi, Tadatsugu

    2003-09-01

    A complex mechanism may be operational for dendritic cell (DC) maturation, wherein Toll-like receptor and other signaling pathways may be coordinated differently depending on the nature of the pathogens, in order for DC maturation to be most effective to a given threat. Here, we show that IFN-/ signaling is selectively required for the maturation of DCs induced by double-stranded RNA or viral infection in vitro. Interestingly, the maturation is still observed in the absence of either of the two target genes of IFN-/, TLR3 and PKR (double-stranded-RNA-dependent protein kinase R), indicating the complexity of the IFN-/-induced transcriptional program in DCs. We also show that the DCs stimulated in vivo by these agents can migrate into the T cell zone of the spleen but fail to mature without the IFN signal. The immune system may have acquired the selective utilization of this cytokine system, which is essential for innate antiviral immunity, to effectively couple with the induction of adaptive immunity.

  19. DNA double-strand breaks with 5' adducts are efficiently channeled to the DNA2-mediated resection pathway.

    PubMed

    Tammaro, Margaret; Liao, Shuren; Beeharry, Neil; Yan, Hong

    2016-01-01

    DNA double-strand breaks (DSBs) with 5' adducts are frequently formed from many nucleic acid processing enzymes, in particular DNA topoisomerase 2 (TOP2). The key intermediate of TOP2 catalysis is the covalent complex (TOP2cc), consisting of two TOP2 subunits covalently linked to the 5' ends of the nicked DNA. In cells, TOP2ccs can be trapped by cancer drugs such as etoposide and then converted into DNA double-strand breaks (DSBs) that carry adducts at the 5' end. The repair of such DSBs is critical to the survival of cells, but the underlying mechanism is still not well understood. We found that etoposide-induced DSBs are efficiently resected into 3' single-stranded DNA in cells and the major nuclease for resection is the DNA2 protein. DNA substrates carrying model 5' adducts were efficiently resected in Xenopus egg extracts and immunodepletion of Xenopus DNA2 also strongly inhibited resection. These results suggest that DNA2-mediated resection is a major mechanism for the repair of DSBs with 5' adducts.

  20. Contribution of DNA double-strand break repair gene XRCC3 genotypes to oral cancer susceptibility in Taiwan.

    PubMed

    Tsai, Chia-Wen; Chang, Wen-Shin; Liu, Juhn-Cherng; Tsai, Ming-Hsui; Lin, Cheng-Chieh; Bau, Da-Tian

    2014-06-01

    The DNA repair gene X-ray repair cross complementing protein 3 (XRCC3) is thought to play a major role in double-strand break repair and in maintaining genomic stability. Very possibly, defective double-strand break repair of cells can lead to carcinogenesis. Therefore, a case-control study was performed to reveal the contribution of XRCC3 genotypes to individual oral cancer susceptibility. In this hospital-based research, the association of XRCC3 rs1799794, rs45603942, rs861530, rs3212057, rs1799796, rs861539, rs28903081 genotypes with oral cancer risk in a Taiwanese population was investigated. In total, 788 patients with oral cancer and 956 age- and gender-matched healthy controls were genotyped. The results showed that there was significant differential distribution among oral cancer and controls in the genotypic (p=0.001428) and allelic (p=0.0013) frequencies of XRCC3 rs861539. As for the other polymorphisms, there was no difference between case and control groups. In gene-lifestyle interaction analysis, we have provided the first evidence showing that there is an obvious joint effect of XRCC3 rs861539 genotype with individual areca chewing habits on oral cancer risk. In conclusion, the T allele of XRCC3 rs861539, which has an interaction with areca chewing habit in oral carcinogenesis, may be an early marker for oral cancer in Taiwanese.

  1. Double strand breaks may be a missing link between entropy and aging.

    PubMed

    Lenart, Peter; Bienertová-Vašků, Julie

    2016-07-01

    It has been previously suggested that an increase in entropy production leads to aging. However, the mechanisms linking increased entropy production in living mass to aging are currently unclear. Even though entropy cannot be easily associated with any specific molecular damage, the increase of entropy in structural mass may be connected with heat stress, which is known to generate double strand breaks. Double strand breaks, which are in turn known to play an important role in process of aging, are thus connected to both aging and an increase of entropy. In view of these associations, we propose a new model where the increase of entropy leads to the formation of double strand breaks, resulting in an aging phenotype. This not only offers a new perspective on aging research and facilitates experimental validation, but could also serve as a useful explanatory tool.

  2. A complex between replication factor A (SSB) and DNA helicase stimulates DNA synthesis of DNA polymerase alpha on double-stranded DNA.

    PubMed

    Zhang, S; Grosse, F

    1992-11-01

    A helicase-like DNA unwinding activity was found in highly purified fractions of the calf thymus single-stranded DNA binding protein (ctSSB), also known as replication protein A (RP-A) or replication factor A (RF-A). This activity depended on the hydrolysis of ATP or dATP, and used CTP with a lower efficiency. ctSSB promoted the homologous DNA polymerase alpha to perform DNA synthesis on double-stranded templates containing replication fork-like structures. The rate and amount of DNA synthesis was found to be dependent on the concentration of ctSSB. At a 10-fold mass excess of ctSSB over double-stranded DNA, products of 200-600 nucleotides in length were obtained. This comprises or even exceeds the length of a eukaryotic Okazaki fragment. The ctSSB-associated DNA helicase activity is most likely a distinct protein rather than an inherent property of SSB, as inferred from titration experiments between SSB and DNA. The association of a helicase with SSB and the stimulatory action of this complex to the DNA polymerase alpha-catalyzed synthesis of double-stranded DNA suggests a cooperative function of the three enzymatic activities in the process of eukaryotic DNA replication.

  3. Use of double-stranded RNA interference in Drosophila cell lines to dissect signal transduction pathways

    PubMed Central

    Clemens, James C.; Worby, Carolyn A.; Simonson-Leff, Nancy; Muda, Marco; Maehama, Tomohiko; Hemmings, Brian A.; Dixon, Jack E.

    2000-01-01

    We demonstrate the efficacy of double-stranded RNA-mediated interference (RNAi) of gene expression in generating “knock-out” phenotypes for specific proteins in several Drosophila cell lines. We prove the applicability of this technique for studying signaling cascades by dissecting the well-characterized insulin signal transduction pathway. Specifically, we demonstrate that inhibiting the expression of the DSOR1 (mitogen-activated protein kinase kinase, MAPKK) prevents the activation of the downstream ERK-A (MAPK). In contrast, blocking ERK-A expression results in increased activation of DSOR1. We also show that Drosophila AKT (DAKT) activation depends on the insulin receptor substrate, CHICO (IRS1–4). Finally, we demonstrate that blocking the expression of Drosophila PTEN results in the activation of DAKT. In all cases, the interference of the biochemical cascade by RNAi is consistent with the known steps in the pathway. We extend this powerful technique to study two proteins, DSH3PX1 and Drosophila ACK (DACK). DSH3PX1 is an SH3, phox homology domain-containing protein, and DACK is homologous to the mammalian activated Cdc42 tyrosine kinase, ACK. Using RNAi, we demonstrate that DACK is upstream of DSH3PX1 phosphorylation, making DSH3PX1 an identified downstream target/substrate of ACK-like tyrosine kinases. These experiments highlight the usefulness of RNAi in dissecting complex biochemical signaling cascades and provide a highly effective method for determining the function of the identified genes arising from the Drosophila genome sequencing project. PMID:10823906

  4. High-throughput detection of DNA double-strand breaks using image cytometry

    PubMed Central

    Fowler, Tyler L.; Bailey, Alison M.; Bednarz, Bryan P.; Kimple, Randall J.

    2015-01-01

    Assessment of γH2AX expression for studying DNA double-strand break formation is often performed by manual counting of foci using immunofluorescence microscopy, an approach that is laborious and subject to significant foci selection bias. Here we present a novel high-throughput method for detecting DNA double-strand breaks using automated image cytometry assessment of cell average γH2AX immunofluorescence. Our technique provides an expedient, high-throughput, objective, and cost-effective method for γH2AX analysis. PMID:25605579

  5. A Tightly Stretched Ultralong Supramolecular Multiporphyrin Array Propagated by Double-Strand Formation.

    PubMed

    Morisue, Mitsuhiko; Hoshino, Yuki; Shimizu, Masaki; Uemura, Shinobu; Sakurai, Shinichi

    2016-09-01

    A shape-programmed linearity through supramolecular polymerization is demonstrated by a step-growth double-strand formation of a telechelic oligomeric porphyrin array in which two alternating pyridyl-porphyrin sequenced units are held together by self-complementary ligand-to-metal coordination. The stiff rod-like structure and sufficiently large binding constant of the double-strand unit considerably extended a supramolecular array in the one dimension, which produced a tightly stretched string with a length that exceeded several micrometers. PMID:27490938

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

    NASA Technical Reports Server (NTRS)

    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.

  7. The Transcriptional Response to DNA-Double-Strand Breaks in Physcomitrella patens

    PubMed Central

    Kamisugi, Yasuko; Whitaker, John W.

    2016-01-01

    The model bryophyte Physcomitrella patens is unique among plants in supporting the generation of mutant alleles by facile homologous recombination-mediated gene targeting (GT). Reasoning that targeted transgene integration occurs through the capture of transforming DNA by the homology-dependent pathway for DNA double-strand break (DNA-DSB) repair, we analysed the genome-wide transcriptomic response to bleomycin-induced DNA damage and generated mutants in candidate DNA repair genes. Massively parallel (Illumina) cDNA sequencing identified potential participants in gene targeting. Transcripts encoding DNA repair proteins active in multiple repair pathways were significantly up-regulated. These included Rad51, CtIP, DNA ligase 1, Replication protein A and ATR in homology-dependent repair, Xrcc4, DNA ligase 4, Ku70 and Ku80 in non-homologous end-joining and Rad1, Tebichi/polymerase theta, PARP in microhomology-mediated end-joining. Differentially regulated cell-cycle components included up-regulated Rad9 and Hus1 DNA-damage-related checkpoint proteins and down-regulated D-type cyclins and B-type CDKs, commensurate with the imposition of a checkpoint at G2 of the cell cycle characteristic of homology-dependent DNA-DSB repair. Candidate genes, including ATP-dependent chromatin remodelling helicases associated with repair and recombination, were knocked out and analysed for growth defects, hypersensitivity to DNA damage and reduced GT efficiency. Targeted knockout of PpCtIP, a cell-cycle activated mediator of homology-dependent DSB resection, resulted in bleomycin-hypersensitivity and greatly reduced GT efficiency. PMID:27537368

  8. The Transcriptional Response to DNA-Double-Strand Breaks in Physcomitrella patens.

    PubMed

    Kamisugi, Yasuko; Whitaker, John W; Cuming, Andrew C

    2016-01-01

    The model bryophyte Physcomitrella patens is unique among plants in supporting the generation of mutant alleles by facile homologous recombination-mediated gene targeting (GT). Reasoning that targeted transgene integration occurs through the capture of transforming DNA by the homology-dependent pathway for DNA double-strand break (DNA-DSB) repair, we analysed the genome-wide transcriptomic response to bleomycin-induced DNA damage and generated mutants in candidate DNA repair genes. Massively parallel (Illumina) cDNA sequencing identified potential participants in gene targeting. Transcripts encoding DNA repair proteins active in multiple repair pathways were significantly up-regulated. These included Rad51, CtIP, DNA ligase 1, Replication protein A and ATR in homology-dependent repair, Xrcc4, DNA ligase 4, Ku70 and Ku80 in non-homologous end-joining and Rad1, Tebichi/polymerase theta, PARP in microhomology-mediated end-joining. Differentially regulated cell-cycle components included up-regulated Rad9 and Hus1 DNA-damage-related checkpoint proteins and down-regulated D-type cyclins and B-type CDKs, commensurate with the imposition of a checkpoint at G2 of the cell cycle characteristic of homology-dependent DNA-DSB repair. Candidate genes, including ATP-dependent chromatin remodelling helicases associated with repair and recombination, were knocked out and analysed for growth defects, hypersensitivity to DNA damage and reduced GT efficiency. Targeted knockout of PpCtIP, a cell-cycle activated mediator of homology-dependent DSB resection, resulted in bleomycin-hypersensitivity and greatly reduced GT efficiency. PMID:27537368

  9. The Double-Stranded DNA Virosphere as a Modular Hierarchical Network of Gene Sharing

    PubMed Central

    Iranzo, Jaime

    2016-01-01

    ABSTRACT Virus genomes are prone to extensive gene loss, gain, and exchange and share no universal genes. Therefore, in a broad-scale study of virus evolution, gene and genome network analyses can complement traditional phylogenetics. We performed an exhaustive comparative analysis of the genomes of double-stranded DNA (dsDNA) viruses by using the bipartite network approach and found a robust hierarchical modularity in the dsDNA virosphere. Bipartite networks consist of two classes of nodes, with nodes in one class, in this case genomes, being connected via nodes of the second class, in this case genes. Such a network can be partitioned into modules that combine nodes from both classes. The bipartite network of dsDNA viruses includes 19 modules that form 5 major and 3 minor supermodules. Of these modules, 11 include tailed bacteriophages, reflecting the diversity of this largest group of viruses. The module analysis quantitatively validates and refines previously proposed nontrivial evolutionary relationships. An expansive supermodule combines the large and giant viruses of the putative order “Megavirales” with diverse moderate-sized viruses and related mobile elements. All viruses in this supermodule share a distinct morphogenetic tool kit with a double jelly roll major capsid protein. Herpesviruses and tailed bacteriophages comprise another supermodule, held together by a distinct set of morphogenetic proteins centered on the HK97-like major capsid protein. Together, these two supermodules cover the great majority of currently known dsDNA viruses. We formally identify a set of 14 viral hallmark genes that comprise the hubs of the network and account for most of the intermodule connections. PMID:27486193

  10. The Enzyme and the cDNA Sequence of a Thermolabile and Double-Strand Specific DNase from Northern Shrimps (Pandalus borealis)

    PubMed Central

    Nilsen, Inge W.; Øverbø, Kersti; Jensen Havdalen, Linda; Elde, Morten; Gjellesvik, Dag Rune; Lanes, Olav

    2010-01-01

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

  11. Significant Correlation of Species Longevity with DNA Double Strand Break-Recognition but not with Telomere Length

    PubMed Central

    Lorenzini, Antonello; Johnson, F. Brad; Oliver, Anthony; Tresini, Maria; Smith, Jasmine S.; Hdeib, Mona; Sell, Christian; Cristofalo, Vincent J.; Stamato, Thomas D.

    2009-01-01

    Summary The identification of the cellular mechanisms responsible for the wide differences in species lifespan remains one of the major unsolved problems of the biology of aging. We measured the capacity of nuclear protein to recognize DNA double strand breaks (DSB) and telomere length of skin fibroblasts derived from mammalian species that exhibit wide differences in longevity. Our results indicate DNA DSB recognition increases exponentially with longevity. Further, an analysis of the level of Ku80 protein in human, cow, and mouse suggests that Ku levels vary dramatically between species and these levels are strongly correlated with longevity. In contrast mean telomere length appears to decrease with increasing longevity of the species, although not significantly. These findings suggest that an enhanced ability to bind to DNA-ends may be important for longevity. A number of possible roles for increased levels of Ku and DNA-PKcs are discussed. PMID:19896964

  12. Role of the silkworm argonaute2 homolog gene in double-strand break repair of extrachromosomal DNA.

    PubMed

    Tsukioka, Haruna; Takahashi, Masateru; Mon, Hiroaki; Okano, Kazuhiro; Mita, Kazuei; Shimada, Toru; Lee, Jae Man; Kawaguchi, Yutaka; Koga, Katsumi; Kusakabe, Takahiro

    2006-01-01

    The argonaute protein family provides central components for RNA interference (RNAi) and related phenomena in a wide variety of organisms. Here, we isolated, from a Bombyx mori cell, a cDNA clone named BmAGO2, which is homologous to Drosophila ARGONAUTE2, the gene encoding a repressive factor for the recombination repair of extrachromosomal double-strand breaks (DSBs). RNAi-mediated silencing of the BmAGO2 sequence markedly increased homologous recombination (HR) repair of DSBs in episomal DNA, but had no effect on that in chromosomes. Moreover, we found that RNAi for BmAGO2 enhanced the integration of linearized DNA into a silkworm chromosome via HR. These results suggested that BmAgo2 protein plays an indispensable role in the repression of extrachromosomal DSB repair.

  13. Binding of undamaged double stranded DNA to vaccinia virus uracil-DNA glycosylase

    SciTech Connect

    Schormann, Norbert; Banerjee, Surajit; Ricciardi, Robert; Chattopadhyay, Debasish

    2015-06-02

    Background: Uracil-DNA glycosylases are evolutionarily conserved DNA repair enzymes. However, vaccinia virus uracil-DNA glycosylase (known as D4), also serves as an intrinsic and essential component of the processive DNA polymerase complex during DNA replication. In this complex D4 binds to a unique poxvirus specific protein A20 which tethers it to the DNA polymerase. At the replication fork the DNA scanning and repair function of D4 is coupled with DNA replication. So far, DNA-binding to D4 has not been structurally characterized. Results: This manuscript describes the first structure of a DNA-complex of a uracil-DNA glycosylase from the poxvirus family. This also represents the first structure of a uracil DNA glycosylase in complex with an undamaged DNA. In the asymmetric unit two D4 subunits bind simultaneously to complementary strands of the DNA double helix. Each D4 subunit interacts mainly with the central region of one strand. DNA binds to the opposite side of the A20-binding surface on D4. In comparison of the present structure with the structure of uracil-containing DNA-bound human uracil-DNA glycosylase suggests that for DNA binding and uracil removal D4 employs a unique set of residues and motifs that are highly conserved within the poxvirus family but different in other organisms. Conclusion: The first structure of D4 bound to a truly non-specific undamaged double-stranded DNA suggests that initial binding of DNA may involve multiple non-specific interactions between the protein and the phosphate backbone.

  14. Binding of undamaged double stranded DNA to vaccinia virus uracil-DNA glycosylase

    DOE PAGES

    Schormann, Norbert; Banerjee, Surajit; Ricciardi, Robert; Chattopadhyay, Debasish

    2015-06-02

    Background: Uracil-DNA glycosylases are evolutionarily conserved DNA repair enzymes. However, vaccinia virus uracil-DNA glycosylase (known as D4), also serves as an intrinsic and essential component of the processive DNA polymerase complex during DNA replication. In this complex D4 binds to a unique poxvirus specific protein A20 which tethers it to the DNA polymerase. At the replication fork the DNA scanning and repair function of D4 is coupled with DNA replication. So far, DNA-binding to D4 has not been structurally characterized. Results: This manuscript describes the first structure of a DNA-complex of a uracil-DNA glycosylase from the poxvirus family. This alsomore » represents the first structure of a uracil DNA glycosylase in complex with an undamaged DNA. In the asymmetric unit two D4 subunits bind simultaneously to complementary strands of the DNA double helix. Each D4 subunit interacts mainly with the central region of one strand. DNA binds to the opposite side of the A20-binding surface on D4. In comparison of the present structure with the structure of uracil-containing DNA-bound human uracil-DNA glycosylase suggests that for DNA binding and uracil removal D4 employs a unique set of residues and motifs that are highly conserved within the poxvirus family but different in other organisms. Conclusion: The first structure of D4 bound to a truly non-specific undamaged double-stranded DNA suggests that initial binding of DNA may involve multiple non-specific interactions between the protein and the phosphate backbone.« less

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

    PubMed

    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

  16. Deficiency in the response to DNA double-strand breaks in mouse early preimplantation embryos

    SciTech Connect

    Yukawa, Masashi; Oda, Shoji; Mitani, Hiroshi; Nagata, Masao; Aoki, Fugaku . E-mail: aokif@k.u-tokyo.ac.jp

    2007-06-29

    DNA double-strand breaks (DSBs) are caused by various environmental stresses, such as ionizing radiation and DNA-damaging agents. When DSBs occur, cell cycle checkpoint mechanisms function to stop the cell cycle until all DSBs are repaired; the phosphorylation of H2AX plays an important role in this process. Mouse preimplantation-stage embryos are hypersensitive to ionizing radiation, and X-irradiated mouse zygotes are arrested at the G2 phase of the first cell cycle. To investigate the mechanisms responding to DNA damage at G2 in mouse preimplantation embryos, we examined G2/M checkpoint and DNA repair mechanisms in these embryos. Most of the one- and two-cell embryos in which DSBs had been induced by {gamma}-irradiation underwent a delay in cleavage and ceased development before the blastocyst stage. In these embryos, phosphorylated H2AX ({gamma}-H2AX) was not detected in the one- or two-cell stages by immunocytochemistry, although it was detected after the two-cell stage during preimplantation development. These results suggest that the G2/M checkpoint and DNA repair mechanisms have insufficient function in one- and two-cell embryos, causing hypersensitivity to {gamma}-irradiation. In addition, phosphorylated ataxia telangiectasia mutated protein and DNA protein kinase catalytic subunits, which phosphorylate H2AX, were detected in the embryos at one- and two-cell stages, as well as at other preimplantation stages, suggesting that the absence of {gamma}-H2AX in one- and two-cell embryos depends on some factor(s) other than these kinases.

  17. The Double-Stranded RNA Bluetongue Virus Induces Type I Interferon in Plasmacytoid Dendritic Cells via a MYD88-Dependent TLR7/8-Independent Signaling Pathway

    PubMed Central

    Ruscanu, Suzana; Pascale, Florentina; Bourge, Mickael; Hemati, Behzad; Elhmouzi-Younes, Jamila; Urien, Céline; Bonneau, Michel; Takamatsu, Haru; Hope, Jayne; Mertens, Peter; Meyer, Gilles; Stewart, Meredith; Roy, Polly; Meurs, Eliane F.; Dabo, Stéphanie; Zientara, Stéphan; Breard, Emmanuel; Sailleau, Corinne; Chauveau, Emilie; Vitour, Damien; Charley, Bernard

    2012-01-01

    Dendritic cells (DCs), especially plasmacytoid DCs (pDCs), produce large amounts of alpha/beta interferon (IFN-α/β) upon infection with DNA or RNA viruses, which has impacts on the physiopathology of the viral infections and on the quality of the adaptive immunity. However, little is known about the IFN-α/β production by DCs during infections by double-stranded RNA (dsRNA) viruses. We present here novel information about the production of IFN-α/β induced by bluetongue virus (BTV), a vector-borne dsRNA Orbivirus of ruminants, in sheep primary DCs. We found that BTV induced IFN-α/β in skin lymph and in blood in vivo. Although BTV replicated in a substantial fraction of the conventional DCs (cDCs) and pDCs in vitro, only pDCs responded to BTV by producing a significant amount of IFN-α/β. BTV replication in pDCs was not mandatory for IFN-α/β production since it was still induced by UV-inactivated BTV (UV-BTV). Other inflammatory cytokines, including tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), and IL-12p40, were also induced by UV-BTV in primary pDCs. The induction of IFN-α/β required endo-/lysosomal acidification and maturation. However, despite being an RNA virus, UV-BTV did not signal through Toll-like receptor 7 (TLR7) for IFN-α/β induction. In contrast, pathways involving the MyD88 adaptor and kinases dsRNA-activated protein kinase (PKR) and stress-activated protein kinase (SAPK)/Jun N-terminal protein kinase (JNK) were implicated. This work highlights the importance of pDCs for the production of innate immunity cytokines induced by a dsRNA virus, and it shows that a dsRNA virus can induce IFN-α/β in pDCs via a novel TLR-independent and Myd88-dependent pathway. These findings have implications for the design of efficient vaccines against dsRNA viruses. PMID:22438548

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  19. Nanoconstructions based on double-stranded DNA molecules and their applications as optical biosensing units

    NASA Astrophysics Data System (ADS)

    Zakharov, M. A.; Kazankov, G. M.; Sergeeva, V. S.; Yevdokimov, Yu. M.

    2006-02-01

    We describe the formation and the properties of biosensing units based on the cholesteric liquid-crystalline dispersions of the double-stranded nucleic acid molecules. The resulting biosensing units are proved to be sensitive to the presence of some relevant chemical or biological compounds in a liquid to be analyzed.

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

    PubMed Central

    Konovalovas, Aleksandras

    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

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

    PubMed

    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

  2. Metallization of double-stranded DNA triggered by bound galactose-modified naphthalene diimide.

    PubMed

    Komizo, Kohei; Ikedo, Hideyuki; Sato, Shinobu; Takenaka, Shigeori

    2014-08-20

    Naphthalene diimide (NDI) derivatives bearing galactose moieties through different spacers, NDI-DS1 and NDI-DS2, were synthesized by the click reaction of the acetylene derivatives of NDI with galactose azide. They bound to double-stranded DNA with threading intercalation, as confirmed by the topoisomerase I assay and circular dichroism spectroscopy. The binding affinities of these ligands were on the order of 10(5) M(-1) with several-fold higher affinity for double-stranded DNA than for single-stranded DNA. The silver mirror reaction on the double-stranded DNA bound to these ligands afforded silver nanowires that were converted to gold nanowires. In the atomic force microscopy measurements, the increased height of DNA areas on a mica plate was observed in the case of double-stranded DNA after NDI-DS2 treatment and subsequently silver mirror reaction, whereas the increased height of DNA areas was not observed in the case of single-stranded DNA after the same treatment. PMID:25011665

  3. Evolution of Susceptibility to Ingested Double-Stranded RNAs in Caenorhabditis Nematodes

    PubMed Central

    Nuez, Isabelle; Félix, Marie-Anne

    2012-01-01

    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. PMID:22253787

  4. Evolution of double-stranded DNA viruses of eukaryotes: from bacteriophages to transposons to giant viruses.

    PubMed

    Koonin, Eugene V; Krupovic, Mart; Yutin, Natalya

    2015-04-01

    Diverse eukaryotes including animals and protists are hosts to a broad variety of viruses with double-stranded (ds) DNA genomes, from the largest known viruses, such as pandoraviruses and mimiviruses, to tiny polyomaviruses. Recent comparative genomic analyses have revealed many evolutionary connections between dsDNA viruses of eukaryotes, bacteriophages, transposable elements, and linear DNA plasmids. These findings provide an evolutionary scenario that derives several major groups of eukaryotic dsDNA viruses, including the proposed order "Megavirales," adenoviruses, and virophages from a group of large virus-like transposons known as Polintons (Mavericks). The Polintons have been recently shown to encode two capsid proteins, suggesting that these elements lead a dual lifestyle with both a transposon and a viral phase and should perhaps more appropriately be named polintoviruses. Here, we describe the recently identified evolutionary relationships between bacteriophages of the family Tectiviridae, polintoviruses, adenoviruses, virophages, large and giant DNA viruses of eukaryotes of the proposed order "Megavirales," and linear mitochondrial and cytoplasmic plasmids. We outline an evolutionary scenario under which the polintoviruses were the first group of eukaryotic dsDNA viruses that evolved from bacteriophages and became the ancestors of most large DNA viruses of eukaryotes and a variety of other selfish elements. Distinct lines of origin are detectable only for herpesviruses (from a different bacteriophage root) and polyoma/papillomaviruses (from single-stranded DNA viruses and ultimately from plasmids). Phylogenomic analysis of giant viruses provides compelling evidence of their independent origins from smaller members of the putative order "Megavirales," refuting the speculations on the evolution of these viruses from an extinct fourth domain of cellular life.

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

    PubMed

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

    2013-03-01

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

  6. The tight linkage between DNA replication and double-strand break repair in bacteriophage T4

    PubMed Central

    George, James W.; Stohr, Bradley A.; Tomso, Daniel J.; Kreuzer, Kenneth N.

    2001-01-01

    Double-strand break (DSB) repair and DNA replication are tightly linked in the life cycle of bacteriophage T4. Indeed, the major mode of phage DNA replication depends on recombination proteins and can be stimulated by DSBs. DSB-stimulated DNA replication is dramatically demonstrated when T4 infects cells carrying two plasmids that share homology. A DSB on one plasmid triggered extensive replication of the second plasmid, providing a useful model for T4 recombination-dependent replication (RDR). This system also provides a view of DSB repair in T4-infected cells and revealed that the DSB repair products had been replicated in their entirety by the T4 replication machinery. We analyzed the detailed structure of these products, which do not fit the simple predictions of any of three models for DSB repair. We also present evidence that the T4 RDR system functions to restart stalled or inactivated replication forks. First, we review experiments involving antitumor drug-stabilized topoisomerase cleavage complexes. The results suggest that forks blocked at cleavage complexes are resolved by recombinational repair, likely involving RDR. Second, we show here that the presence of a T4 replication origin on one plasmid substantially stimulated recombination events between it and a homologous second plasmid that did not contain a T4 origin. Furthermore, replication of the second plasmid was increased when the first plasmid contained the T4 origin. Our interpretation is that origin-initiated forks become inactivated at some frequency during replication of the first plasmid and are then restarted via RDR on the second plasmid. PMID:11459966

  7. BRUCE regulates DNA double-strand break response by promoting USP8 deubiquitination of BRIT1

    PubMed Central

    Ge, Chunmin; Che, Lixiao; Ren, Jinyu; Pandita, Raj K.; Lu, Jing; Li, Kaiyi; Pandita, Tej K.; Du, Chunying

    2015-01-01

    The DNA damage response (DDR) is crucial for genomic integrity. BRIT1 (breast cancer susceptibility gene C terminus-repeat inhibitor of human telomerase repeat transcriptase expression), a tumor suppressor and early DDR factor, is recruited to DNA double-strand breaks (DSBs) by phosphorylated H2A histone family, member X (γ-H2AX), where it promotes chromatin relaxation by recruiting the switch/sucrose nonfermentable (SWI–SNF) chromatin remodeler to facilitate DDR. However, regulation of BRIT1 recruitment is not fully understood. The baculovirus IAP repeat (BIR)-containing ubiquitin-conjugating enzyme (BRUCE) is an inhibitor of apoptosis protein (IAP). Here, we report a non-IAP function of BRUCE in the regulation of the BRIT1–SWI–SNF DSB-response pathway and genomic stability. We demonstrate that BRIT1 is K63 ubiquitinated in unstimulated cells and that deubiquitination of BRIT1 is a prerequisite for its recruitment to DSB sites by γ-H2AX. We show mechanistically that BRUCE acts as a scaffold, bridging the ubiquitin-specific peptidase 8 (USP8) and BRIT1 in a complex to coordinate USP8-catalyzed deubiquitination of BRIT1. Loss of BRUCE or USP8 impairs BRIT1 deubiquitination, BRIT1 binding with γ-H2AX, the formation of BRIT1 DNA damage foci, and chromatin relaxation. Moreover, BRUCE-depleted cells display reduced homologous recombination repair, and BRUCE-mutant mice exhibit repair defects and genomic instability. These findings identify BRUCE and USP8 as two hitherto uncharacterized critical DDR regulators and uncover a deubiquitination regulation of BRIT1 assembly at damaged chromatin for efficient DDR and genomic stability. PMID:25733871

  8. Evolution of double-stranded DNA viruses of eukaryotes: from bacteriophages to transposons to giant viruses

    PubMed Central

    Koonin, Eugene V; Krupovic, Mart; Yutin, Natalya

    2015-01-01

    Diverse eukaryotes including animals and protists are hosts to a broad variety of viruses with double-stranded (ds) DNA genomes, from the largest known viruses, such as pandoraviruses and mimiviruses, to tiny polyomaviruses. Recent comparative genomic analyses have revealed many evolutionary connections between dsDNA viruses of eukaryotes, bacteriophages, transposable elements, and linear DNA plasmids. These findings provide an evolutionary scenario that derives several major groups of eukaryotic dsDNA viruses, including the proposed order “Megavirales,” adenoviruses, and virophages from a group of large virus-like transposons known as Polintons (Mavericks). The Polintons have been recently shown to encode two capsid proteins, suggesting that these elements lead a dual lifestyle with both a transposon and a viral phase and should perhaps more appropriately be named polintoviruses. Here, we describe the recently identified evolutionary relationships between bacteriophages of the family Tectiviridae, polintoviruses, adenoviruses, virophages, large and giant DNA viruses of eukaryotes of the proposed order “Megavirales,” and linear mitochondrial and cytoplasmic plasmids. We outline an evolutionary scenario under which the polintoviruses were the first group of eukaryotic dsDNA viruses that evolved from bacteriophages and became the ancestors of most large DNA viruses of eukaryotes and a variety of other selfish elements. Distinct lines of origin are detectable only for herpesviruses (from a different bacteriophage root) and polyoma/papillomaviruses (from single-stranded DNA viruses and ultimately from plasmids). Phylogenomic analysis of giant viruses provides compelling evidence of their independent origins from smaller members of the putative order “Megavirales,” refuting the speculations on the evolution of these viruses from an extinct fourth domain of cellular life. PMID:25727355

  9. Targeting abnormal DNA double strand break repair in tyrosine kinase inhibitor-resistant chronic myeloid leukemias

    PubMed Central

    Tobin, Lisa A.; Robert, Carine; Rapoport, Aaron P.; Gojo, Ivana; Baer, Maria R.; Tomkinson, Alan E.; Rassool, Feyruz V.

    2013-01-01

    Resistance to imatinib (IM) and other BCR-ABL1 tyrosine kinase inhibitors (TKI)s is an increasing problem in leukemias caused by expression of BCR-ABL1. Since chronic myeloid leukemia (CML) cell lines expressing BCR-ABL1 utilize an alternative non-homologous end-joining pathway (ALT NHEJ) to repair DNA double strand breaks (DSB)s, we asked whether this repair pathway is a novel therapeutic target in TKI-resistant disease. Notably, the steady state levels of two ALT NHEJ proteins, poly-(ADP-ribose) polymerase 1 (PARP1) and DNA ligase IIIα were increased in the BCR-ABL1-positive CML cell line K562 and, to a greater extent, in its imatinib resistant (IMR) derivative. Incubation of these cell lines with a combination of DNA ligase and PARP inhibitors inhibited ALT NHEJ and selectively decreased survival with the effect being greater in the IMR derivative. Similar results were obtained with TKI-resistant derivatives of two hematopoietic cell lines that had been engineered to stably express BCR-ABL1. Together our results show that the sensitivity of cell lines expressing BCR-ABL1 to the combination of DNA ligase and PARP inhibitors correlates with the steady state levels of PARP1 and DNA ligase IIIα, and ALT NHEJ activity. Importantly, analysis of clinical samples from CML patients confirmed that the expression levels of PARP1 and DNA ligase IIIα correlated with sensitivity to the DNA repair inhibitor combination. Thus, the expression levels of PARP1 and DNA ligase IIIα serve as biomarkers to identify a subgroup of CML patients who may be candidates for therapies that target the ALT NHEJ pathway when treatment with TKIs has failed. PMID:22641215

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

    PubMed Central

    Wang, Luan; Zhang, Jingze; Zhang, Hailong; Qiu, Dewen; Guo, Lihua

    2016-01-01

    Two novel double-stranded RNA (dsRNA) mycoviruses, termed Fusarium poae dsRNA virus 2 (FpV2) and Fusarium poae dsRNA virus 3 (FpV3), were isolated from the plant pathogenic fungus, Fusarium poae strain SX63, and molecularly characterized. FpV2 and FpV3, with respective genome sequences of 9518 and 9419 base pairs (bps), are both predicted to contain two discontinuous open reading frames (ORFs), ORF1 and ORF2. A hypothetical polypeptide (P1) and a RNA-dependent RNA polymerase (RdRp) are encoded by ORF1 and ORF2, respectively. Phytoreo_S7 domain (pfam07236) homologs were detected downstream of the RdRp domain (RdRp_4; pfam02123) of the ORF2-coded proteins of both FpV2 and FpV3. The same shifty heptamers (GGAAAAC) were both found immediately before the stop codon UAG of ORF1 in FpV2 and FpV3, which could mediate programmed –1 ribosomal frameshifting (–1 PRF). Phylogenetic analysis based on RdRp sequences clearly place FpV2 and FpV3 in a taxonomically unassigned dsRNA mycovirus group. Together, with a comparison of genome organization, a new taxonomic family termed Fusagraviridae is proposed to be created to include FpV2- and FpV3-related dsRNA mycoviruses, within which FpV2 and FpV3 would represent two distinct virus species. PMID:27144564

  11. Illegitimate recombination mediated by double-strand break and end-joining in Escherichia coli.

    PubMed

    Ikeda, Hideo; Shiraishi, Kouya; Ogata, Yasuyuki

    2004-01-01

    The frequency of illegitimate recombination has been measured by a lambdabio transducing phage assay during the induction of the E. coli lambda c1857 lysogen. Illegitimate recombination falls into two classes, short homology-independent and short homology-dependent illegitimate recombination. The former involves sequences with virtually no homology, and is mediated by DNA topoisomerases and controlled by the DNA binding protein HU. The latter is induced by UV irradiation or other DNA damaging agents and requires short regions of homology, usually contain 4 to 13 base pairs, at sites involved in recombination. It has been shown that the RecJ exonuclease promotes short homology-dependent illegitimate recombination, but that the RecQ helicase suppresses it. In addition, we have shown that the overexpression of RecE and RecT enhances the frequencies of spontaneous and UV-induced illegitimate recombination and that the RecJ, RecF, RecO, and RecR functions are required for this RecE-mediated illegitimate recombination. Moreover, we have also indicated that RecQ plays a role in the suppression of RecEmediated illegitimate recombination, with the participation of DnaB, Fis, Exol, and H-NS. Models have been proposed for these modes of recombination: the DNA gyrase subunit exchange model for short homology-independent illegitimate recombination and the "double-strand break and join" model for short homologydependent illegitimate recombination. Many features of these models remain to be tested in future studies. PMID:15476890

  12. Illegitimate recombination mediated by double-strand break and end-joining in Escherichia coli.

    PubMed

    Ikeda, Hideo; Shiraishi, Kouya; Ogata, Yasuyuki

    2004-01-01

    The frequency of illegitimate recombination has been measured by a lambda bio transducing phage assay during the induction of the E. coli lambda cI857 lysogen. Illegitimate recombination falls into two classes, short homology-independent and short homology-dependent illegitimate recombination. The former involves sequences with virtually no homology, and is mediated by DNA topoisomerases and controlled by the DNA binding protein HU. The latter is induced by UV irradiation or other DNA damaging agents and requires short regions of homology, usually contain 4 to 13 base pairs, at sites involved in recombination. It has been shown that the RecJ exonuclease promotes short homology-dependent illegitimate recombination, but that the RecQ helicase suppresses it. In addition, we have shown that the overexpression of RecE and RecT enhances the frequencies of spontaneous and UV-induced illegitimate recombination and that the RecJ, RecF, RecO, and RecR functions are required for this RecE-mediated illegitimate recombination. Moreover, we have also indicated that RecQ plays a role in the suppression of RecE-mediated illegitimate recombination, with the participation of DnaB, Fis, ExoI, and H-NS. Models have been proposed for these modes of recombination: the DNA gyrase subunit exchange model for short homology-independent illegitimate recombination and the "double-strand break and join" model for short homology-dependent illegitimate recombination. Many features of these models remain to be tested in future studies. PMID:15493325

  13. Mycobacteria exploit three genetically distinct DNA double-strand break repair pathways

    PubMed Central

    Gupta, Richa; Barkan, Daniel; Redelman-Sidi, Gil; Shuman, Stewart; Glickman, Michael S.

    2013-01-01

    Bacterial pathogens rely on their DNA repair pathways to resist genomic damage inflicted by the host. DNA double-strand breaks (DSBs) are especially threatening to bacterial viability. DSB repair by homologous recombination (HR) requires nucleases that resect DSB ends and a strand exchange protein that facilitates homology search. RecBCD and RecA perform these functions in E. coli and constitute the major pathway of error free DSB repair. Mycobacteria, including the human pathogen M. tuberculosis, elaborate an additional error-prone pathway of DSB repair via nonhomologous end-joining (NHEJ) catalyzed by Ku and DNA ligase D (LigD). Little is known about the relative contributions of HR and NHEJ to mycobacterial chromosome repair, the factors that dictate pathway choice, or the existence of additional DSB repair pathways. Here we demonstrate that Mycobacterium smegmatis has three DSB repair pathway options: HR, NHEJ, and a novel mechanism of single-strand annealing (SSA). Inactivation of NHEJ or SSA is compensated by elevated HR. We find that mycobacterial RecBCD does not participate in HR or confer resistance to ionizing radiation (IR), but is required for the RecA-independent SSA pathway. In contrast, the mycobacterial helicase-nuclease AdnAB participates in the RecA-dependent HR pathway, and is a major determinant of resistance to IR and oxidative DNA damage. These findings reveal distinctive features of mycobacterial DSB repair, most notably the dedication of the RecBCD and AdnAB helicase-nuclease machines to distinct repair pathways. PMID:21219454

  14. Double-stranded RNAs induce a pattern-triggered immune signaling pathway in plants.

    PubMed

    Niehl, Annette; Wyrsch, Ines; Boller, Thomas; Heinlein, Manfred

    2016-08-01

    Pattern-triggered immunity (PTI) is a plant defense response that relies on the perception of conserved microbe- or pathogen-associated molecular patterns (MAMPs or PAMPs, respectively). Recently, it has been recognized that PTI restricts virus infection in plants; however, the nature of the viral or infection-induced PTI elicitors and the underlying signaling pathways are still unknown. As double-stranded RNAs (dsRNAs) are conserved molecular patterns associated with virus replication, we applied dsRNAs or synthetic dsRNA analogs to Arabidopsis thaliana and investigated PTI responses. We show that in vitro-generated dsRNAs, dsRNAs purified from virus-infected plants and the dsRNA analog polyinosinic-polycytidylic acid (poly(I:C)) induce typical PTI responses dependent on the co-receptor SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE 1 (SERK1), but independent of dicer-like (DCL) proteins in Arabidopsis. Moreover, dsRNA treatment of Arabidopsis induces SERK1-dependent antiviral resistance. Screening of Arabidopsis wild accessions demonstrates natural variability in dsRNA sensitivity. Our findings suggest that dsRNAs represent genuine PAMPs in plants, which induce a signaling cascade involving SERK1 and a specific dsRNA receptor. The dependence of dsRNA-mediated PTI on SERK1, but not on DCLs, implies that dsRNA-mediated PTI involves membrane-associated processes and operates independently of RNA silencing. dsRNA sensitivity may represent a useful trait to increase antiviral resistance in cultivated plants.

  15. Double-stranded RNAs induce a pattern-triggered immune signaling pathway in plants.

    PubMed

    Niehl, Annette; Wyrsch, Ines; Boller, Thomas; Heinlein, Manfred

    2016-08-01

    Pattern-triggered immunity (PTI) is a plant defense response that relies on the perception of conserved microbe- or pathogen-associated molecular patterns (MAMPs or PAMPs, respectively). Recently, it has been recognized that PTI restricts virus infection in plants; however, the nature of the viral or infection-induced PTI elicitors and the underlying signaling pathways are still unknown. As double-stranded RNAs (dsRNAs) are conserved molecular patterns associated with virus replication, we applied dsRNAs or synthetic dsRNA analogs to Arabidopsis thaliana and investigated PTI responses. We show that in vitro-generated dsRNAs, dsRNAs purified from virus-infected plants and the dsRNA analog polyinosinic-polycytidylic acid (poly(I:C)) induce typical PTI responses dependent on the co-receptor SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE 1 (SERK1), but independent of dicer-like (DCL) proteins in Arabidopsis. Moreover, dsRNA treatment of Arabidopsis induces SERK1-dependent antiviral resistance. Screening of Arabidopsis wild accessions demonstrates natural variability in dsRNA sensitivity. Our findings suggest that dsRNAs represent genuine PAMPs in plants, which induce a signaling cascade involving SERK1 and a specific dsRNA receptor. The dependence of dsRNA-mediated PTI on SERK1, but not on DCLs, implies that dsRNA-mediated PTI involves membrane-associated processes and operates independently of RNA silencing. dsRNA sensitivity may represent a useful trait to increase antiviral resistance in cultivated plants. PMID:27030513

  16. Effect of pH and Salt on Adsorption of Double-Stranded DNA on Graphene Oxide.

    PubMed

    Kim, Seyeon; Park, Chanoong; Gang, Jongback

    2015-10-01

    Graphene oxide (GO) has a large surface-to-volume ratio and hydrophobic hexagonal rings that can interact with biomolecules. Single-stranded DNA adsorbs strongly to the surface of GO via hydrophobic interactions. GO has been used in optical biosensors and biomedical platforms for the detection of DNA, proteins, and small molecules. This study was designed to measure the adsorption of double-stranded DNA (dsDNA) onto GO according to DNA length, salt concentration, and pH of the reaction. Results showed that dsDNA molecules were adsorbed progressively as the pH changed from 6.0 to 4.0. At high pH, dsDNA adsorption was enhanced by the presence of MgCl2 rather than NaCl. Desorption of DNA from GO, with triton X-100 led to the rapid release of DNA from GO in the presence of MgCl2. PMID:26726439

  17. Double-strand-break repair recombination in Escherichia coli: physical evidence for a DNA replication mechanism in vivo

    PubMed Central

    Motamedi, Mohammad R.; Szigety, Susan K.; Rosenberg, Susan M.

    1999-01-01

    DNA double-strand-break repair (DSBR) is, in many organisms, accomplished by homologous recombination. In Escherichia coli DSBR was thought to result from breakage and reunion of parental DNA molecules, assisted by known endonucleases, the Holliday junction resolvases. Under special circumstances, for example, SOS induction, recombination forks were proposed to initiate replication. We provide physical evidence that this is a major alternative mechanism in which replication copies information from one chromosome to another generating recombinant chromosomes in normal cells in vivo. This alternative mechanism can occur independently of known Holliday junction cleaving proteins, requires DNA polymerase III, and produces recombined DNA molecules that carry newly replicated DNA. The replicational mechanism underlies about half the recombination of linear DNA in E. coli; the other half occurs by breakage and reunion, which we show requires resolvases, and is replication-independent. The data also indicate that accumulation of recombination intermediates promotes replication dramatically. PMID:10557215

  18. Effect of pH and Salt on Adsorption of Double-Stranded DNA on Graphene Oxide.

    PubMed

    Kim, Seyeon; Park, Chanoong; Gang, Jongback

    2015-10-01

    Graphene oxide (GO) has a large surface-to-volume ratio and hydrophobic hexagonal rings that can interact with biomolecules. Single-stranded DNA adsorbs strongly to the surface of GO via hydrophobic interactions. GO has been used in optical biosensors and biomedical platforms for the detection of DNA, proteins, and small molecules. This study was designed to measure the adsorption of double-stranded DNA (dsDNA) onto GO according to DNA length, salt concentration, and pH of the reaction. Results showed that dsDNA molecules were adsorbed progressively as the pH changed from 6.0 to 4.0. At high pH, dsDNA adsorption was enhanced by the presence of MgCl2 rather than NaCl. Desorption of DNA from GO, with triton X-100 led to the rapid release of DNA from GO in the presence of MgCl2.

  19. Atrazine Triggers DNA Damage Response and Induces DNA Double-Strand Breaks in MCF-10A Cells.

    PubMed

    Huang, Peixin; Yang, John; Ning, Jie; Wang, Michael; Song, Qisheng

    2015-01-01

    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. PMID:26114388

  20. Atrazine Triggers DNA Damage Response and Induces DNA Double-Strand Breaks in MCF-10A Cells

    PubMed Central

    Huang, Peixin; Yang, John; Ning, Jie; Wang, Michael; Song, Qisheng

    2015-01-01

    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. PMID:26114388

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

    PubMed

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

    2003-05-23

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

  2. Hidden anti-double stranded DNA antibodies in autoimmune mice.

    PubMed Central

    Fish, F; Ziff, M

    1982-01-01

    When MRL/l mouse spleen cell culture supernatants were incubated with normal mouse spleen cells, a two-50-fold increase in anti-dsDNA activity was noted. A smaller increase in anti-ssDNA and no change in anti-TNP antibody activity were observed. This 'hidden' antibody in the MRL/l supernatants could not be revealed by DNAse digestion and could not be absorbed by a DNA cellulose column. Hidden antibody was removed from supernatants by sepharose-anti Ig. After DNAase digestion of the MRL/l supernatants, hidden anti-dsDNA could not be revealed by incubation with spleen cells. All the hidden activity was excluded by gel filtration on Sephacryl S-300 (mol. wt greater than 300,000) but was banded in the low density protein area of caesium chloride equilibrium density gradients. It was also noted that MRL/l mouse sera had hidden anti-dsDNA antibodies. Hidden antibodies were present in both the IgG and IgM classes. The revealed antibodies demonstrated impaired ability to bind Fc specific anti-Ig reagents suggesting that they were partially degraded during the incubation with mouse spleen cells. The hidden anti-dsDNA thus appears to represent a DNA-anti-dsDNA complex, perhaps of very high affinity. It may explain why anti-dsDNA but not anti-ssDNA antibodies are of pathological importance in SLE. PMID:6756722

  3. Uptake of extracellular double-stranded RNA by SID-2

    PubMed Central

    McEwan, Deborah L; Weisman, Alexandra S; Hunter, Craig P

    2012-01-01

    Summary Ingested dsRNAs trigger RNA interference (RNAi) in many invertebrates including the nematode Caenorhabditis elegans. Here we show that the C. elegans apical intestinal membrane protein SID-2 is required in C. elegans for the import of ingested dsRNA and, when expressed in Drosophila S2 cells, SID-2 enables the uptake of dsRNAs. SID-2-dependent dsRNA transport requires an acidic extracellular environment and is selective for dsRNAs with at least 50 base pairs. Through structure-function analysis, we identify several SID-2 regions required for this activity including three extracellular, positively-charged, histidines. Finally, we find that SID-2-dependent transport is inhibited by drugs that interfere with vesicle transport. Therefore, we propose that environmental dsRNAs are imported from the acidic intestinal lumen by SID-2 via endocytosis and are released from internalized vesicles in a secondary step mediated by the dsRNA-channel SID-1. Similar multistep mechanisms may underlie the widespread observations of environmental RNAi. PMID:22902558

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

    PubMed Central

    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 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

  5. Physical properties of single- and double-stranded coliphage ribonucleic acid

    PubMed Central

    Bishop, D. H. L.

    1966-01-01

    1. The physical characteristics of single- and double-stranded coliphage RNA with regard to their sedimentation behaviour in gradients of sucrose in high or low ionic conditions were examined. The effect of heat on their sedimentation characteristics was also determined. 2. Single-stranded coliphage RNA was found to exist in three different forms having sedimentation coefficients 28s, 20s and 12s. The latter two were interchangeable, depending on ionic strength. All three were almost equally infectious to spheroplasts. 3. Double-stranded coliphage RNA was found to be non-infectious to spheroplasts and had sedimentation coefficients 15s and 12s. Thermal denaturation gave rise to infectious single-stranded 12s RNA. 4. Four possible hypotheses on the mechanism of replication of coliphage RNA are discussed. PMID:4165511

  6. Millisecond analysis of double stranded DNA with fluorescent intercalator by micro-thermocontrol-device.

    PubMed

    Arata, Hideyuki F; Gillot, Frederic; Collard, Dominique; Fujita, Hiroyuki

    2009-08-15

    Study of interaction between DNA and intercalator at molecular level is important to understand the mechanisms of DNA replication and repair. A micro-fabricated local heating thermodevice was adapted to perform denaturation experiments of DNA with fluorescent intercalator on millisecond time scale. Response time of complete unzipping of double stranded DNA, 16 microm in length, was measured to be around 5 min by commercial thermocycler. Response time of quenching of double stranded DNA with fluorescent intercalator SYBR Green was measured to be 10 ms. Thus, quenching properties owing to strand unzipping and denaturation at base pair level were distinguished. This method has provided easy access to measure this parameter and may be a powerful methodology in analyzing biomolecules on millisecond time scale.

  7. Spin transport and spin polarization properties in double-stranded DNA

    SciTech Connect

    Simchi, Hamidreza; Esmaeilzadeh, Mahdi Mazidabadi, Hossein

    2013-11-21

    We study the spin-dependent electron transport through a double-stranded DNA (dsDNA) using the Bogoliubov-de Gennes equations and non-equilibrium Green's function method. We calculate the spin-dependent electron conductance and spin-polarization for different lengths, helix angles, twist angles of dsDNA, the environment-induced dephasing factors, and hopping integral. It is shown that the conductance decreases by increasing the length and dephasing factor. Also, we show that the spin-polarization depends on the helical symmetry and the length of DNA. It is shown that the double-stranded DNA can act as a perfect spin filter. Finally, we show that the sign of spin polarization can be inverted from +1 (−1) to −1 (+1) for some values of hopping integral.

  8. Cell transcriptional state alters genomic patterns of DNA double-strand break repair in human astrocytes.

    PubMed

    Yong, Raymund L; Yang, Chunzhang; Lu, Jie; Wang, Huaien; Schlaff, Cody D; Tandle, Anita; Graves, Christian A; Elkahloun, Abdel G; Chen, Xiaoyuan; Zhuang, Zhengping; Lonser, Russell R

    2014-01-01

    The misrepair of DNA double-strand breaks in close spatial proximity within the nucleus can result in chromosomal rearrangements that are important in the pathogenesis of haematopoietic and solid malignancies. It is unknown why certain epigenetic states, such as those found in stem or progenitor cells, appear to facilitate neoplastic transformation. Here we show that altering the transcriptional state of human astrocytes alters patterns of DNA damage repair from ionizing radiation at a gene locus-specific and genome-wide level. Astrocytes induced into a reactive state exhibit increased DNA repair, compared with non-reactive cells, in actively transcribed chromatin after irradiation. In mapping these repair sites, we identify misrepair events and repair hotspots that are unique to each state. The precise characterization of genomic regions susceptible to mutation in specific transcriptional states provides new opportunities for addressing clonal evolution in solid cancers, in particular those where double-strand break induction is a cornerstone of clinical intervention. PMID:25517576

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

    PubMed

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

    1994-10-01

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

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

    PubMed Central

    2016-01-01

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

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

    NASA Astrophysics Data System (ADS)

    Zimmermann, H.; Schäfer, M.; Schmitz, C.; Bücker, 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.

  12. Choreographing the Double Strand Break Response: Ubiquitin and SUMO Control of Nuclear Architecture

    PubMed Central

    Harding, Shane M.; Greenberg, Roger A.

    2016-01-01

    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. PMID:27375678

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

    PubMed

    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

  14. Double-strand end repair via the RecBC pathway in Escherichia coli primes DNA replication

    PubMed Central

    Kuzminov, Andrei; Stahl, Franklin W.

    1999-01-01

    To study the relationship between homologous recombination and DNA replication in Escherichia coli, we monitored the behavior of phage λ chromosomes, repressed or not for λ gene activities. Recombination in our system is stimulated both by DNA replication and by experimentally introduced double-strand ends, supporting the idea that DNA replication generates occasional double-strand ends. We report that the RecBC recombinational pathway of E. coli uses double-strand ends to prime DNA synthesis, implying a circular relationship between DNA replication and recombination and suggesting that the primary role of recombination is in the repair of disintegrated replication forks arising during vegetative reproduction. PMID:9990858

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

    NASA Technical Reports Server (NTRS)

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

    PubMed Central

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

    2008-01-01

    Gene promoter hypermethylation in sputum is a promising biomarker for predicting lung cancer. Identifying factors that predispose smokers to methylation of multiple gene promoters in the lung could impact strategies for early detection and chemoprevention. This study evaluated the hypothesis that double-strand break repair capacity and sequence variation in genes in this pathway are associated with a high methylation index in a cohort of current and former cancer-free smokers. A 50% reduction in the mean level of double-strand break repair capacity was seen in lymphocytes from smokers with a high methylation index, defined as ≥ 3 of 8 genes methylated in sputum, compared to smokers with no genes methylated. The classification accuracy for predicting risk for methylation was 88%. Single nucleotide polymorphisms within the MRE11A, CHEK2, XRCC3, DNA-Pkc, and NBN DNA repair genes were highly associated with the methylation index. A 14.5-fold increased odds for high methylation was seen for persons with ≥ 7 risk alleles of these genes. Promoter activity of the MRE11A gene that plays a critical role in recognition of DNA damage and activation of ATM was reduced in persons with the risk allele. Collectively, ours is the first population-based study to identify double-strand break DNA repair capacity and specific genes within this pathway as critical determinants for gene methylation in sputum, that is, in turn, associated with elevated risk for lung cancer. PMID:18413776

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

    PubMed

    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.

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

    PubMed Central

    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. 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

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

    PubMed

    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

  20. The effect of a magnetic field on the spin-selective transport in double-stranded DNA

    SciTech Connect

    Simchi, Hamidreza; Esmaeilzadeh, Mahdi Mazidabadi, Hossein

    2014-05-28

    Spin-polarization in double-stranded DNA is studied in the presence of a magnetic field applied along its helix axis using the non-equilibrium Green's function method. The spin-polarization could be tuned by changing the magnetic field. In some special cases, the double-stranded DNA behaved as a perfect spin-filter. Furthermore, the dependency of the spin-polarization on the spin-orbit strength and dephasing strength is studied.

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

    PubMed Central

    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

  2. Myricetin induces apoptosis via endoplasmic reticulum stress and DNA double-strand breaks in human ovarian cancer cells

    PubMed Central

    XU, YE; XIE, QI; WU, SHAOHUA; YI, DAN; YU, YANG; LIU, SHIBING; LI, SONGYAN; LI, ZHIXIN

    2016-01-01

    The mechanisms underlying myricetin-induced cancer cell apoptosis remain to be elucidated. Certain previous studies have shown that myricetin induces apoptosis through the mitochondrial pathway. Apoptosis, however, can also be induced by other classical pathways, including endoplasmic reticulum (ER) stress and DNA double-strand breaks (DSBs). The aim of the present study was to assess whether these two apoptotic pathways are involved in myricetin-induced cell death in SKOV3 ovarian cancer cells. The results revealed that treatment with myricetin inhibited viability of SKOV3 cells in a dose-dependent manner. Myricetin induced nuclear chromatin condensation and fragmentation, and also upregulated the protein levels of active caspase 3 in a time-dependent manner. In addition, myricetin upregulated ER stress-associated proteins, glucose-regulated protein-78 and C/EBP homologous protein in SKOV3 cells. Phosphorylation of H2AX, a marker of DNA DSBs, was revealed to be upregulated in myricetin-treated cells. The data indicated that myricetin induces DNA DSBs and ER stress, which leads to apoptosis in SKOV3 cells. PMID:26782830

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

    PubMed

    He, Yi; Wang, Chong; Higgins, James D; Yu, Junping; Zong, Jie; Lu, Pingli; Zhang, Dabing; Liang, Wanqi

    2016-08-01

    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

  4. Age-dependent change of HMGB1 and DNA double-strand break accumulation in mouse brain

    SciTech Connect

    Enokido, Yasushi; Yoshitake, Ayaka; Ito, Hikaru; Okazawa, Hitoshi

    2008-11-07

    HMGB1 is an evolutionarily conserved non-histone chromatin-associated protein with key roles in maintenance of nuclear homeostasis; however, the function of HMGB1 in the brain remains largely unknown. Recently, we found that the reduction of nuclear HMGB1 protein level in the nucleus associates with DNA double-strand break (DDSB)-mediated neuronal damage in Huntington's disease [M.L. Qi, K. Tagawa, Y. Enokido, N. Yoshimura, Y. Wada, K. Watase, S. Ishiura, I. Kanazawa, J. Botas, M. Saitoe, E.E. Wanker, H. Okazawa, Proteome analysis of soluble nuclear proteins reveals that HMGB1/2 suppress genotoxic stress in polyglutamine diseases, Nat. Cell Biol. 9 (2007) 402-414]. In this study, we analyze the region- and cell type-specific changes of HMGB1 and DDSB accumulation during the aging of mouse brain. HMGB1 is localized in the nuclei of neurons and astrocytes, and the protein level changes in various brain regions age-dependently. HMGB1 reduces in neurons, whereas it increases in astrocytes during aging. In contrast, DDSB remarkably accumulates in neurons, but it does not change significantly in astrocytes during aging. These results indicate that HMGB1 expression during aging is differentially regulated between neurons and astrocytes, and suggest that the reduction of nuclear HMGB1 might be causative for DDSB in neurons of the aged brain.

  5. RecA binding to a single double-stranded DNA molecule: a possible role of DNA conformational fluctuations.

    PubMed

    Leger, J F; Robert, J; Bourdieu, L; Chatenay, D; Marko, J F

    1998-10-13

    Most genetic regulatory mechanisms involve protein-DNA interactions. In these processes, the classical Watson-Crick DNA structure sometimes is distorted severely, which in turn enables the precise recognition of the specific sites by the protein. Despite its key importance, very little is known about such deformation processes. To address this general question, we have studied a model system, namely, RecA binding to double-stranded DNA. Results from micromanipulation experiments indicate that RecA binds strongly to stretched DNA; based on this observation, we propose that spontaneous thermal stretching fluctuations may play a role in the binding of RecA to DNA. This has fundamental implications for the protein-DNA binding mechanism, which must therefore rely in part on a combination of flexibility and thermal fluctuations of the DNA structure. We also show that this mechanism is sequence sensitive. Theoretical simulations support this interpretation of our experimental results, and it is argued that this is of broad relevance to DNA-protein interactions.

  6. RecA Binding to a Single Double-Stranded DNA Molecule: A Possible Role of DNA Conformational Fluctuations

    NASA Astrophysics Data System (ADS)

    Leger, J. F.; Robert, J.; Bourdieu, L.; Chatenay, D.; Marko, J. F.

    1998-10-01

    Most genetic regulatory mechanisms involve protein-DNA interactions. In these processes, the classical Watson-Crick DNA structure sometimes is distorted severely, which in turn enables the precise recognition of the specific sites by the protein. Despite its key importance, very little is known about such deformation processes. To address this general question, we have studied a model system, namely, RecA binding to double-stranded DNA. Results from micromanipulation experiments indicate that RecA binds strongly to stretched DNA; based on this observation, we propose that spontaneous thermal stretching fluctuations may play a role in the binding of RecA to DNA. This has fundamental implications for the protein-DNA binding mechanism, which must therefore rely in part on a combination of flexibility and thermal fluctuations of the DNA structure. We also show that this mechanism is sequence sensitive. Theoretical simulations support this interpretation of our experimental results, and it is argued that this is of broad relevance to DNA-protein interactions.

  7. Members of the RAD52 Epistasis Group Contribute to Mitochondrial Homologous Recombination and Double-Strand Break Repair in Saccharomyces cerevisiae.

    PubMed

    Stein, Alexis; Kalifa, Lidza; Sia, Elaine A

    2015-11-01

    Mitochondria contain an independently maintained genome that encodes several proteins required for cellular respiration. Deletions in the mitochondrial genome have been identified that cause several maternally inherited diseases and are associated with certain cancers and neurological disorders. The majority of these deletions in human cells are flanked by short, repetitive sequences, suggesting that these deletions may result from recombination events. Our current understanding of the maintenance and repair of mtDNA is quite limited compared to our understanding of similar events in the nucleus. Many nuclear DNA repair proteins are now known to also localize to mitochondria, but their function and the mechanism of their action remain largely unknown. This study investigated the contribution of the nuclear double-strand break repair (DSBR) proteins Rad51p, Rad52p and Rad59p in mtDNA repair. We have determined that both Rad51p and Rad59p are localized to the matrix of the mitochondria and that Rad51p binds directly to mitochondrial DNA. In addition, a mitochondrially-targeted restriction endonuclease (mtLS-KpnI) was used to produce a unique double-strand break (DSB) in the mitochondrial genome, which allowed direct analysis of DSB repair in vivo in Saccharomyces cerevisiae. We find that loss of these three proteins significantly decreases the rate of spontaneous deletion events and the loss of Rad51p and Rad59p impairs the repair of induced mtDNA DSBs.

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

    PubMed Central

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

    2011-01-01

    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

  9. OsRDR6 plays role in host defense against double-stranded RNA virus, Rice Dwarf Phytoreovirus

    PubMed Central

    Hong, Wei; Qian, Dan; Sun, Runhong; Jiang, Lin; Wang, Yu; Wei, Chunhong; Zhang, Zhongkai; Li, Yi

    2015-01-01

    RNAi is a major antiviral defense response in plant and animal model systems. RNA-dependent RNA polymerase 6 (RDR6) is an essential component of RNAi, which plays an important role in the resistance against viruses in the model plants. We found previously that rice RDR6 (OsRDR6) functioned in the defense against Rice stripe virus (RSV), and Rice Dwarf Phytoreovirus (RDV) infection resulted in down-regulation of expression of RDR6. Here we report our new findings on the function of OsRDR6 against RDV. Our result showed that down-regulation of OsRDR6 through the antisense (OsRDR6AS) strategy increased rice susceptibility to RDV infection while over-expression of OsRDR6 had no effect on RDV infection. The accumulation of RDV vsiRNAs was reduced in the OsRDR6AS plants. In the OsRDR6 over-expressed plants, the levels of OsRDR6 RNA transcript and protein were much higher than that in the control plants. Interestingly, the accumulation level of OsRDR6 protein became undetectable after RDV infection. This finding indicated that the translation and/or stability of OsRDR6 protein were negatively impacted upon RDV infection. This new finding provides a new light on the function of RDR6 in plant defense response and the cross-talking between factors encoded by host plant and double-stranded RNA viruses. PMID:26165755

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

    PubMed

    Cheng, Xiaofei; Deng, Ping; Cui, Hongguang; Wang, Aiming

    2015-11-01

    Double-stranded RNA (dsRNA) is an important type of RNA that plays essential roles in diverse cellular processes in eukaryotic organisms and a hallmark in infections by positive-sense RNA viruses. Currently, no in vivo technology has been developed for visualizing dsRNA in living cells. Here, we report a dsRNA binding-dependent fluorescence complementation (dRBFC) assay that can be used to efficiently monitor dsRNA distribution and dynamics in vivo. The system consists of two dsRNA-binding proteins, which are fused to the N- and C-terminal halves of the yellow fluorescent protein (YFP). Binding of the two fusion proteins to a common dsRNA brings the split YFP halves in close proximity, leading to the reconstitution of the fluorescence-competent structure and restoration of fluorescence. Using this technique, we were able to visualize the distribution and trafficking of the replicative RNA intermediates of positive-sense RNA viruses in living cells. PMID:26351203

  11. RuvABC-dependent double-strand breaks in dnaBts mutants require recA.

    PubMed

    Seigneur, M; Ehrlich, S D; Michel, B

    2000-11-01

    Replication fork arrest can cause DNA double-strand breaks (DSBs). These DSBs are caused by the action of the Holliday junction resolvase RuvABC, indicating that they are made by resolution of Holliday junctions formed at blocked forks. In this work, we study the homologous recombination functions required for RuvABC-mediated breakage in cells deficient for the accessory replicative helicase Rep or deficient for the main Escherichia coli replicative helicase DnaB. We show that, in the rep mutant, RuvABC-mediated breakage occurs in the absence of the homologous recombination protein RecA. In contrast, in dnaBts mutants, most of the RuvABC-mediated breakage depends on the presence of RecA, which suggests that RecA participates in the formation of Holliday junctions at forks blocked by the inactivation of DnaB. This action of RecA does not involve the induction of the SOS response and does not require any of the recombination proteins essential for the presynaptic step of homologous recombination, RecBCD, RecF or RecO. Consequently, our observations suggest a new function for RecA at blocked replication forks, and we propose that RecA acts by promoting homologous recombination without the assistance of known presynaptic proteins. PMID:11069680

  12. Dynamic formation of RecA filaments at DNA double strand break repair centers in live cells.

    PubMed

    Kidane, Dawit; Graumann, Peter L

    2005-08-01

    We show that RecN protein is recruited to a defined DNA double strand break (DSB) in Bacillus subtilis cells at an early time point during repair. Because RecO and RecF are successively recruited to DSBs, it is now clear that dynamic DSB repair centers (RCs) exist in prokaryotes. RecA protein was also recruited to RCs and formed highly dynamic filamentous structures, which we term threads, across the nucleoids. Formation of RecA threads commenced approximately 30 min after the induction of DSBs, after RecN recruitment to RCs, and disassembled after 2 h. Time-lapse microscopy showed that the threads rapidly changed in length, shape, and orientation within minutes and can extend at 1.02 microm/min. The formation of RecA threads was abolished in recJ addAB mutant cells but not in each of the single mutants, suggesting that RecA filaments can be initiated via two pathways. Contrary to proteins forming RCs, DNA polymerase I did not form foci but was present throughout the nucleoids (even after induction of DSBs or after UV irradiation), suggesting that it continuously scans the chromosome for DNA lesions. PMID:16061691

  13. Detection and sequence analysis of two novel co-infecting double-strand RNA mycoviruses in Ustilaginoidea virens.

    PubMed

    Zhong, Jie; Lei, Xiang Hua; Zhu, Jun Zi; Song, Ge; Zhang, Ya Dong; Chen, Yi; Gao, Bi Da

    2014-11-01

    Four novel double-stranded RNA molecules, named dsRNA 1 (5124 bp), dsRNA 2(1711 bp), dsRNA 3 (1423 bp) and dsRNA 4 (855 bp), were detected in strain HNHS-1 of Ustilaginoidea virens, the causal agent of rice false smut disease. Sequence analysis showed that the dsRNA1 contains two overlapping open reading frames (ORF) potentially encoding proteins with modest levels of sequence similarity to the coat protein (CP) and putative RNA-dependent RNA polymerase (RdRp), respectively, of viruses of the family Totiviridae. The deduced gene product of the ORF encoded by dsRNA2 is homologous to putative RdRp of viruses in the family Partitiviridae; the ORF encoded by dsRNA3 shares some similarity to a hypothetical protein with unknown function. It is noteworthy that the dsRNA4 lacked integrated ORFs. Isomeric viral particles of about 40 nm in diameter were observed by transmission electron microscopy in a mycelium tissue preparation of strain HNHS-1-R1, a single-spore subculture of strain HNHS-1 containing only the dsRNA1 segment. Phylogenetic analysis and examination of the organization of the two putative RdRp sequences both indicated that there are at least two novel virus species present in strain HNHS-1. We named the two novel viruses Ustilaginoidea virens RNA virus 2 and Ustilaginoidea virens partitivirus 4, respectively.

  14. Vilya, a component of the recombination nodule, is required for meiotic double-strand break formation in Drosophila

    PubMed Central

    Lake, Cathleen M; Nielsen, Rachel J; Guo, Fengli; Unruh, Jay R; Slaughter, Brian D; Hawley, R Scott

    2015-01-01

    Meiotic recombination begins with the induction of programmed double-strand breaks (DSBs). In most organisms only a fraction of DSBs become crossovers. Here we report a novel meiotic gene, vilya, which encodes a protein with homology to Zip3-like proteins shown to determine DSB fate in other organisms. Vilya is required for meiotic DSB formation, perhaps as a consequence of its interaction with the DSB accessory protein Mei-P22, and localizes to those DSB sites that will mature into crossovers. In early pachytene Vilya localizes along the central region of the synaptonemal complex and to discrete foci. The accumulation of Vilya at foci is dependent on DSB formation. Immuno-electron microscopy demonstrates that Vilya is a component of recombination nodules, which mark the sites of crossover formation. Thus Vilya links the mechanism of DSB formation to either the selection of those DSBs that will become crossovers or to the actual process of crossing over. DOI: http://dx.doi.org/10.7554/eLife.08287.001 PMID:26452093

  15. Directional recombination is initiated at a double strand break in human nuclear extracts.

    PubMed Central

    Lopez, B S; Corteggiani, E; Bertrand-Mercat, P; Coppey, J

    1992-01-01

    The involvement of a double strand break in the initiation of homologous recombination was examined in human nuclear extracts. M13 duplex derivatives, containing inserts in the LacZ' region (producing white plaques), were cleaved by restriction enzymes and coincubated in the extracts with a circular plasmid containing the LacZ' region without insert, and unable to produce plaques. Repair was estimated by the ability to produce plaques after transfection into JM109 (recA1) bacteria. Recombination with the plasmid enhances the number of plaques and also the frequency of M13 producing blue plaques. Heterologous insertions in the region surrounding the break were analyzed for their effects on initiation of recombination. The extent of repair by recombination (number of plaques) was compared with the number of blue plaques among the repaired population. Initiation of recombination is inhibited when heterologous insertions are located at 7bp from the break, on the right side as well as on the left side. A low level of recombination is measurable for 27 bp of homology but the maximum efficiency of recombination occurred with homologies of 165 or 320 bp from the break to the heterologous insertion. At 320 bp, the extent of recombinational repair remained at a plateau level but the frequency of blue plaques progressively decreases. We have also analyzed the effect of different sizes of inserts. With longer inserts, a longer length of homology adjacent to the break is required for optimum recombination. However, the size of the insert does not affect the low level of recombination that occurred with a short homology (27 bp). The results indicate that the process is initiated at or near the break, requires homology on both sides of the break and is followed by an elongation from the double strand break to the distal regions of the DNA. Our data provide some support to the double-strand-break repair model established for meiotic recombination in yeast. PMID:1311076

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

    PubMed

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

    1998-02-01

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

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

    NASA Astrophysics Data System (ADS)

    Cheon, Taksu; Poghosyan, Sergey S.

    2015-06-01

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

  18. Single- and double-strand photocleavage of DNA by YO, YOYO and TOTO.

    PubMed Central

    Akerman, B; Tuite, E

    1996-01-01

    Photocleavage of dsDNA by the fluorescent DNA stains oxazole yellow (YO), its dimer YOYO) and the dimer TOTO of thiazole orange (TO) has been investigated as a function of binding ratio. On visible illumination, both YO and YOYO cause single-strand cleavage, with an efficiency that varies with the dye/DNA binding ratio in a manner which can be rationalized in terms of free dye being an inefficient photocleavage reagent and externally bound dye being more efficient than intercalated dye. Moreover, the photocleavage mechanism changes with binding mode. Photocleavage by externally bound dye is, at least partly, oxygen dependent with scavenger studies implicating singlet oxygen as the activated oxygen intermediate. Photocleavage by intercalated dye is essentially oxygen-independent but can be inhibited by moderate concentrations of beta- mercaptoethanol--direct attack on the phosphoribose backbone is a possible mechanism. TOTO causes single-strand cleavage approximately five times less efficiently than YOYO. No direct double-strand breaks (dsb) are detected with YO or YOYO, but in both cases single-strand breaks (ssb) are observed to accumulate to eventually produce double-strand cleavage. With intercalated YO the accumulation occurs in a manner consistent with random generation of strand lesions, while with bisintercalated YOYO the yield of double-strand cleavage (per ssb) is 5-fold higher. A contributing factor is the slow dissociation of the bis-intercalated dimer, which allows for repeated strand-attack at the same binding site, but the observation that the dsb/ssb yield is considerably lower for externally bound than for bis-intercalated YOYO at low dye/DNA ratios indicates that the binding geometry and/or the cleavage mechanism are also important for the high dsb-efficiency. In fact, double-strand cleavage yields with bis-intercalated YOYO are higher than those predicted by simple models, implying a greater than statistical probability for a second cleavage event

  19. Sources of DNA Double-Strand Breaks and Models of Recombinational DNA Repair

    PubMed Central

    Mehta, Anuja; Haber, James E.

    2014-01-01

    DNA is subject to many endogenous and exogenous insults that impair DNA replication and proper chromosome segregation. DNA double-strand breaks (DSBs) are one of the most toxic of these lesions and must be repaired to preserve chromosomal integrity. Eukaryotes are equipped with several different, but related, repair mechanisms involving homologous recombination, including single-strand annealing, gene conversion, and break-induced replication. In this review, we highlight the chief sources of DSBs and crucial requirements for each of these repair processes, as well as the methods to identify and study intermediate steps in DSB repair by homologous recombination. PMID:25104768

  20. Real-time monitoring of double-stranded DNA cleavage using molecular beacons.

    PubMed

    Ma, Changbei; Tang, Zhiwen; Huo, Xiqin; Yang, Xiaohai; Li, Wei; Tan, Weihong

    2008-07-15

    Traditional methods to assay enzymatic cleavage of DNA are discontinuous, time-consuming and laborious. Here, we report a new approach for real-time monitoring of double-stranded DNA cleavage by restriction endonuclease based on nucleic acid ligation using molecular beacon. Upon cleavage of DNA, the cleavage product can be ligated by DNA ligase, which results in a fluorescence enhancement of the molecular beacon. This method permits real-time monitoring of DNA cleavage and makes it easy to characterize the activity of restriction endonuclease and to study the cleavage reaction kinetics.

  1. RSC facilitates Rad59-dependent homologous recombination between sister chromatids by promoting cohesin loading at DNA double-strand breaks.

    PubMed

    Oum, Ji-Hyun; Seong, Changhyun; Kwon, Youngho; Ji, Jae-Hoon; Sid, Amy; Ramakrishnan, Sreejith; Ira, Grzegorz; Malkova, Anna; Sung, Patrick; Lee, Sang Eun; Shim, Eun Yong

    2011-10-01

    Homologous recombination repairs DNA double-strand breaks by searching for, invading, and copying information from a homologous template, typically the homologous chromosome or sister chromatid. Tight wrapping of DNA around histone octamers, however, impedes access of repair proteins to DNA damage. To facilitate DNA repair, modifications of histones and energy-dependent remodeling of chromatin are required, but the precise mechanisms by which chromatin modification and remodeling enzymes contribute to homologous DNA repair are unknown. Here we have systematically assessed the role of budding yeast RSC (remodel structure of chromatin), an abundant, ATP-dependent chromatin-remodeling complex, in the cellular response to spontaneous and induced DNA damage. RSC physically interacts with the recombination protein Rad59 and functions in homologous recombination. Multiple recombination assays revealed that RSC is uniquely required for recombination between sister chromatids by virtue of its ability to recruit cohesin at DNA breaks and thereby promoting sister chromatid cohesion. This study provides molecular insights into how chromatin remodeling contributes to DNA repair and maintenance of chromatin fidelity in the face of DNA damage.

  2. Pre-Exposure to Ionizing Radiation Stimulates DNA Double Strand Break End Resection, Promoting the Use of Homologous Recombination Repair

    PubMed Central

    Oike, Takahiro; Okayasu, Ryuichi; Murakami, Takeshi; Nakano, Takashi; Shibata, Atsushi

    2015-01-01

    The choice of DNA double strand break (DSB) repair pathway is determined at the stage of DSB end resection. Resection was proposed to control the balance between the two major DSB repair pathways, homologous recombination (HR) and non-homologous end joining (NHEJ). Here, we examined the regulation of DSB repair pathway choice at two-ended DSBs following ionizing radiation (IR) in G2 phase of the cell cycle. We found that cells pre-exposed to low-dose IR preferred to undergo HR following challenge IR in G2, whereas NHEJ repair kinetics in G1 were not affected by pre-IR treatment. Consistent with the increase in HR usage, the challenge IR induced Replication protein A (RPA) foci formation and RPA phosphorylation, a marker of resection, were enhanced by pre-IR. However, neither major DNA damage signals nor the status of core NHEJ proteins, which influence the choice of repair pathway, was significantly altered in pre-IR treated cells. Moreover, the increase in usage of HR due to pre-IR exposure was prevented by treatment with ATM inhibitor during the incubation period between pre-IR and challenge IR. Taken together, the results of our study suggest that the ATM-dependent damage response after pre-IR changes the cellular environment, possibly by regulating gene expression or post-transcriptional modifications in a manner that promotes resection. PMID:25826455

  3. Clioquinol induces DNA double-strand breaks, activation of ATM, and subsequent activation of p53 signaling.

    PubMed

    Katsuyama, Masato; Iwata, Kazumi; Ibi, Masakazu; Matsuno, Kuniharu; Matsumoto, Misaki; Yabe-Nishimura, Chihiro

    2012-09-01

    Clioquinol, a Cu²⁺/Zn²⁺/Fe²⁺ chelator/ionophor, was used extensively in the mid 1900s as an amebicide for treating indigestion and diarrhea. It was eventually withdrawn from the market because of a link to subacute myelo-optic neuropathy (SMON) in Japan. The pathogenesis of SMON, however, is not fully understood. To clarify the molecular mechanisms of clioquinol-induced neurotoxicity, a global analysis using DNA chips was carried out on human neuroblastoma cells. The global analysis and quantitative PCR demonstrated that mRNA levels of p21(Cip1), an inhibitor of cyclins D and E, and of GADD45α, a growth arrest and DNA damage-inducible protein, were significantly increased by clioquinol treatment in SH-SY5Y and IMR-32 neuroblastoma cells. Activation of p53 by clioquinol was suggested, since clioquinol induced phosphorylation of p53 at Ser15 to enhance its stabilization. The phosphorylation of p53 was inhibited by KU-55933, an inhibitor of ataxia-telangiectasia mutated kinase (ATM), but not by NU7026, an inhibitor of DNA-dependent protein kinase (DNA-PK). Clioquinol in fact induced phosphorylation of ATM and histone H2AX, a marker of DNA double-strand breaks (DSBs). These results suggest that clioquinol-induced neurotoxicity is mediated by DSBs and subsequent activation of ATM/p53 signaling. PMID:22627294

  4. Changes in the expression of DNA double strand break repair genes in primordial follicles from immature and aged rats.

    PubMed

    Govindaraj, Vijayakumar; Keralapura Basavaraju, Rajani; Rao, Addicam Jagannadha

    2015-03-01

    Oocytes present at birth undergo a progressive process of apoptosis in humans and other mammals as they age. Accepted opinion is that no fresh oocytes are produced other than those present at the time of birth. Studies have shown that DNA repair genes in oocytes of mice and women decline with age, and lack of these genes show higher DNA breaks and increased oocyte death rates. In contrast to the ethical problems associated with monitoring the changes in DNA double-strand breaks in oocytes from young and old humans, it is relatively easy to carry out such a study using a rodent model. In this study, the mRNA levels of DNA repair genes are compared with protein products of some of the genes in the primordial follicles isolated from immature (18-20 days) and aged (400-450 days) female rats. Results revealed a significant decline in mRNA levels of BRAC1 (P < 0.01), RAD51 (P < 0.05), ERCC2 (P < 0.05), and H2AX (P < 0.01) of DNA repair genes and phospho-protein levels of BRAC1 (P < 0.01) and H2AX (P < 0.05) in primordial follicles of aged rats. Impaired DNA repair is confirmed as a mechanism of oocyte ageing.

  5. Two different routes for double-stranded DNA transfer in natural and artificial transformation of Escherichia coli.

    PubMed

    Sun, Dongchang

    2016-02-26

    Escherichia coli is naturally transformable, independent on the conserved DNA uptake machinery for single-stranded DNA (ssDNA) integration. The transfer of double-stranded DNA (dsDNA) during natural transformation of E. coli is regulated by the alternative sigma factor σ(S). However, it remains mysterious how dsDNA transfers across the membranes and how σ(S) regulates natural transformation of E. coli. Here, I screened for σ(S)-regulated genes for dsDNA transfer in E. coli. The screening identified the σ(S)-regulated genes ydcS and ydcV, both locate on the putative ABC transporter ydcSTUV operon. Considering that ydcS and ydcV are predicted to encode a periplasmic protein and an inner membrane protein for substrate binding and translocation respectively, I propose that they may mediate dsDNA translocation across the inner membrane during natural transformation. In chemical transformation of E. coli, ydcS was but ydcV was not required. Thus, YdcV should not be the channel for dsDNA translocation in artificial transformation. Together with the previous observation that the outer membrane porin OmpA mediates dsDNA transfer across the outer membrane in chemical transformation but not in natural transformation, I conclude that dsDNA transfers across the two membranes through different routes in natural and artificial transformation of E. coli.

  6. Real Estate in the DNA Damage Response: Ubiquitin and SUMO Ligases Home in on DNA Double-Strand Breaks

    PubMed Central

    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

  7. Real Estate in the DNA Damage Response: Ubiquitin and SUMO Ligases Home in on DNA Double-Strand Breaks.

    PubMed

    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

  8. DNA double strand break end-processing and RecA induce RecN expression levels in Bacillus subtilis.

    PubMed

    Cardenas, Paula P; Gándara, Carolina; Alonso, Juan C

    2014-02-01

    Bacillus subtilis cells respond to double strand breaks (DSBs) with an ordered recruitment of repair proteins to the site lesion, being RecN one of the first responders. In B. subtilis, one of the responses to DSBs is to increase RecN expression rather than modifying its turnover rate. End-processing activities and the RecA protein itself contribute to increase RecN levels after DNA DSBs. RecO is required for RecA filament formation and full SOS induction, but its absence did not significantly affect RecN expression. Neither the absence of LexA nor the phosphorylation state of RecA or SsbA significantly affect RecN expression levels. These findings identify two major mechanisms (SOS and DSB response) used to respond to DSBs, with LexA required for one of them (SOS response). The DSB response, which requires end-processing and RecA or short RecO-independent RecA filaments, highlights the importance of guarding genome stability by modulating the DNA damage responses. PMID:24373815

  9. Role for Artemis nuclease in the repair of radiation-induced DNA double strand breaks by alternative end joining.

    PubMed

    Moscariello, Mario; Wieloch, Radi; Kurosawa, Aya; Li, Fanghua; Adachi, Noritaka; Mladenov, Emil; Iliakis, George

    2015-07-01

    Exposure of cells to ionizing radiation or radiomimetic drugs generates DNA double-strand breaks that are processed either by homologous recombination repair (HRR), or by canonical, DNA-PKcs-dependent non-homologous end-joining (C-NHEJ). Chemical or genetic inactivation of factors involved in C-NHEJ or HRR, but also their local failure in repair proficient cells, promotes an alternative, error-prone end-joining pathway that serves as backup (A-EJ). There is evidence for the involvement of Artemis endonuclease, a protein deficient in a human radiosensitivity syndrome associated with severe immunodeficiency (RS-SCID), in the processing of subsets of DSBs by HRR or C-NHEJ. It is thought that within HRR or C-NHEJ Artemis processes DNA termini at complex DSBs. Whether Artemis has a role in A-EJ remains unknown. Here, we analyze using pulsed-field gel electrophoresis (PFGE) and specialized reporter assays, DSB repair in wild-type pre-B NALM-6 lymphocytes, as well as in their Artemis(-/-), DNA ligase 4(-/-) (LIG4(-/-)), and LIG4(-/-)/Artemis(-/-) double mutant counterparts, under conditions allowing evaluation of A-EJ. Our results substantiate the suggested roles of Artemis in C-NHEJ and HRR, but also demonstrate a role for the protein in A-EJ that is confirmed in Artemis deficient normal human fibroblasts. We conclude that Artemis is a nuclease participating in DSB repair by all major repair pathways.

  10. Real Estate in the DNA Damage Response: Ubiquitin and SUMO Ligases Home in on DNA Double-Strand Breaks.

    PubMed

    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.

  11. RecA binding to a single double-stranded DNA molecule: A possible role of DNA conformational fluctuations

    PubMed Central

    Leger, J. F.; Robert, J.; Bourdieu, L.; Chatenay, D.; Marko, J. F.

    1998-01-01

    Most genetic regulatory mechanisms involve protein–DNA interactions. In these processes, the classical Watson–Crick DNA structure sometimes is distorted severely, which in turn enables the precise recognition of the specific sites by the protein. Despite its key importance, very little is known about such deformation processes. To address this general question, we have studied a model system, namely, RecA binding to double-stranded DNA. Results from micromanipulation experiments indicate that RecA binds strongly to stretched DNA; based on this observation, we propose that spontaneous thermal stretching fluctuations may play a role in the binding of RecA to DNA. This has fundamental implications for the protein–DNA binding mechanism, which must therefore rely in part on a combination of flexibility and thermal fluctuations of the DNA structure. We also show that this mechanism is sequence sensitive. Theoretical simulations support this interpretation of our experimental results, and it is argued that this is of broad relevance to DNA–protein interactions. PMID:9770480

  12. In vitro topological loading of bacterial condensin MukB on DNA, preferentially single-stranded DNA rather than double-stranded DNA.

    PubMed

    Niki, Hironori; Yano, Koichi

    2016-01-01

    Condensin is the major driving force in the segregation of daughter chromosomes in prokaryotes. Core subunits of condensin belong to the SMC protein family, whose members are characterized by a unique ATPase activity and dimers with a V-shaped structure. The V-shaped dimers might close between head domains, forming a ring structure that can encircle DNA. Indeed, cohesin, which is a subfamily of SMC proteins, encircles double-stranded DNA to hold sister chromatids in eukaryotes. However, the question of whether or not condensin encircles the chromosomal DNA remains highly controversial. Here we report that MukB binds topologically to DNA in vitro, and this binding is preferentially single-stranded DNA (ssDNA) rather than double-stranded DNA. The binding of MukB to ssDNA does not require ATP. In fact, thermal energy enhances the binding. The non-SMC subunits MukF and MukE did stimulate the topological binding of MukB, although they hindered DNA-binding of MukB. Recent reports on the distribution of condensin in genomes reveal that actively transcribed genes in yeast and humans are enriched in condensin. In consideration of all these results, we propose that the binding specificity of condensin to chromosome is provided not by the DNA sequence but by the DNA structure, which is ssDNA. PMID:27387439

  13. In vitro topological loading of bacterial condensin MukB on DNA, preferentially single-stranded DNA rather than double-stranded DNA

    PubMed Central

    Niki, Hironori; Yano, Koichi

    2016-01-01

    Condensin is the major driving force in the segregation of daughter chromosomes in prokaryotes. Core subunits of condensin belong to the SMC protein family, whose members are characterized by a unique ATPase activity and dimers with a V-shaped structure. The V-shaped dimers might close between head domains, forming a ring structure that can encircle DNA. Indeed, cohesin, which is a subfamily of SMC proteins, encircles double-stranded DNA to hold sister chromatids in eukaryotes. However, the question of whether or not condensin encircles the chromosomal DNA remains highly controversial. Here we report that MukB binds topologically to DNA in vitro, and this binding is preferentially single-stranded DNA (ssDNA) rather than double-stranded DNA. The binding of MukB to ssDNA does not require ATP. In fact, thermal energy enhances the binding. The non-SMC subunits MukF and MukE did stimulate the topological binding of MukB, although they hindered DNA-binding of MukB. Recent reports on the distribution of condensin in genomes reveal that actively transcribed genes in yeast and humans are enriched in condensin. In consideration of all these results, we propose that the binding specificity of condensin to chromosome is provided not by the DNA sequence but by the DNA structure, which is ssDNA. PMID:27387439

  14. RecR-mediated modulation of RecF dimer specificity for single- and double-stranded DNA.

    PubMed

    Makharashvili, Nodar; Mi, Tian; Koroleva, Olga; Korolev, Sergey

    2009-01-16

    RecF pathway proteins play an important role in the restart of stalled replication and DNA repair in prokaryotes. Following DNA damage, RecF, RecR, and RecO initiate homologous recombination (HR) by loading of the RecA recombinase on single-stranded (ss) DNA, protected by ssDNA-binding protein. The specific role of RecF in this process is not well understood. Previous studies have proposed that RecF directs the RecOR complex to boundaries of damaged DNA regions by recognizing single-stranded/double-stranded (ss/ds) DNA junctions. RecF belongs to ABC-type ATPases, which function through an ATP-dependent dimerization. Here, we demonstrate that the RecF of Deinococcus radiodurans interacts with DNA as an ATP-dependent dimer, and that the DNA binding and ATPase activity of RecF depend on both the structure of DNA substrate, and the presence of RecR. We found that RecR interacts as a tetramer with the RecF dimer. RecR increases the RecF affinity to dsDNA without stimulating ATP hydrolysis but destabilizes RecF binding to ssDNA and dimerization, likely due to increasing the ATPase rate. The DNA-dependent binding of RecR to the RecF-DNA complex occurs through specific protein-protein interactions without significant contributions from RecR-DNA interactions. Finally, RecF neither alone nor in complex with RecR preferentially binds to the ss/dsDNA junction. Our data suggest that the specificity of the RecFOR complex toward the boundaries of DNA damaged regions may result from a network of protein-protein and DNA-protein interactions, rather than a simple recognition of the ss/dsDNA junction by RecF. PMID:19017635

  15. Overlapping mechanisms promote postsynaptic RAD-51 filament disassembly during meiotic double-strand break repair.

    PubMed

    Ward, Jordan D; Muzzini, Diego M; Petalcorin, Mark I R; Martinez-Perez, Enrique; Martin, Julie S; Plevani, Paolo; Cassata, Giuseppe; Marini, Federica; Boulton, Simon J

    2010-01-29

    Homologous recombination (HR) is essential for repair of meiotic DNA double-strand breaks (DSBs). Although the mechanisms of RAD-51-DNA filament assembly and strand exchange are well characterized, the subsequent steps of HR are less well defined. Here, we describe a synthetic lethal interaction between the C. elegans helicase helq-1 and RAD-51 paralog rfs-1, which results in a block to meiotic DSB repair after strand invasion. Whereas RAD-51-ssDNA filaments assemble at meiotic DSBs with normal kinetics in helq-1, rfs-1 double mutants, persistence of RAD-51 foci and genetic interactions with rtel-1 suggest a failure to disassemble RAD-51 from strand invasion intermediates. Indeed, purified HELQ-1 and RFS-1 independently bind to and promote the disassembly of RAD-51 from double-stranded, but not single-stranded, DNA filaments via distinct mechanisms in vitro. These results indicate that two compensating activities are required to promote postsynaptic RAD-51 filament disassembly, which are collectively essential for completion of meiotic DSB repair.

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

    PubMed

    Kurian, P; Dunston, G; Lindesay, J

    2016-02-21

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

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

    PubMed

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

    2014-09-15

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

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

    PubMed

    Stevens, Deanna; Oegema, Karen; Desai, Arshad

    2013-12-01

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

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

    PubMed Central

    2016-01-01

    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

  20. DNA double strand breaks in the acute phase after synchrotron pencilbeam irradiation

    NASA Astrophysics Data System (ADS)

    Fernandez-Palomo, C.; Bräuer-Krisch, E.; Trippel, M.; Schroll, C.; Requardt, H.; Bartzsch, S.; Nikkhah, G.; Schültke, E.

    2013-07-01

    Introduction. At the biomedical beamline of the European Synchrotron Radiation Facility (ESRF), we have established a method to study pencilbeam irradiation in-vivoin small animal models. The pencilbeam irradiation technique is based on the principle of microbeam irradiation, a concept of spatially fractionated high-dose irradiation. Using γH2AX as marker, we followed the development of DNA double strand breaks over 48 hrs after whole brain irradiation with the pencilbeam technique. Method. Almost square pencilbeams with an individual size of 51 × 50 μm were produced with an MSC collimator using a step and shoot approach, while the animals were moved vertically through the beam. The center-to-center distance (ctc) was 400 μm, with a peak-to-valley dose ratio (PVDR) of about 400. Five groups of healthy adult mice received peak irradiation doses of either 330 Gy or 2,460 Gy and valley doses of 0.82 Gy and 6.15 Gy, respectively. Animals were sacrificed at 2, 12 and 48 hrs after irradiation. Results. DNA double strand breaks are observed in the path of the pencilbeam. The size of the damaged volume undergoes changes within the first 48 hours after irradiation. Conclusions. The extent of DNA damage caused by pencilbeam irradiation, as assessed by H2AX antibody staining, is dose- dependent.

  1. Ingestion of bacterially expressed double-stranded RNA inhibits gene expression in planarians

    PubMed Central

    Newmark, Phillip A.; Reddien, Peter W.; Cebrià, Francesc; Alvarado, Alejandro Sánchez

    2003-01-01

    Freshwater planarian flatworms are capable of regenerating complete organisms from tiny fragments of their bodies; the basis for this regenerative prowess is an experimentally accessible stem cell population that is present in the adult planarian. The study of these organisms, classic experimental models for investigating metazoan regeneration, has been revitalized by the application of modern molecular biological approaches. The identification of thousands of unique planarian ESTs, coupled with large-scale whole-mount in situ hybridization screens, and the ability to inhibit planarian gene expression through double-stranded RNA-mediated genetic interference, provide a wealth of tools for studying the molecular mechanisms that regulate tissue regeneration and stem cell biology in these organisms. Here we show that, as in Caenorhabditis elegans, ingestion of bacterially expressed double-stranded RNA can inhibit gene expression in planarians. This inhibition persists throughout the process of regeneration, allowing phenotypes with disrupted regenerative patterning to be identified. These results pave the way for large-scale screens for genes involved in regenerative processes. PMID:12917490

  2. Double-strand gap repair in a mammalian gene targeting reaction.

    PubMed Central

    Valancius, V; Smithies, O

    1991-01-01

    To better understand the mechanism of homologous recombination in mammalian cells that facilitates gene targeting, we have analyzed the recombination reaction that inserts a plasmid into a homologous chromosomal locus in mouse embryonic stem cells. A partially deleted HPRT gene was targeted with various plasmids capable of correcting the mutation at this locus, and HPRT+ recombinants were directly selected in HAT medium. The structures of the recombinant loci were then determined by genomic Southern blot hybridizations. We demonstrate that plasmid gaps of 200, 600, and 2,500 bp are efficiently repaired during the integrative recombination reaction. Targeting plasmids that carry a double-strand break or gap in the region of DNA homologous to the target locus produce 33- to 140-fold more hypoxanthine-aminopterin-thymidine-resistant recombinants than did these same plasmids introduced in their uncut (supercoiled) forms. Our data suggest that double-strand gaps and breaks may be enlarged prior to the repair reaction since sequence heterologies carried by the incoming plasmids located close to them are often lost. These results extend the known similarities between mammalian and yeast recombination mechanisms and suggest several features of the insertional (O-type) gene targeting reaction that should be considered when one is designing mammalian gene targeting experiments. Images PMID:1875928

  3. Gamma-irradiated quiescent cells repair directly induced double-strand breaks but accumulate persistent double-strand breaks during subsequent DNA replication.

    PubMed

    Minakawa, Yusuke; Atsumi, Yuko; Shinohara, Akira; Murakami, Yasufumi; Yoshioka, Ken-Ichi

    2016-07-01

    H2AX is expressed at very low levels in quiescent normal cells in vivo and in vitro. Such cells repair DNA double-strand breaks (DSBs) induced by γ-irradiation through a transient stabilization of H2AX. However, the resultant cells accumulate small numbers of irreparable (or persistent) DSBs via an unknown mechanism. We found that quiescent cells that had repaired DSBs directly induced by γ-rays were prone to accumulate DSBs during the subsequent DNA replication. Unlike directly induced DSBs, secondary DSBs were not efficiently repaired, although Rad51 and 53BP1 were recruited to these sites. H2AX was dramatically stabilized in response to DSBs directly caused by γ-rays, enabling γH2AX foci formation and DSB repair, whereas H2AX was barely stabilized in response to secondary DSBs, in which γH2AX foci were small and DSBs were not efficiently repaired. Our results show a pathway that leads to the persistent DSB formation after γ-irradiation. PMID:27251002

  4. Gamma-irradiated quiescent cells repair directly induced double-strand breaks but accumulate persistent double-strand breaks during subsequent DNA replication.

    PubMed

    Minakawa, Yusuke; Atsumi, Yuko; Shinohara, Akira; Murakami, Yasufumi; Yoshioka, Ken-Ichi

    2016-07-01

    H2AX is expressed at very low levels in quiescent normal cells in vivo and in vitro. Such cells repair DNA double-strand breaks (DSBs) induced by γ-irradiation through a transient stabilization of H2AX. However, the resultant cells accumulate small numbers of irreparable (or persistent) DSBs via an unknown mechanism. We found that quiescent cells that had repaired DSBs directly induced by γ-rays were prone to accumulate DSBs during the subsequent DNA replication. Unlike directly induced DSBs, secondary DSBs were not efficiently repaired, although Rad51 and 53BP1 were recruited to these sites. H2AX was dramatically stabilized in response to DSBs directly caused by γ-rays, enabling γH2AX foci formation and DSB repair, whereas H2AX was barely stabilized in response to secondary DSBs, in which γH2AX foci were small and DSBs were not efficiently repaired. Our results show a pathway that leads to the persistent DSB formation after γ-irradiation.

  5. Three-dimensional structure of penicillium chrysogenum virus: a double-stranded RNA virus with a genuine T=1 capsid.

    PubMed

    Castón, José R; Ghabrial, Said A; Jiang, Daohong; Rivas, Germán; Alfonso, Carlos; Roca, Ramón; Luque, Daniel; Carrascosa, José L

    2003-08-01

    Although double-stranded (ds) RNA viruses are a rather diverse group, they share general architectural principles and numerous functional features. All dsRNA viruses, from the mammalian reoviruses to the bacteriophage phi6, including fungal viruses, share a specialized capsid involved in transcription and replication of the dsRNA genome, and release of the viral plus strand RNA. This ubiquitous capsid consists of 120 protein subunits in a so-called T=2 organization. The stringent requirements of dsRNA metabolism may explain the similarities observed in capsid architecture among a broad spectrum of dsRNA viruses. We have used cryo-electron microscopy combined with three-dimensional reconstruction techniques and complementary biophysical techniques, to determine the structure at 26A resolution of the Penicillium chrysogenum virus (PcV) capsid. In contrast to all previous studies of dsRNA viruses, PcV capsid is an authentic T=1 capsid with 60 equivalent protein subunits. This T=1 capsid is built with the largest structural protein (110 kDa). Structural comparison between viral particles and capsids devoid of RNA show changes along the inner surface of the capsid, mostly located around the icosahedral 5 and 3-fold axis. Considering that there may be numerous interactions between the inner surface of the protein shell and the underlying RNA, the genome could have an important role in the conformation of the structural subunits. The empty capsid structure suggests a mechanism for transcript release from actively transcribing particles. Furthermore, sequence analysis of the PcV coat protein revealed that both halves of the protein share numerous regions of similar amino acid residues. These results open new perspectives when considering the structural organization of dsRNA virus capsids.

  6. Preventing Damage Limitation: Targeting DNA-PKcs and DNA Double-Strand Break Repair Pathways for Ovarian Cancer Therapy

    PubMed Central

    Dungl, Daniela A.; Maginn, Elaina N.; Stronach, Euan A.

    2015-01-01

    Platinum-based chemotherapy is the cornerstone of ovarian cancer treatment, and its efficacy is dependent on the generation of DNA damage, with subsequent induction of apoptosis. Inappropriate or aberrant activation of the DNA damage response network is associated with resistance to platinum, and defects in DNA repair pathways play critical roles in determining patient response to chemotherapy. In ovarian cancer, tumor cell defects in homologous recombination – a repair pathway activated in response to double-strand DNA breaks (DSB) – are most commonly associated with platinum-sensitive disease. However, despite initial sensitivity, the emergence of resistance is frequent. Here, we review strategies for directly interfering with DNA repair pathways, with particular focus on direct inhibition of non-homologous end joining (NHEJ), another DSB repair pathway. DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is a core component of NHEJ and it has shown considerable promise as a chemosensitization target in numerous cancer types, including ovarian cancer where it functions to promote platinum-induced survival signaling, via AKT activation. The development of pharmacological inhibitors of DNA-PKcs is on-going, and clinic-ready agents offer real hope to patients with chemoresistant disease. PMID:26579492

  7. Non-redundant Functions of ATM and DNA-PKcs in Response to DNA Double-Strand Breaks

    PubMed Central

    Caron, Pierre; Choudjaye, Jonathan; Clouaire, Thomas; Bugler, Béatrix; Daburon, Virginie; Aguirrebengoa, Marion; Mangeat, Thomas; Iacovoni, Jason S.; Álvarez-Quilón, Alejandro; Cortés-Ledesma, Felipe; Legube, Gaëlle

    2015-01-01

    Summary DNA double-strand breaks (DSBs) elicit the so-called DNA damage response (DDR), largely relying on ataxia telangiectasia mutated (ATM) and DNA-dependent protein kinase (DNA-PKcs), two members of the PI3K-like kinase family, whose respective functions during the sequential steps of the DDR remains controversial. Using the DIvA system (DSB inducible via AsiSI) combined with high-resolution mapping and advanced microscopy, we uncovered that both ATM and DNA-PKcs spread in cis on a confined region surrounding DSBs, independently of the pathway used for repair. However, once recruited, these kinases exhibit non-overlapping functions on end joining and γH2AX domain establishment. More specifically, we found that ATM is required to ensure the association of multiple DSBs within “repair foci.” Our results suggest that ATM acts not only on chromatin marks but also on higher-order chromatin organization to ensure repair accuracy and survival. PMID:26586426

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

    PubMed

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

    2014-07-01

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

  9. Chemotherapeutic Compounds Targeting the DNA Double-Strand Break Repair Pathways: The Good, the Bad, and the Promising

    PubMed Central

    Jekimovs, Christian; Bolderson, Emma; Suraweera, Amila; Adams, Mark; O’Byrne, Kenneth J.; Richard, Derek J.

    2014-01-01

    The repair of DNA double-strand breaks (DSBs) is a critical cellular mechanism that exists to ensure genomic stability. DNA DSBs are the most deleterious type of insult to a cell’s genetic material and can lead to genomic instability, apoptosis, or senescence. Incorrectly repaired DNA DSBs have the potential to produce chromosomal translocations and genomic instability, potentially leading to cancer. The prevalence of DNA DSBs in cancer due to unregulated growth and errors in repair opens up a potential therapeutic window in the treatment of cancers. The cellular response to DNA DSBs is comprised of two pathways to ensure DNA breaks are repaired: homologous recombination and non-homologous end joining. Identifying chemotherapeutic compounds targeting proteins involved in these DNA repair pathways has shown promise as a cancer therapy for patients, either as a monotherapy or in combination with genotoxic drugs. From the beginning, there have been a number of chemotherapeutic compounds that have yielded successful responses in the clinic, a number that have failed (CGK-733 and iniparib), and a number of promising targets for future studies identified. This review looks in detail at how the cell responds to these DNA DSBs and investigates the chemotherapeutic avenues that have been and are currently being explored to target this repair process. PMID:24795863

  10. MEI4 – a central player in the regulation of meiotic DNA double-strand break formation in the mouse

    PubMed Central

    Kumar, Rajeev; Ghyselinck, Norbert; Ishiguro, Kei-ichiro; Watanabe, Yoshinori; Kouznetsova, Anna; Höög, Christer; Strong, Edward; Schimenti, John; Daniel, Katrin; Toth, Attila; de Massy, Bernard

    2015-01-01

    The formation of programmed DNA double-strand breaks (DSBs) at the beginning of meiotic prophase marks the initiation of meiotic recombination. Meiotic DSB formation is catalyzed by SPO11 and their repair takes place on meiotic chromosome axes. The evolutionarily conserved MEI4 protein is required for meiotic DSB formation and is localized on chromosome axes. Here, we show that HORMAD1, one of the meiotic chromosome axis components, is required for MEI4 localization. Importantly, the quantitative correlation between the level of axis-associated MEI4 and DSB formation suggests that axis-associated MEI4 could be a limiting factor for DSB formation. We also show that MEI1, REC8 and RAD21L are important for proper MEI4 localization. These findings on MEI4 dynamics during meiotic prophase suggest that the association of MEI4 to chromosome axes is required for DSB formation, and that the loss of this association upon DSB repair could contribute to turning off meiotic DSB formation. PMID:25795304

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

    PubMed Central

    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

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

    SciTech Connect

    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. - 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.

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

    PubMed

    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

  14. Activation of innate antiviral immune response via double-stranded RNA-dependent RLR receptor-mediated necroptosis

    PubMed Central

    Wang, Wei; Wang, Wei-Hua; Azadzoi, Kazem M.; Su, Ning; Dai, Peng; Sun, Jianbin; Wang, Qin; Liang, Ping; Zhang, Wentao; Lei, Xiaoying; Yan, Zhen; Yang, Jing-Hua

    2016-01-01

    Viruses induce double-stranded RNA (dsRNA) in the host cells. The mammalian system has developed dsRNA-dependent recognition receptors such as RLRs that recognize the long stretches of dsRNA as PAMPs to activate interferon-mediated antiviral pathways and apoptosis in severe infection. Here we report an efficient antiviral immune response through dsRNA-dependent RLR receptor-mediated necroptosis against infections from different classes of viruses. We demonstrated that virus-infected A549 cells were efficiently killed in the presence of a chimeric RLR receptor, dsCARE. It measurably suppressed the interferon antiviral pathway but promoted IL-1β production. Canonical cell death analysis by morphologic assessment, phosphatidylserine exposure, caspase cleavage and chemical inhibition excluded the involvement of apoptosis and consistently suggested RLR receptor-mediated necroptosis as the underlying mechanism of infected cell death. The necroptotic pathway was augmented by the formation of RIP1-RIP3 necrosome, recruitment of MLKL protein and the activation of cathepsin D. Contributing roles of RIP1 and RIP3 were confirmed by gene knockdown. Furthermore, the necroptosis inhibitor necrostatin-1 but not the pan-caspase inhibitor zVAD impeded dsCARE-dependent infected cell death. Our data provides compelling evidence that the chimeric RLR receptor shifts the common interferon antiviral responses of infected cells to necroptosis and leads to rapid death of the virus-infected cells. This mechanism could be targeted as an efficient antiviral strategy. PMID:26935990

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

    SciTech Connect

    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.

    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.

  16. Mek1 Kinase Is Regulated To Suppress Double-Strand Break Repair between Sister Chromatids during Budding Yeast Meiosis▿

    PubMed Central

    Niu, Hengyao; Li, Xue; Job, Emily; Park, Caroline; Moazed, Danesh; Gygi, Steven P.; Hollingsworth, Nancy M.

    2007-01-01

    Mek1 is a meiosis-specific kinase in budding yeast which promotes recombination between homologous chromosomes by suppressing double-strand break (DSB) repair between sister chromatids. Previous work has shown that in the absence of the meiosis-specific recombinase gene, DMC1, cells arrest in prophase due to unrepaired DSBs and that Mek1 kinase activity is required in this situation to prevent repair of the breaks using sister chromatids. This work demonstrates that Mek1 is activated in response to DSBs by autophosphorylation of two conserved threonines, T327 and T331, in the Mek1 activation loop. Using a version of Mek1 that can be conditionally dimerized during meiosis, Mek1 function was shown to be promoted by dimerization, perhaps as a way of enabling autophosphorylation of the activation loop in trans. A putative HOP1-dependent dimerization domain within the C terminus of Mek1 has been identified. Dimerization alone, however, is insufficient for activation, as DSBs and Mek1 recruitment to the meiosis-specific chromosomal core protein Red1 are also necessary. Phosphorylation of S320 in the activation loop inhibits sister chromatid repair specifically in dmc1Δ-arrested cells. Ectopic dimerization of Mek1 bypasses the requirement for S320 phosphorylation, suggesting this phosphorylation is necessary for maintenance of Mek1 dimers during checkpoint-induced arrest. PMID:17526735

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

    PubMed

    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

  18. Enrichment of Cdk1-cyclins at DNA double-strand breaks stimulates Fun30 phosphorylation and DNA end resection

    PubMed Central

    Chen, Xuefeng; Niu, Hengyao; Yu, Yang; Wang, Jingjing; Zhu, Shuangyi; Zhou, Jianjie; Papusha, Alma; Cui, Dandan; Pan, Xuewen; Kwon, Youngho; Sung, Patrick; Ira, Grzegorz

    2016-01-01

    DNA double-strand breaks (DSBs) are one of the most cytotoxic types of DNA lesion challenging genome integrity. The activity of cyclin-dependent kinase Cdk1 is essential for DSB repair by homologous recombination and for DNA damage signaling. Here we identify the Fun30 chromatin remodeler as a new target of Cdk1. Fun30 is phosphorylated by Cdk1 on Serine 28 to stimulate its functions in DNA damage response including resection of DSB ends. Importantly, Cdk1-dependent phosphorylation of Fun30-S28 increases upon DNA damage and requires the recruitment of Fun30 to DSBs, suggesting that phosphorylation increases in situ at the DNA damage. Consistently, we find that Cdk1 and multiple cyclins become highly enriched at DSBs and that the recruitment of Cdk1 and cyclins Clb2 and Clb5 ensures optimal Fun30 phosphorylation and checkpoint activation. We propose that the enrichment of Cdk1-cyclin complexes at DSBs serves as a mechanism for enhanced targeting and modulating of the activity of DNA damage response proteins. PMID:26801641

  19. Selective structural change of bulged-out region of double-stranded RNA containing bulged nucleotides by spermidine

    SciTech Connect

    Higashi, Kyohei; Terui, Yusuke; Inomata, Emire; Katagiri, Daisuke; Nomura, Yusuke; Someya, Tatsuhiko; Nishimura, Kazuhiro; Kashiwagi, Keiko; Kawai, Gota; Igarashi, Kazuei

    2008-06-13

    Polyamines are essential for cell growth due to effects mainly at the level of translation. These effects likely involve a structural change, induced by polyamines, of the bulged-out region of double-stranded RNA that is different from changes induced by Mg{sup 2+}. Structural changes were studied using U6-34, a model RNA of U6 small nuclear RNA containing bulged nucleotides. Binding of NS1-2 peptide derived from the RNA binding site of NS1 protein, to U6-34 was inhibited by spermidine but not by Mg{sup 2+}. A selective conformational change of the bases in the bulged-out region of U6-34 induced by spermidine was observed by NMR. The selective effect of spermidine was lost when the bulged-out region of U6-34 was removed in U6-34({delta}5). The binding of NS1-2 peptide to U6-34({delta}5) was inhibited both by spermidine and Mg{sup 2+}. The selective structural change of U6-34 by spermidine was confirmed by circular dichroism.

  20. The Importance of becoming double-stranded: innate immunity and the kinetic model of HIV-1 central plus strand synthesis

    PubMed Central

    Poeschla, Eric

    2013-01-01

    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

  1. Molecular Dynamics Simulations of Double-Stranded DNA in an Explicit Solvent Model with the Zero-Dipole Summation Method

    PubMed Central

    Arakawa, Takamasa; Kamiya, Narutoshi; Nakamura, Haruki; Fukuda, Ikuo

    2013-01-01

    Molecular dynamics (MD) simulations of a double-stranded DNA with explicit water and small ions were performed with the zero-dipole summation (ZD) method, which was recently developed as one of the non-Ewald methods. Double-stranded DNA is highly charged and polar, with phosphate groups in its backbone and their counterions, and thus precise treatment for the long-range electrostatic interactions is always required to maintain the stable and native double-stranded form. A simple truncation method deforms it profoundly. On the contrary, the ZD method, which considers the neutralities of charges and dipoles in a truncated subset, well reproduced the electrostatic energies of the DNA system calculated by the Ewald method. The MD simulations using the ZD method provided a stable DNA system, with similar structures and dynamic properties to those produced by the conventional Particle mesh Ewald method. PMID:24124577

  2. DNA double-strand-break sensitivity, DNA replication, and cell cycle arrest phenotypes of Ku-deficient Saccharomyces cerevisiae

    PubMed Central

    Barnes, Georjana; Rio, Donald

    1997-01-01

    In mammalian cells, the Ku heterodimer is involved in DNA double-strand-break recognition and repair. We have established in yeast a connection between Ku activity and DNA double-strand-break damage repair, and a connection between Ku activity and commitment to DNA replication. We generated double-stranded DNA breaks in yeast cells in vivo by expressing a restriction endonuclease and have shown that yeast mutants lacking Ku p70 activity died while isogenic wild-type cells did not. Moreover, we have discovered that DNA damage occurs spontaneously during normal yeast mitotic growth, and that Ku functions in repair of this damage. We also observed that mitotically growing Ku p70 mutants have an anomalously high DNA content, suggesting a role for Ku in regulation of DNA synthesis. Finally, we present evidence that Ku p70 function is conserved between yeast, Drosophila, and humans. PMID:9023348

  3. A major role of the RecFOR pathway in DNA double-strand-break repair through ESDSA in Deinococcus radiodurans.

    PubMed

    Bentchikou, Esma; Servant, Pascale; Coste, Geneviève; Sommer, Suzanne

    2010-01-01

    In Deinococcus radiodurans, the extreme resistance to DNA-shattering treatments such as ionizing radiation or desiccation is correlated with its ability to reconstruct a functional genome from hundreds of chromosomal fragments. The rapid reconstitution of an intact genome is thought to occur through an extended synthesis-dependent strand annealing process (ESDSA) followed by DNA recombination. Here, we investigated the role of key components of the RecF pathway in ESDSA in this organism naturally devoid of RecB and RecC proteins. We demonstrate that inactivation of RecJ exonuclease results in cell lethality, indicating that this protein plays a key role in genome maintenance. Cells devoid of RecF, RecO, or RecR proteins also display greatly impaired growth and an important lethal sectoring as bacteria devoid of RecA protein. Other aspects of the phenotype of recFOR knock-out mutants paralleled that of a DeltarecA mutant: DeltarecFOR mutants are extremely radiosensitive and show a slow assembly of radiation-induced chromosomal fragments, not accompanied by DNA synthesis, and reduced DNA degradation. Cells devoid of RecQ, the major helicase implicated in repair through the RecF pathway in E. coli, are resistant to gamma-irradiation and have a wild-type DNA repair capacity as also shown for cells devoid of the RecD helicase; in contrast, DeltauvrD mutants show a markedly decreased radioresistance, an increased latent period in the kinetics of DNA double-strand-break repair, and a slow rate of fragment assembly correlated with a slow rate of DNA synthesis. Combining RecQ or RecD deficiency with UvrD deficiency did not significantly accentuate the phenotype of DeltauvrD mutants. In conclusion, RecFOR proteins are essential for DNA double-strand-break repair through ESDSA whereas RecJ protein is essential for cell viability and UvrD helicase might be involved in the processing of double stranded DNA ends and/or in the DNA synthesis step of ESDSA. PMID:20090937

  4. Characterization of a Novel Bipartite Double-Stranded RNA Mycovirus Conferring Hypovirulence in the Phytopathogenic Fungus Botrytis porri

    PubMed Central

    Wu, Mingde; Jin, Fengyin; Zhang, Jing; Yang, Long; Jiang, Daohong

    2012-01-01

    The ascomycete Botrytis porri causes clove rot and leaf blight of garlic worldwide. We report here the biological and molecular features of a novel bipartite double-stranded RNA (dsRNA) mycovirus named Botrytis porri RNA virus 1 (BpRV1) from the hypovirulent strain GarlicBc-72 of B. porri. The BpRV1 genome comprises two dsRNAs, dsRNA-1 (6,215 bp) and dsRNA-2 (5,879 bp), which share sequence identities of 62 and 95% at the 3′- and 5′-terminal regions, respectively. Two open reading frames (ORFs), ORF I (dsRNA-1) and ORF II (dsRNA-2), were detected. The protein encoded by the 3′-proximal coding region of ORF I shows sequence identities of 19 to 23% with RNA-dependent RNA polymerases encoded by viruses in the families Totiviridae, Chrysoviridae, and Megabirnaviridae. However, the proteins encoded by the 5′-proximal coding region of ORF I and by the entire ORF II lack sequence similarities to any reported virus proteins. Phylogenetic analysis showed that BpRV1 belongs to a separate clade distinct from those of other known RNA mycoviruses. Purified virions of ∼35 nm in diameter encompass dsRNA-1 and dsRNA-2, and three structural proteins (SPs) of 70, 80, and 85 kDa, respectively. Peptide mass fingerprinting analysis revealed that the 80- and 85-kDa SPs are encoded by ORF I, while the 70-kDa SP is encoded by ORF II. Introducing BpRV1 purified virions into the virulent strain GarlicBc-38 of B. porri caused derivative 38T reduced mycelial growth and hypovirulence. These combined results suggest that BpRV1 is a novel bipartite dsRNA virus that possibly belongs to a new virus family. PMID:22496220

  5. A computational approach to the relationship between radiation induced double strand breaks and translocations

    NASA Technical Reports Server (NTRS)

    Holley, W. R.; Chatterjee, A.

    1994-01-01

    A theoretical framework is presented which provides a quantitative analysis of radiation induced translocations between the ab1 oncogene on CH9q34 and a breakpoint cluster region, bcr, on CH 22q11. Such translocations are associated frequently with chronic myelogenous leukemia. The theory is based on the assumption that incorrect or unfaithful rejoining of initial double strand breaks produced concurrently within the 200 kbp intron region upstream of the second abl exon, and the 16.5 kbp region between bcr exon 2 and exon 6 interact with each other, resulting in a fusion gene. for an x-ray dose of 100 Gy, there is good agreement between the theoretical estimate and the one available experimental result. The theory has been extended to provide dose response curves for these types of translocations. These curves are quadratic at low doses and become linear at high doses.

  6. Translocation of double-strand DNA through a silicon oxide nanopore.

    PubMed

    Storm, A J; Chen, J H; Zandbergen, H W; Dekker, C

    2005-05-01

    We report double-strand DNA translocation experiments using silicon oxide nanopores with a diameter of about 10 nm . By monitoring the conductance of a voltage-biased pore, we detect molecules with a length ranging from 6557 to 48 500 base pairs. We find that the molecules can pass the pore both in a straight linear fashion and in a folded state. Experiments on circular DNA further support this picture. We sort the molecular events according to their folding state and estimate the folding position. As a proof-of-principle experiment, we show that a nanopore can be used to distinguish the lengths of DNA fragments present in a mixture. These experiments pave the way for quantitative analytical techniques with solid-state nanopores.

  7. Repairing DNA double-strand breaks by the prokaryotic non-homologous end-joining pathway.

    PubMed

    Brissett, Nigel C; Doherty, Aidan J

    2009-06-01

    The NHEJ (non-homologous end-joining) pathway is one of the major mechanisms for repairing DSBs (double-strand breaks) that occur in genomic DNA. In common with eukaryotic organisms, many prokaryotes possess a conserved NHEJ apparatus that is essential for the repair of DSBs arising in the stationary phase of the cell cycle. Although the bacterial NHEJ complex is much more minimal than its eukaryotic counterpart, both pathways share a number of common mechanistic features. The relative simplicity of the prokaryotic NHEJ complex makes it a tractable model system for investigating the cellular and molecular mechanisms of DSB repair. The present review describes recent advances in our understanding of prokaryotic end-joining, focusing primarily on biochemical, structural and cellular aspects of the mycobacterial NHEJ repair pathway.

  8. DNA double-strand break formation and repair in Tetrahymena meiosis.

    PubMed

    Loidl, Josef; Lorenz, Alexander

    2016-06-01

    The molecular details of meiotic recombination have been determined for a small number of model organisms. From these studies, a general picture has emerged that shows that most, if not all, recombination is initiated by a DNA double-strand break (DSB) that is repaired in a recombinogenic process using a homologous DNA strand as a template. However, the details of recombination vary between organisms, and it is unknown which variant is representative of evolutionarily primordial meiosis or most prevalent among eukaryotes. To answer these questions and to obtain a better understanding of the range of recombination processes among eukaryotes, it is important to study a variety of different organisms. Here, the ciliate Tetrahymena thermophila is introduced as a versatile meiotic model system, which has the additional bonus of having the largest phylogenetic distance to all of the eukaryotes studied to date. Studying this organism can contribute to our understanding of the conservation and diversification of meiotic recombination processes.

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

    PubMed Central

    Kato, Akihiro; Komatsu, Kenshi

    2015-01-01

    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. PMID:26184323

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

    PubMed

    Xiao, Xiao; Gang, Yi; Wang, Honghong; Wang, Jiayin; Zhao, Lina; Xu, Li; Liu, Zhiguo

    2015-02-01

    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.

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

    NASA Astrophysics Data System (ADS)

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

    2000-05-01

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

  12. Allosteric Regulation of Unidirectional Spring-like Motion of Double-Stranded Helicates.

    PubMed

    Suzuki, Yoshimasa; Nakamura, Taiki; Iida, Hiroki; Ousaka, Naoki; Yashima, Eiji

    2016-04-13

    We report the unprecedented allosteric regulation of the extension and contraction motions of double-stranded spiroborate helicates composed of 4,4'-linked 2,2'-bipyridine (bpy) and its N,N'-dioxide units in the middle of ortho-linked tetraphenol strands. NMR and circular dichroism measurements and an X-ray crystallographic analysis along with theoretical calculations revealed that enantiomeric helicates contract and extend upon the binding and release of protons and/or metal ions at the covalently linked two binding bpy or N,N'-dioxide moieties without racemization, respectively, regulated by a cooperative anti-syn conformational change of the two bpy or N,N'-dioxide moieties. These anti-syn conformational changes that occurred at the linkages are amplified into a large-scale molecular motion of the helicates leading to reversible spring-like motions coupled with twisting in one direction in a highly homotropic allosteric fashion. PMID:26910831

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

    PubMed Central

    2016-01-01

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

  14. Site-Selective Binding of Nanoparticles to Double-Stranded DNA via Peptide Nucleic Acid "Invasion"

    SciTech Connect

    Stadler, A.L.; van der Lelie, D.; Sun, D.; Maye, M. M.; Gang, O.

    2011-04-01

    We demonstrate a novel method for by-design placement of nano-objects along double-stranded (ds) DNA. A molecular intercalator, designed as a peptide nucleic acid (PNA)-DNA chimera, is able to invade dsDNA at the PNA-side due to the hybridization specificity between PNA and one of the duplex strands. At the same time, the single-stranded (ss) DNA tail of the chimera, allows for anchoring of nano-objects that have been functionalized with complementary ssDNA. The developed method is applied for interparticle attachment and for the fabrication of particle clusters using a dsDNA template. This method significantly broadens the molecular toolbox for constructing nanoscale systems by including the most conventional not yet utilized DNA motif, double helix DNA.

  15. Double-stranded DNA organization in bacteriophage heads: An alternative toroid-based model

    SciTech Connect

    Hud, N.V.

    1995-10-01

    Studies of the organization of double-stranded DNA within bacteriophage heads during the past four decades have produced a wealth of data. However, despite the presentation of numerous models, the true organization of DNA within phage heads remains unresolved. The observations of toroidal DNA structures in electron micrographs of phage lysates have long been cited as support for the organization of DNA in a spool-like fashion. This particular model, like all other models, has not been found to be consistent with all available data. Recently, the authors proposed that DNA within toroidal condensates produced in vitro is organized in a manner significantly different from that suggested by the spool model. This new toroid model has allowed the development of an alternative model for DNA organization within bacteriophage heads that is consistent with a wide range of biophysical data. Here the authors propose that bacteriophage DNA is packaged in a toroid that is folded into a highly compact structure.

  16. Estimating the number of double-strand breaks formed during meiosis from partial observation.

    PubMed

    Toyoizumi, Hiroshi; Tsubouchi, Hideo

    2012-12-01

    Analyzing the basic mechanism of DNA double-strand breaks (DSB) formation during meiosis is important for understanding sexual reproduction and genetic diversity. The location and amount of meiotic DSBs can be examined by using a common molecular biological technique called Southern blotting, but only a subset of the total DSBs can be observed; only DSB fragments still carrying the region recognized by a Southern blot probe are detected. With the assumption that DSB formation follows a nonhomogeneous Poisson process, we propose two estimators of the total number of DSBs on a chromosome: (1) an estimator based on the Nelson-Aalen estimator, and (2) an estimator based on a record value process. Further, we compared their asymptotic accuracy.

  17. Conservative Inheritance of Newly Synthesized DNA in Double-Strand Break-Induced Gene Conversion▿

    PubMed Central

    Ira, Grzegorz; Satory, Dominik; Haber, James E.

    2006-01-01

    To distinguish among possible mechanisms of repair of a double-strand break (DSB) by gene conversion in budding yeast, Saccharomyces cerevisiae, we employed isotope density transfer to analyze budding yeast mating type (MAT) gene switching in G2/M-arrested cells. Both of the newly synthesized DNA strands created during gene conversion are found at the repaired locus, leaving the donor unchanged. These results support suggestions that mitotic DSBs are primarily repaired by a synthesis-dependent strand-annealing mechanism. We also show that the proportion of crossing-over associated with DSB-induced ectopic recombination is not affected by the presence of nonhomologous sequences at one or both ends of the DSB or the presence of additional sequences that must be copied from the donor. PMID:17030630

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

    PubMed

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

    2015-12-01

    An innovative single-stranded DNA (ssDNA) library preparation method has sparked great interest among ancient DNA (aDNA) researchers, especially after reports of endogenous DNA content increases >20-fold in some samples. To investigate the behavior of this method, we generated ssDNA and conventional double-stranded DNA (dsDNA) libraries from 23 ancient and historic plant and animal specimens. We found ssDNA library preparation substantially increased endogenous content when dsDNA libraries contained <3% endogenous DNA, but this enrichment is less pronounced when dsDNA preparations successfully recover short endogenous DNA fragments (mean size < 70 bp). Our findings can help researchers determine when to utilize the time- and resource-intensive ssDNA library preparation method.

  19. DNA phosphorothioate modifications influence the global transcriptional response and protect DNA from double-stranded breaks

    PubMed Central

    Gan, Rui; Wu, Xiaolin; He, Wei; Liu, Zhenhua; Wu, Shuangju; Chen, Chao; Chen, Si; Xiang, Qianrong; Deng, Zixin; Liang, Dequan; Chen, Shi; Wang, Lianrong

    2014-01-01

    The modification of DNA by phosphorothioate (PT) occurs when the non-bridging oxygen in the sugar-phosphate backbone of DNA is replaced with sulfur. This DNA backbone modification was recently discovered and is governed by the dndABCDE genes in a diverse group of bacteria and archaea. However, the biological function of DNA PT modifications is poorly understood. In this study, we employed the RNA-seq analysis to characterize the global transcriptional changes in response to PT modifications. Our results show that DNA without PT protection is susceptible to DNA damage caused by the dndFGHI gene products. The DNA double-stranded breaks then trigger the SOS response, cell filamentation and prophage induction. Heterologous expression of dndBCDE conferring DNA PT modifications at GPSA and GPST prevented the damage in Salmonella enterica. Our data provide insights into the physiological role of the DNA PT system. PMID:25319634

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

    PubMed

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

    2011-08-15

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

  1. Visualization of complex DNA double-strand breaks in a tumor treated with carbon ion radiotherapy

    PubMed Central

    Oike, Takahiro; Niimi, Atsuko; Okonogi, Noriyuki; Murata, Kazutoshi; Matsumura, Akihiko; Noda, Shin-Ei; Kobayashi, Daijiro; Iwanaga, Mototaro; Tsuchida, Keisuke; Kanai, Tatsuaki; Ohno, Tatsuya; Shibata, Atsushi; Nakano, Takashi

    2016-01-01

    Carbon ion radiotherapy shows great potential as a cure for X-ray-resistant tumors. Basic research suggests that the strong cell-killing effect induced by carbon ions is based on their ability to cause complex DNA double-strand breaks (DSBs). However, evidence supporting the formation of complex DSBs in actual patients is lacking. Here, we used advanced high-resolution microscopy with deconvolution to show that complex DSBs are formed in a human tumor clinically treated with carbon ion radiotherapy, but not in a tumor treated with X-ray radiotherapy. Furthermore, analysis using a physics model suggested that the complexity of radiotherapy-induced DSBs is related to linear energy transfer, which is much higher for carbon ion beams than for X-rays. Visualization of complex DSBs in clinical specimens will help us to understand the anti-tumor effects of carbon ion radiotherapy. PMID:26925533

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

    PubMed

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

    2011-01-01

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

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

    PubMed

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

    2016-01-01

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

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

    NASA Technical Reports Server (NTRS)

    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.

  5. A Monte Carlo Study of Knots in Long Double-Stranded DNA Chains

    PubMed Central

    Rieger, Florian C.; Virnau, Peter

    2016-01-01

    We determine knotting probabilities and typical sizes of knots in double-stranded DNA for chains of up to half a million base pairs with computer simulations of a coarse-grained bead-stick model: Single trefoil knots and composite knots which include at least one trefoil as a prime factor are shown to be common in DNA chains exceeding 250,000 base pairs, assuming physiologically relevant salt conditions. The analysis is motivated by the emergence of DNA nanopore sequencing technology, as knots are a potential cause of erroneous nucleotide reads in nanopore sequencing devices and may severely limit read lengths in the foreseeable future. Even though our coarse-grained model is only based on experimental knotting probabilities of short DNA strands, it reproduces the correct persistence length of DNA. This indicates that knots are not only a fine gauge for structural properties, but a promising tool for the design of polymer models. PMID:27631891

  6. Proximity-induced superconductivity effect in a double-stranded DNA

    SciTech Connect

    Simchi, Hamidreza; Esmaeilzadeh, Mahdi Mazidabadi, Hossein

    2014-02-07

    We study the proximity-induced superconductivity effect in a double-stranded DNA by solving the Bogoliubov-de Gennes equations and taking into account the effect of thermal fluctuations of the twist angle between neighboring base pairs. We show that the electron conductance is spin-dependent and the conductance of spin up (down) increases (decreases) due to the spin-orbit coupling (SOC). It is found that, for T < 100 K, the band gap energy is temperature-independent and it decreases due to the SOC. In addition, by solving the Bogoliubov-de Gennes equations and local gap parameter equation self-consistently, we find the critical temperature at which transition to superconductivity can take place.

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

    PubMed

    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

  8. A Monte Carlo Study of Knots in Long Double-Stranded DNA Chains.

    PubMed

    Rieger, Florian C; Virnau, Peter

    2016-09-01

    We determine knotting probabilities and typical sizes of knots in double-stranded DNA for chains of up to half a million base pairs with computer simulations of a coarse-grained bead-stick model: Single trefoil knots and composite knots which include at least one trefoil as a prime factor are shown to be common in DNA chains exceeding 250,000 base pairs, assuming physiologically relevant salt conditions. The analysis is motivated by the emergence of DNA nanopore sequencing technology, as knots are a potential cause of erroneous nucleotide reads in nanopore sequencing devices and may severely limit read lengths in the foreseeable future. Even though our coarse-grained model is only based on experimental knotting probabilities of short DNA strands, it reproduces the correct persistence length of DNA. This indicates that knots are not only a fine gauge for structural properties, but a promising tool for the design of polymer models. PMID:27631891

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

    SciTech Connect

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

    2011-03-15

    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 {lambda}-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.

  10. Horizontal transfer and hypovirulence associated with double-stranded RNA in Beauveria bassiana.

    PubMed

    Dalzoto, Patricia R; Glienke-Blanco, Chirlei; Kava-Cordeiro, Vanessa; Ribeiro, Juliana Z; Kitajima, Elliot Watanabe; Azevedo, João Lúcio

    2006-12-01

    Beauveria bassiana strains from different hosts and geographic origins were assayed for the presence of double-stranded RNA (dsRNA). Two of them (15.4%) showed extra bands, with approximately 4.0-3.5 kb and 2-0.7 kb, respectively, after electrophoretic separation of undigested nucleic acids. Virus-like particles were approximately 28-30 nm diam. The dsRNA was maintained after conidiogenesis (vertical transmission) and was transmitted horizontally by hyphal anastomosis. Strains purged of dsRNA obtained after cycloheximide treatment showed increased conidial production when compared with strains carrying dsRNA particles. Bioassays demonstrated hypovirulence associated with dsRNA. The mean mortality against the insect Euschistus heros was reduced in strains containing dsRNA when compared with the isogenic dsRNA-free ones.

  11. Apparent Epigenetic Meiotic Double-Strand-Break Disparity in Saccharomyces cerevisiae: A Meta-Analysis

    PubMed Central

    Stahl, Franklin W.; Rehan, Maryam Binti Mohamed; Foss, Henriette M.; Borts, Rhona H.

    2016-01-01

    Previously published, and some unpublished, tetrad data from budding yeast (Saccharomyces cerevisiae) are analyzed for disparity in gene conversion, in which one allele is more often favored than the other (conversion disparity). One such disparity, characteristic of a bias in the frequencies of meiotic double-strand DNA breaks at the hotspot near the His4 locus, is found in diploids that undergo meiosis soon after their formation, but not in diploids that have been cloned and frozen. Altered meiotic DNA breakability associated with altered metabolism-related chromatin states has been previously reported. However, the above observations imply that such differing parental chromatin states can persist through at least one chromosome replication, and probably more, in a common environment. This conclusion may have implications for interpreting changes in allele frequencies in populations. PMID:27356614

  12. The cytotoxicity of (-)-lomaiviticin A arises from induction of double-strand breaks in DNA

    NASA Astrophysics Data System (ADS)

    Colis, Laureen C.; Woo, Christina M.; Hegan, Denise C.; Li, Zhenwu; Glazer, Peter M.; Herzon, Seth B.

    2014-06-01

    The metabolite (-)-lomaiviticin A, which contains two diazotetrahydrobenzo[b]fluorene (diazofluorene) functional groups, inhibits the growth of cultured human cancer cells at nanomolar-picomolar concentrations; however, the mechanism responsible for the potent cytotoxicity of this natural product is not known. Here we report that (-)-lomaiviticin A nicks and cleaves plasmid DNA by a pathway that is independent of reactive oxygen species and iron, and that the potent cytotoxicity of (-)-lomaiviticin A arises from the induction of DNA double-strand breaks (dsbs). In a plasmid cleavage assay, the ratio of single-strand breaks (ssbs) to dsbs is 5.3 ± 0.6:1. Labelling studies suggest that this cleavage occurs via a radical pathway. The structurally related isolates (-)-lomaiviticin C and (-)-kinamycin C, which contain one diazofluorene, are demonstrated to be much less effective DNA cleavage agents, thereby providing an explanation for the enhanced cytotoxicity of (-)-lomaiviticin A compared to that of other members of this family.

  13. Probing 3D Collective Cancer Invasion Using Double-Stranded Locked Nucleic Acid Biosensors.

    PubMed

    Dean, Zachary S; Elias, Paul; Jamilpour, Nima; Utzinger, Urs; Wong, Pak Kin

    2016-09-01

    Cancer is a leading cause of death worldwide and metastases are responsible for over 90% of human cancer deaths. There is an urgent need to develop novel therapeutics for suppressing cancer invasion, the initial step of metastasis. Nevertheless, the regulation of cancer invasion is poorly understood due to a paucity of tools for monitoring the invasion process in 3D microenvironments. Here, we report a double-stranded locked nucleic acid (dsLNA) biosensor for investigating 3D collective cancer invasion. By incorporating multiphoton microscopy and the dsLNA biosensor, we perform dynamic single cell gene expression analysis while simultaneously characterizing the biomechanical interaction between the invading sprouts and the extracellular matrix. Gene profiling of invasive leader cells and detached cells suggest distinctive signaling mechanisms involved in collective and individual invasion in the 3D microenvironment. Our results underscore the involvement of Notch signaling in 3D collective cancer invasion, which warrants further investigation toward antimetastasis therapy in the future.

  14. A Unified Sensor Architecture for Isothermal Detection of Double-Stranded DNA, Oligonucleotides, and Small Molecules

    PubMed Central

    Brown, Carl W.; Lakin, Matthew R.; Fabry-Wood, Aurora; Horwitz, Eli K.; Baker, Nicholas A.; Stefanovic, Darko; Graves, Steven W.

    2015-01-01

    Pathogen detection is an important problem in many areas of medicine and agriculture, which may involve genomic or transcriptomic signatures, or small molecule metabolites. We report a unified, DNA-based sensor architecture capable of isothermal detection of double-stranded DNA targets, single-stranded oligonucleotides, and small molecules. Each sensor contains independent target detection and reporter modules, enabling rapid design. We detected gene variants on plasmids via a straightforward isothermal denaturation protocol. The sensors were highly specific, even with a randomized DNA background. We achieved a limit of detection of ~15 pM for single-stranded targets and ~5 nM for targets on denatured plasmids. By incorporating a blocked aptamer sequence, we also detected small molecules using the same sensor architecture. This work provides a starting point for multiplexed detection of multi-strain pathogens, and disease states caused by genetic variants (e.g., sickle cell anemia). PMID:25663617

  15. RNA-Binding Proteins in the Regulation of miRNA Activity: A Focus on Neuronal Functions

    PubMed Central

    Loffreda, Alessia; Rigamonti, Aurora; Barabino, Silvia M. L.; Lenzken, Silvia C.

    2015-01-01

    Posttranscriptional modifications of messenger RNAs (mRNAs) are key processes in the fine-tuning of cellular homeostasis. Two major actors in this scenario are RNA binding proteins (RBPs) and microRNAs (miRNAs) that together play important roles in the biogenesis, turnover, translation and localization of mRNAs. This review will highlight recent advances in the understanding of the role of RBPs in the regulation of the maturation and the function of miRNAs. The interplay between miRNAs and RBPs is discussed specifically in the context of neuronal development and function. PMID:26437437

  16. A Structural Model of the Genome Packaging Process in a Membrane-Containing Double Stranded DNA Virus

    PubMed Central

    Hong, Chuan; Oksanen, Hanna M.; Liu, Xiangan; Jakana, Joanita; Bamford, Dennis H.; Chiu, Wah

    2014-01-01

    Two crucial steps in the virus life cycle are genome encapsidation to form an infective virion and genome exit to infect the next host cell. In most icosahedral double-stranded (ds) DNA viruses, the viral genome enters and exits the capsid through a unique vertex. Internal membrane-containing viruses possess additional complexity as the genome must be translocated through the viral membrane bilayer. Here, we report the structure of the genome packaging complex with a membrane conduit essential for viral genome encapsidation in the tailless icosahedral membrane-containing bacteriophage PRD1. We utilize single particle electron cryo-microscopy (cryo-EM) and symmetry-free image reconstruction to determine structures of PRD1 virion, procapsid, and packaging deficient mutant particles. At the unique vertex of PRD1, the packaging complex replaces the regular 5-fold structure and crosses the lipid bilayer. These structures reveal that the packaging ATPase P9 and the packaging efficiency factor P6 form a dodecameric portal complex external to the membrane moiety, surrounded by ten major capsid protein P3 trimers. The viral transmembrane density at the special vertex is assigned to be a hexamer of heterodimer of proteins P20 and P22. The hexamer functions as a membrane conduit for the DNA and as a nucleating site for the unique vertex assembly. Our structures show a conformational alteration in the lipid membrane after the P9 and P6 are recruited to the virion. The P8-genome complex is then packaged into the procapsid through the unique vertex while the genome terminal protein P8 functions as a valve that closes the channel once the genome is inside. Comparing mature virion, procapsid, and mutant particle structures led us to propose an assembly pathway for the genome packaging apparatus in the PRD1 virion. PMID:25514469

  17. The Fun30 ATP-dependent nucleosome remodeler promotes resection of DNA double-strand break ends

    PubMed Central

    Chen, Xuefeng; Cui, Dandan; Papusha, Alma; Zhang, Xiaotian; Chu, Chia-Dwo; Tang, Jiangwu; Chen, Kaifu; Pan, Xuewen; Ira, Grzegorz

    2013-01-01

    Chromosomal double-strand breaks (DSBs) are resected by 5′-nucleases to form 3′ single-strand DNA (ssDNA) substrates for binding by homologous recombination and DNA damage checkpoint proteins. Two redundant pathways of extensive resection were described both in cells 1-3 and in vitro 4-6, one relying on Exo1 exonuclease and the other on Sgs1 helicase and Dna2 nuclease. However, it remains unknown how resection proceeds within the context of chromatin where histones and histone-bound proteins represent barriers for resection enzymes. Here, we have identified the yeast nucleosome remodeling enzyme Fun30 as novel factor promoting DSB end resection. Fun30 is the major nucleosome remodeler promoting extensive Exo1- and Sgs1-dependent resection of DSBs while the RSC and INO80 chromatin remodeling complexes play redundant roles with Fun30 in resection adjacent to DSB ends. ATPase and helicase domains of Fun30, which are needed for nucleosome remodeling 7, are also required for resection. Fun30 is robustly recruited to DNA breaks and spreads around the DSB coincident with resection. Fun30 becomes less important for resection in the absence of the histone-bound Rad9 checkpoint adaptor protein known to block 5′ strand processing 8 and in the absence of either histone H3 K79 methylation or γ-H2A, which mediate recruitment of the Rad9 9, 10. Together these data suggest that Fun30 helps to overcome the inhibitory effect of Rad9 on DNA resection. PMID:22960743

  18. Synthetic double-stranded RNA enhances airway inflammation and remodelling in a rat model of asthma.

    PubMed

    Takayama, Satoshi; Tamaoka, Meiyo; Takayama, Koji; Okayasu, Kaori; Tsuchiya, Kimitake; Miyazaki, Yasunari; Sumi, Yuki; Martin, James G; Inase, Naohiko

    2011-10-01

    Respiratory viral infections are frequently associated with exacerbations of asthma. Double-stranded RNA (dsRNA) produced during viral infections may be one of the stimuli for exacerbation. We aimed to assess the potential effect of dsRNA on certain aspects of chronic asthma through the administration of polyinosine-polycytidylic acid (poly I:C), synthetic dsRNA, to a rat model of asthma. Brown Norway rats were sensitized to ovalbumin and challenged three times to evoke airway remodelling. The effect of poly I:C on the ovalbumin-induced airway inflammation and structural changes was assessed from bronchoalveolar lavage fluid and histological findings. The expression of cytokines and chemokines was evaluated by real-time quantitative reverse transcription PCR and ELISA. Ovalbumin-challenged animals showed an increased number of total cells and eosinophils in bronchoalveolar lavage fluid compared with PBS-challenged controls. Ovalbumin-challenged animals treated with poly I:C showed an increased number of total cells and neutrophils in bronchoalveolar lavage fluid compared with those without poly I:C treatment. Ovalbumin-challenged animals showed goblet cell hyperplasia, increased airway smooth muscle mass, and proliferation of both airway epithelial cells and airway smooth muscle cells. Treatment with poly I:C enhanced these structural changes. Among the cytokines and chemokines examined, the expression of interleukins 12 and 17 and of transforming growth factor-β(1) in ovalbumin-challenged animals treated with poly I:C was significantly increased compared with those of the other groups. Double-stranded RNA enhanced airway inflammation and remodelling in a rat model of bronchial asthma. These observations suggest that viral infections may promote airway remodelling.

  19. Dicopper double-strand helicates held together by additional π-π interactions.

    PubMed

    Boiocchi, Massimo; Brega, Valentina; Ciarrocchi, Carlo; Fabbrizzi, Luigi; Pallavicini, Piersandro

    2013-09-16

    The bis-bidentate ligand, obtained from Schiff base condensation of RR-1,2-cyclohexanediamine and 8-naphthylmethoxyquinoline-2-carbaldehyde (L-L), forms with [Cu(I)(MeCN)4]ClO4 a double strand helicate complex, made especially stable by the presence of four definite interstrand π-π interactions involving a quinoline subunit and a naphthylmethoxy substituent of the two strands. The [Cu(I)2(L-L)2](2+) complex, which does not decompose even on excess addition of either L-L or Cu(I), undergoes a two electron oxidation in MeCN, through two one-electron fully reversible steps, separated by 260 mV, as shown by cyclic voltammetry (CV) studies. The high stability of the mixed valence complex [Cu(I)Cu(II)(L-L)2](3+) with respect to disproportionation to [Cu(I)2(L-L)2](2+) and [Cu(II)2(L-L)2](4+) is essentially due to a favorable electrostatic term. Cu(II) forms with L-L a stable species, with a 1:1 stoichiometric ratio, but, in the absence of crystallographic data, it was impossible to assess whether it is of mono- or dinuclear nature. However, CV studies on an MeCN solution containing equimolar amounts of Cu(II) and L-L showed the presence in the reduction scan of two fully reversible waves, separated by about 250 mV, which indicated the presence in solution of a dicopper(II) double strand helicate complex, [Cu(II)2(L-L)2](4+). This work demonstrates that additional interstrand π-π interactions can favor the formation of unusually stable dicopper(I) and dicopper(II) helicate complexes. PMID:24003965

  20. An Examination of the Effects of Double-Strand Breaks on Extrachromosomal Recombination in Mammalian Cells

    PubMed Central

    Yang, D.; Waldman, A. S.

    1992-01-01

    We studied the effects of double-strand breaks on intramolecular extrachromosomal homologous recombination in mammalian cells. Pairs of defective herpes thymidine kinase (tk) sequences were introduced into mouse Ltk(-) cells on a DNA molecule that also contained a neo gene under control of the SV40 early promoter/enhancer. With the majority of the constructs used, gene conversions or double crossovers, but not single crossovers, were recoverable. DNA was linearized with various restriction enzymes prior to transfection. Recombination events producing a functional tk gene were monitored by selecting for tk-positive colonies. For double-strand breaks placed outside of the region of homology, maximal recombination frequencies were measured when a break placed the two tk sequences downstream from the SV40 early promoter/enhancer. We observed no relationship between recombination frequency and either the distance between a break and the tk sequences or the distance between the tk sequences. The quantitative effects of the breaks appeared to depend on the degree of homology between the tk sequences. We also observed that inverted repeats recombined as efficiently as direct repeats. The data indicated that the breaks influenced recombination indirectly, perhaps by affecting the binding of a factor(s) to the SV40 promoter region which in turn stimulated or inhibited recombination of the tk sequences. Taken together, we believe that our results provide strong evidence for the existence of a pathway for extrachromosomal homologous recombination in mammalian cells that is distinct from single-strand annealing. We discuss the possibility that intrachromosomal and extrachromosomal recombination have mechanisms in common. PMID:1459429

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

    PubMed

    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

  2. Strand displacement and duplex invasion into double-stranded DNA by pyrrolidinyl peptide nucleic acids.

    PubMed

    Bohländer, Peggy R; Vilaivan, Tirayut; Wagenknecht, Hans-Achim

    2015-09-21

    The so-called acpcPNA system bears a peptide backbone consisting of 4'-substituted proline units with (2'R,4'R) configuration in an alternating combination with (2S)-amino-cyclopentane-(1S)-carboxylic acids. acpcPNA forms exceptionally stable hybrids with complementary DNA. We demonstrate herein (i) strand displacements by single-stranded DNA from acpcPNA-DNA hybrids, and by acpcPNA strands from DNA duplexes, and (ii) strand invasions by acpcPNA into double-stranded DNA. These processes were studied in vitro using synthetic oligonucleotides and by means of our concept of wavelength-shifting fluorescent nucleic acid probes, including fluorescence lifetime measurements that allow quantifying energy transfer efficiencies. The strand displacements of preannealed 14mer acpcPNA-7mer DNA hybrids consecutively by 10mer and 14mer DNA strands occur with rather slow kinetics but yield high fluorescence color ratios (blue : yellow or blue : red), fluorescence intensity enhancements, and energy transfer efficiencies. Furthermore, 14mer acpcPNA strands are able to invade into 30mer double-stranded DNA, remarkably with quantitative efficiency in all studied cases. These processes can also be quantified by means of fluorescence. This remarkable behavior corroborates the extraordinary versatile properties of acpcPNA. In contrast to conventional PNA systems which require 3 or more equivalents PNA, only 1.5 equivalents acpcPNA are sufficient to get efficient double duplex invasion. Invasions also take place even in the presence of 250 mM NaCl which represents an ionic strength nearly twice as high as the physiological ion concentration. These remarkable results corroborate the extraordinary properties of acpcPNA, and thus acpcPNA represents an eligible tool for biological analytics and antigene applications.

  3. Characterization of single- and double-stranded DNA on gold surfaces.

    PubMed

    Moses, Selina; Brewer, Scott H; Lowe, Lisa B; Lappi, Simon E; Gilvey, Lauren B G; Sauthier, Marc; Tenent, Robert C; Feldheim, Daniel L; Franzen, Stefan

    2004-12-01

    Single- and double-stranded deoxy ribonucleic acid (DNA) molecules attached to self-assembled monolayers (SAMs) on gold surfaces were characterized by a number of optical and electronic spectroscopic techniques. The DNA-modified gold surfaces were prepared through the self-assembly of 6-mercapto-1-hexanol and 5'-C(6)H(12)SH -modified single-stranded DNA (ssDNA). Upon hybridization of the surface-bound probe ssDNA with its complimentary target, formation of double-stranded DNA (dsDNA) on the gold surface is observed and in a competing process, probe ssDNA is desorbed from the gold surface. The competition between hybridization of ssDNA with its complimentary target and ssDNA probe desorption from the gold surface has been investigated in this paper using X-ray photoelectron spectroscopy, chronocoulometry, fluorescence, and polarization modulation-infrared reflection absorption spectroscopy (PM-IRRAS). The formation of dsDNA on the surface was identified by PM-IRRAS by a dsDNA IR signature at approximately 1678 cm(-)(1) that was confirmed by density functional theory calculations of the nucleotides and the nucleotides' base pairs. The presence of dsDNA through the specific DNA hybridization was additionally confirmed by atomic force microscopy through colloidal gold nanoparticle labeling of the target ssDNA. Using these methods, strand loss was observed even for DNA hybridization performed at 25 degrees C for the DNA monolayers studied here consisting of attachment to the gold surfaces by single Au-S bonds. This finding has significant consequence for the application of SAM technology in the detection of oligonucleotide hybridization on gold surfaces.

  4. Chromosome thripsis by DNA double strand break clusters causes enhanced cell lethality, chromosomal translocations and 53BP1-recruitment

    PubMed Central

    Schipler, Agnes; Mladenova, Veronika; Soni, Aashish; Nikolov, Vladimir; Saha, Janapriya; Mladenov, Emil; Iliakis, George

    2016-01-01

    Chromosome translocations are hallmark of cancer and of radiation-induced cell killing, reflecting joining of incongruent DNA-ends that alter the genome. Translocation-formation requires DNA end-joining mechanisms and incompletely characterized, permissive chromatin conditions. We show that chromatin destabilization by clusters of DNA double-strand-breaks (DSBs) generated by the I-SceI meganuclease at multiple, appropriately engineered genomic sites, compromises c-NHEJ and markedly increases cell killing and translocation-formation compared to single-DSBs. Translocation-formation from DSB-clusters utilizes Parp1 activity, implicating alt-EJ in their formation. Immunofluorescence experiments show that single-DSBs and DSB-clusters uniformly provoke the formation of single γ-H2AX foci, suggesting similar activation of early DNA damage response (DDR). Live-cell imaging also shows similar single-focus recruitment of the early-response protein MDC1, to single-DSBs and DSB-clusters. Notably, the late DDR protein, 53BP1 shows in live-cell imaging strikingly stronger recruitment to DSB-clusters as compared to single-DSBs. This is the first report that chromatin thripsis, in the form of engineered DSB-clusters, compromises first-line DSB-repair pathways, allowing alt-EJ to function as rescuing-backup. DSB-cluster-formation is indirectly linked to the increased biological effectiveness of high ionization-density radiations, such as the alpha-particles emitted by radon gas or the heavy-ions utilized in cancer therapy. Our observations provide the first direct mechanistic explanation for this long-known effect. PMID:27257076

  5. Chromosome thripsis by DNA double strand break clusters causes enhanced cell lethality, chromosomal translocations and 53BP1-recruitment.

    PubMed

    Schipler, Agnes; Mladenova, Veronika; Soni, Aashish; Nikolov, Vladimir; Saha, Janapriya; Mladenov, Emil; Iliakis, George

    2016-09-19

    Chromosome translocations are hallmark of cancer and of radiation-induced cell killing, reflecting joining of incongruent DNA-ends that alter the genome. Translocation-formation requires DNA end-joining mechanisms and incompletely characterized, permissive chromatin conditions. We show that chromatin destabilization by clusters of DNA double-strand-breaks (DSBs) generated by the I-SceI meganuclease at multiple, appropriately engineered genomic sites, compromises c-NHEJ and markedly increases cell killing and translocation-formation compared to single-DSBs. Translocation-formation from DSB-clusters utilizes Parp1 activity, implicating alt-EJ in their formation. Immunofluorescence experiments show that single-DSBs and DSB-clusters uniformly provoke the formation of single γ-H2AX foci, suggesting similar activation of early DNA damage response (DDR). Live-cell imaging also shows similar single-focus recruitment of the early-response protein MDC1, to single-DSBs and DSB-clusters. Notably, the late DDR protein, 53BP1 shows in live-cell imaging strikingly stronger recruitment to DSB-clusters as compared to single-DSBs. This is the first report that chromatin thripsis, in the form of engineered DSB-clusters, compromises first-line DSB-repair pathways, allowing alt-EJ to function as rescuing-backup. DSB-cluster-formation is indirectly linked to the increased biological effectiveness of high ionization-density radiations, such as the alpha-particles emitted by radon gas or the heavy-ions utilized in cancer therapy. Our observations provide the first direct mechanistic explanation for this long-known effect. PMID:27257076

  6. Gamma irradiation induces DNA double-strand breaks in fibroblasts: a model study for the development of biodosimetry

    NASA Astrophysics Data System (ADS)

    Uttayarat, P.; Tangtong, T.; Sukapirom, K.; Boonsirichai, K.

    2015-05-01

    Double-strand breaks (DSBs) of DNAs induced by ionizing radiation can pose detrimental damages on organisms which include genetic instability and cell death. It is necessary to be able to assess health risks associated with irradiation from both accidental and therapeutic exposures in a timely manner for proper medical treatments. This present study showed the first attempt to develop a biodosimetric measure in Thailand based on the quantification of phosphorylated histone H2AX (γ-H2AX) formed at DSB sites with an aim to establish a dose response curve using a two-dimensional (2D) cell culture model. Human dermal fibroblasts were grown into monolayers before irradiated by gamma rays from a Co-60 source in a custom-made lead chamber at doses 0, 0.2, 1, 2 and 4 Gy and a dose rate of 0.21 Gy/min. After 30 min post exposure, γ-H2AX proteins were immunofluorescently labelled for evaluation by confocal microscopy and flow cytometry. The accumulation of phosphorylated γ-H2AX proteins at DSBs appeared as nuclear foci with the most prominent intensity at 4 Gy. Linear regression analysis of flow cytometric data showed a linear response (R2 = 0.9862) of foci intensity in proportion to irradiation dose. In addition, the fraction of cell viability was shown to decrease at higher doses. This technique can be further developed as a quick assessment tool to identify individuals subjected to accidental radiation in parallel to other established biodosimetric measures.

  7. Computational Study on Full-length Human Ku70 with Double Stranded DNA: Dynamics, Interactions and Functional Implications

    NASA Technical Reports Server (NTRS)

    Hu, Shaowen; Cucinotta, Francis A.

    2009-01-01

    The Ku70/80 heterodimer is the first repair protein in the initial binding of double-strand break (DSB) ends following DNA damage, and is a component of nonhomologous end joining repair, the primary pathway for DSB repair in mammalian cells. In this study we constructed a full-length human Ku70 structure based on its crystal structure, and performed 20 ns conventional molecular dynamic (CMD) simulations on this protein and several other complexes with short DNA duplexes of different sequences. The trajectories of these simulations indicated that, without the topological support of Ku80, the residues in the bridge and C-terminal arm of Ku70 are more flexible than other experimentally identified domains. We studied the two missing loops in the crystal structure and predicted that they are also very flexible. Simulations revealed that they make an important contribution to the Ku70 interaction with DNA. Dislocation of the previously studied SAP domain was observed in several systems, implying its role in DNA binding. Targeted molecular dynamic (TMD) simulation was also performed for one system with a far-away 14bp DNA duplex. The TMD trajectory and energetic analysis disclosed detailed interactions of the DNA-binding residues during the DNA dislocation, and revealed a possible conformational transition for a DSB end when encountering Ku70 in solution. Compared to experimentally based analysis, this study identified more detailed interactions between DNA and Ku70. Free energy analysis indicated Ku70 alone is able to bind DNA with relatively high affinity, with consistent contributions from various domains of Ku70 in different systems. The functional implications of these domains in the processes of Ku heterodimerization and DNA damage recognition and repair can be characterized in detail based upon this analysis.

  8. Template recognition and formation of initiation complexes by the replicase of a segmented double-stranded RNA virus.

    PubMed

    Tortorici, M Alejandra; Broering, Teresa J; Nibert, Max L; Patton, John T

    2003-08-29

    Replication of the segmented double-stranded (ds) RNA genome of viruses belonging to the Reoviridae family requires the RNA-dependent RNA polymerase (RdRP) to use 10-12 different mRNAs as templates for (-) strand synthesis. Rotavirus serves as a model system for study of this process, since its RdRP (VP1) is catalytically active and can specifically recognize template mRNAs in vitro. Here, we have analyzed the requirements for template recognition by the rotavirus RdRP and compared those to the requirements for formation of (-) strand initiation complexes. The results show that multiple functionally independent recognition signals are present at the 3'-end of viral mRNAs, some positioned in nonconserved regions upstream of the highly conserved 3'-terminal consensus sequence. We also found that RdRP recognition signals are distinct from cis-acting signals that promote (-) strand synthesis, because deletions of portions of the 3'-consensus sequence that caused viral mRNAs to be poorly replicated in vitro did not necessarily prevent efficient recognition of the RNA by the RdRP. Although the RdRP alone can specifically bind to viral mRNAs, our analysis reveals that this interaction is not sufficient to generate initiation complexes, even in the presence of nucleotides and divalent cations. Rather, the formation of initiation complexes also requires the core lattice protein (VP2), a virion component that forms a T = 1 icosahedral shell that encapsidates the segmented dsRNA genome. The essential role that the core lattice protein has in (-) strand initiation provides a mechanism for the coordination of genome replication and virion assembly.

  9. Rad54B targeting to DNA double-strand break repair sites requires complex formation with S100A11.

    PubMed

    Murzik, Ulrike; Hemmerich, Peter; Weidtkamp-Peters, Stefanie; Ulbricht, Tobias; Bussen, Wendy; Hentschel, Julia; von Eggeling, Ferdinand; Melle, Christian

    2008-07-01

    S100A11 is involved in a variety of intracellular activities such as growth regulation and differentiation. To gain more insight into the physiological role of endogenously expressed S100A11, we used a proteomic approach to detect and identify interacting proteins in vivo. Hereby, we were able to detect a specific interaction between S100A11 and Rad54B, which could be confirmed under in vivo conditions. Rad54B, a DNA-dependent ATPase, is described to be involved in recombinational repair of DNA damage, including DNA double-strand breaks (DSBs). Treatment with bleomycin, which induces DSBs, revealed an increase in the degree of colocalization between S100A11 and Rad54B. Furthermore, S100A11/Rad54B foci are spatially associated with sites of DNA DSB repair. Furthermore, while the expression of p21(WAF1/CIP1) was increased in parallel with DNA damage, its protein level was drastically down-regulated in damaged cells after S100A11 knockdown. Down-regulation of S100A11 by RNA interference also abolished Rad54B targeting to DSBs. Additionally, S100A11 down-regulated HaCaT cells showed a restricted proliferation capacity and an increase of the apoptotic cell fraction. These observations suggest that S100A11 targets Rad54B to sites of DNA DSB repair sites and identify a novel function for S100A11 in p21-based regulation of cell cycle. PMID:18463164

  10. Rad54B Targeting to DNA Double-Strand Break Repair Sites Requires Complex Formation with S100A11

    PubMed Central

    Murzik, Ulrike; Hemmerich, Peter; Weidtkamp-Peters, Stefanie; Ulbricht, Tobias; Bussen, Wendy; Hentschel, Julia; von Eggeling, Ferdinand

    2008-01-01

    S100A11 is involved in a variety of intracellular activities such as growth regulation and differentiation. To gain more insight into the physiological role of endogenously expressed S100A11, we used a proteomic approach to detect and identify interacting proteins in vivo. Hereby, we were able to detect a specific interaction between S100A11 and Rad54B, which could be confirmed under in vivo conditions. Rad54B, a DNA-dependent ATPase, is described to be involved in recombinational repair of DNA damage, including DNA double-strand breaks (DSBs). Treatment with bleomycin, which induces DSBs, revealed an increase in the degree of colocalization between S100A11 and Rad54B. Furthermore, S100A11/Rad54B foci are spatially associated with sites of DNA DSB repair. Furthermore, while the expression of p21WAF1/CIP1 was increased in parallel with DNA damage, its protein level was drastically down-regulated in damaged cells after S100A11 knockdown. Down-regulation of S100A11 by RNA interference also abolished Rad54B targeting to DSBs. Additionally, S100A11 down-regulated HaCaT cells showed a restricted proliferation capacity and an increase of the apoptotic cell fraction. These observations suggest that S100A11 targets Rad54B to sites of DNA DSB repair sites and identify a novel function for S100A11 in p21-based regulation of cell cycle. PMID:18463164

  11. Increased Expression of the dsRNA-Activated Protein Kinase PKR in Breast Cancer Promotes Sensitivity to Doxorubicin

    PubMed Central

    Bennett, Richard L.; Carruthers, Aubrey L.; Hui, Teng; Kerney, Krystal R.; Liu, Xiangfei; May, W. Stratford

    2012-01-01

    It has been reported that the expression and activity of the interferon-inducible, dsRNA-dependent protein kinase, PKR, is increased in mammary carcinoma cell lines and primary tumor samples. To extend these findings and determine how PKR signaling may affect breast cancer cell sensitivity to chemotherapy, we measured PKR expression by immunohistochemical staining of 538 cases of primary breast cancer and normal tissues. Significantly, PKR expression was elevated in ductal, lobular and squamous cell carcinomas or lymph node metastases but not in either benign tumor specimens or cases of inflammation compared to normal tissues. Furthermore, PKR expression was increased in precancerous stages of mammary cell hyperplasia and dysplasia compared to normal tissues, indicating that PKR expression may be upregulated by the process of tumorigenesis. To test the function of PKR in breast cancer, we generated MCF7, T-47D and MDA-MB-231 breast cancer cell lines with significantly reduced PKR expression by siRNA knockdown. Importantly, while knockdown of PKR expression had no effect on cell proliferation under normal growth conditions, MCF7, T-47D or MDA-MB-231 cells with reduced PKR expression or treated with a small molecule PKR inhibitor were significantly less sensitive to doxorubicin or H2O2-induced toxicity compared to control cells. In addition, the rate of eIF2α phosphorylation following treatment with doxorubicin was delayed in breast cancer cell lines with decreased PKR expression. Significantly, treatment of breast cancer lines with reduced PKR expression with either interferon-α, which increases PKR expression, or salubrinal, which increases eIF2α phosphorylation, restored doxorubicin sensitivity to normal levels. Taken together these results indicate that increased PKR expression in primary breast cancer tissues may serve as a biomarker for response to doxorubicin-containing chemotherapy and that future therapeutic approaches to promote PKR expression

  12. Double Strand Breaks Can Initiate Gene Silencing and SIRT1-Dependent Onset of DNA Methylation in an Exogenous Promoter CpG Island

    PubMed Central

    O'Hagan, Heather M.; Mohammad, Helai P.; Baylin, Stephen B.

    2008-01-01

    Chronic exposure to inducers of DNA base oxidation and single and double strand breaks contribute to tumorigenesis. In addition to the genetic changes caused by this DNA damage, such tumors often contain epigenetically silenced genes with aberrant promoter region CpG island DNA hypermethylation. We herein explore the relationships between such DNA damage and epigenetic gene silencing using an experimental model in which we induce a defined double strand break in an exogenous promoter construct of the E-cadherin CpG island, which is frequently aberrantly DNA hypermethylated in epithelial cancers. Following the onset of repair of the break, we observe recruitment to the site of damage of key proteins involved in establishing and maintaining transcriptional repression, namely SIRT1, EZH2, DNMT1, and DNMT3B, and the appearance of the silencing histone modifications, hypoacetyl H4K16, H3K9me2 and me3, and H3K27me3. Although in most cells selected after the break, DNA repair occurs faithfully with preservation of activity of the promoter, a small percentage of the plated cells demonstrate induction of heritable silencing. The chromatin around the break site in such a silent clone is enriched for most of the above silent chromatin proteins and histone marks, and the region harbors the appearance of increasing DNA methylation in the CpG island of the promoter. During the acute break, SIRT1 appears to be required for the transient recruitment of DNMT3B and subsequent methylation of the promoter in the silent clones. Taken together, our data suggest that normal repair of a DNA break can occasionally cause heritable silencing of a CpG island–containing promoter by recruitment of proteins involved in silencing. Furthermore, with contribution of the stress-related protein SIRT1, the break can lead to the onset of aberrant CpG island DNA methylation, which is frequently associated with tight gene silencing in cancer. PMID:18704159

  13. Treatment of transplanted rat tumours with double-stranded RNA(BRL 5907). II. Treatment of pleural and peritoneal growths.

    PubMed Central

    Pimm, M. V.; Baldwin, R. W.

    1976-01-01

    Intrapleural growth of transplanted rat tumours was prevented or retarded by intrapleural administration of double-stranded RNA. A similar suppression of growth was achieved with peitoneal tumours by the intraperitoneal injection of the compound. These studies indicate the possible potential of this form of treatment of thoracic and peritoneal tumours for clinical application in the treatment of mesothelioma. PMID:177036

  14. Manganese porphyrin-double stranded DNA complex guided in situ deposition of polyaniline for electrochemical thrombin detection.

    PubMed

    Xie, Shunbi; Chai, Yaqin; Yuan, Yali; Yuan, Ruo

    2014-07-11

    In this work, we proposed a novel electrochemical strategy for sensitive detection of thrombin (TB) based on in situ generation of polyaniline (PANI) as a redox mediator by using a manganese porphyrin-double stranded DNA (MnTMPyP-dsDNA) peroxidase-like artificial enzyme mimic as a powerful catalyst and template.

  15. Complementation of Hyponastic Leaves1 by Double-Strand RNA-Binding Domains of Dicer-Like1 in Nuclear Dicing Bodies1[W][OPEN

    PubMed Central

    Liu, Qi; Yan, Qingqing; Liu, Yin; Hong, Fang; Sun, Zhenfei; Shi, Leilei; Huang, Ying; Fang, Yuda

    2013-01-01

    MicroRNAs (miRNAs) are a class of small regulatory RNAs that are found in almost all of the eukaryotes. Arabidopsis (Arabidopsis thaliana) miRNAs are processed from primary miRNAs (pri-miRNAs), mainly by the ribonuclease III-like enzyme DICER-LIKE1 (DCL1) and its specific partner, HYPONASTIC LEAVES1 (HYL1), a double-strand RNA-binding protein, both of which contain two double-strand RNA-binding domains (dsRBDs). These dsRBDs are essential for miRNA processing, but the functions of them are not clear. Here, we report that the two dsRBDs of DCL1 (DCL1-D1D2), and to some extent the second dsRBD (DCL1-D2), complement the hyl1 mutant, but not the first dsRBD of DCL1 (DCL1-D1). DCL1-D1 is diffusely distributed throughout the nucleoplasm, whereas DCL1-D2 and DCL1-D1D2 concentrate in nuclear dicing bodies in which DCL1 and HYL1 colocalize. We show further that protein-protein interaction is mainly mediated by DCL1-D2, while DCL1-D1 plays a major role in binding of pri-miRNAs. These results suggest parallel roles between C-terminal dsRBDs of DCL1 and N-terminal dsRBDs of HYL1 and support a model in which Arabidopsis pri-miRNAs are recruited to dicing bodies through functionally divergent dsRBDs of microprocessor for accurate processing of plant pri-miRNAs. PMID:23886622

  16. Mediation of CTCF transcriptional insulation by DEAD-box RNA-binding protein p68 and steroid receptor RNA activator SRA

    PubMed Central

    Yao, Hongjie; Brick, Kevin; Evrard, Yvonne; Xiao, Tiaojiang; Camerini-Otero, R. Daniel; Felsenfeld, Gary

    2010-01-01

    CCCTC-binding factor (CTCF) is a DNA-binding protein that plays important roles in chromatin organization, although the mechanism by which CTCF carries out these functions is not fully understood. Recent studies show that CTCF recruits the cohesin complex to insulator sites and that cohesin is required for insulator activity. Here we showed that the DEAD-box RNA helicase p68 (DDX5) and its associated noncoding RNA, steroid receptor RNA activator (SRA), form a complex with CTCF that is essential for insulator function. p68 was detected at CTCF sites in the IGF2/H19 imprinted control region (ICR) as well as other genomic CTCF sites. In vivo depletion of SRA or p68 reduced CTCF-mediated insulator activity at the IGF2/H19 ICR, increased levels of IGF2 expression, and increased interactions between the endodermal enhancer and IGF2 promoter. p68/SRA also interacts with members of the cohesin complex. Depletion of either p68 or SRA does not affect CTCF binding to its genomic sites, but does reduce cohesin binding. The results suggest that p68/SRA stabilizes the interaction of cohesin with CTCF by binding to both, and is required for proper insulator function. PMID:20966046

  17. MeHg Developing Exposure Causes DNA Double-Strand Breaks and Elicits Cell Cycle Arrest in Spinal Cord Cells

    PubMed Central

    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

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

    PubMed

    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

  19. The repair of environmentally relevant DNA double strand breaks caused by high linear energy transfer irradiation--no simple task.

    PubMed

    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

  20. Prevention of DNA Double-Strand Breaks Induced by Radioiodide-131I in FRTL-5 Thyroid Cells

    PubMed Central

    Okunyan, Armen; Rivina, Yelena; Cannon, Sophie; Hogen, Victor

    2011-01-01

    Radioiodine-131 released from nuclear reactor accidents has dramatically increased the incidence of papillary thyroid cancer in exposed individuals. 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 cancer. The aims of this study were to develop a method to show DNA DSBs induced by 131I in thyroid cells; to test monovalent anions that are transported by the sodium/iodide symporter to determine whether they prevent 131I-induced DSB; and to test other radioprotective agents for their effect on irradiated thyroid cells. Rat FRTL-5 thyroid cells were incubated with 131I. DSBs were measured by nuclear immunofluorescence using antibodies to p53-binding protein 1 or γH2AX. Incubation with 1–10 μCi 131I per milliliter for 90 min resulted in a dose-related increase of DSBs; the number of DSBs increased from a baseline of 4–15% before radiation to 65–90% after radiation. GH3 or CHO cells that do not transport iodide did not develop DSBs when incubated with 131I. Incubation with 20–100 μm iodide or thiocyanate markedly attenuated DSBs. Perchlorate was about 6 times more potent than iodide or thiocyanate. The effects of the anions were much greater when each was added 30–120 min before the 131I. Two natural organic compounds recently shown to provide radiation protection partially prevented DSBs caused by 131I and had an additive effect with perchlorate. In conclusion, we developed a thyroid cell model to quantify the mitogenic effect of 131I. 131I causes DNA DSBs in FRTL-5 cells and had no effect on cells that do not transport iodide. Perchlorate, iodide, and thiocyanate protect against DSBs induced by 131I. PMID:21190956

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

    PubMed Central

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

    2015-01-01

    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

  2. Impact of Charged Particle Exposure on Homologous DNA Double-Strand Break Repair in Human Blood-Derived Cells

    PubMed Central

    Rall, Melanie; Kraft, Daniela; Volcic, Meta; Cucu, Aljona; Nasonova, Elena; Taucher-Scholz, Gisela; Bönig, Halvard; Wiesmüller, Lisa; Fournier, Claudia

    2015-01-01

    Ionizing radiation generates DNA double-strand breaks (DSB) which, unless faithfully repaired, can generate chromosomal rearrangements in hematopoietic stem and/or progenitor cells (HSPC), potentially priming the cells towards a leukemic phenotype. Using an enhanced green fluorescent protein (EGFP)-based reporter system, we recently identified differences in the removal of enzyme-mediated DSB in human HSPC versus mature peripheral blood lymphocytes (PBL), particularly regarding homologous DSB repair (HR). Assessment of chromosomal breaks via premature chromosome condensation or γH2AX foci indicated similar efficiency and kinetics of radiation-induced DSB formation and rejoining in PBL and HSPC. Prolonged persistence of chromosomal breaks was observed for higher LET charged particles which are known to induce more complex DNA damage compared to X-rays. Consistent with HR deficiency in HSPC observed in our previous study, we noticed here pronounced focal accumulation of 53BP1 after X-ray and carbon ion exposure (intermediate LET) in HSPC versus PBL. For higher LET, 53BP1 foci kinetics was similarly delayed in PBL and HSPC suggesting similar failure to repair complex DNA damage. Data obtained with plasmid reporter systems revealed a dose- and LET-dependent HR increase after X-ray, carbon ion and higher LET exposure, particularly in HR-proficient immortalized and primary lymphocytes, confirming preferential use of conservative HR in PBL for intermediate LET damage repair. HR measured adjacent to the leukemia-associated MLL breakpoint cluster sequence in reporter lines revealed dose dependency of potentially leukemogenic rearrangements underscoring the risk of leukemia-induction by radiation treatment. PMID:26618143

  3. Hybridization of DNA and PNA molecular beacons to single-stranded and double-stranded DNA targets.

    PubMed

    Kuhn, Heiko; Demidov, Vadim V; Coull, James M; Fiandaca, Mark J; Gildea, Brian D; Frank-Kamenetskii, Maxim D

    2002-02-13

    Molecular beacons are sensitive fluorescent probes hybridizing selectively to designated DNA and RNA targets. They have recently become practical tools for quantitative real-time monitoring of single-stranded nucleic acids. Here, we comparatively study the performance of a variety of such probes, stemless and stem-containing DNA and PNA (peptide nucleic acid) beacons, in Tris-buffer solutions containing various concentrations of NaCl and MgCl(2). We demonstrate that different molecular beacons respond differently to the change of salt concentration, which could be attributed to the differences in their backbones and constructions. We have found that the stemless PNA beacon hybridizes rapidly to the complementary oligodeoxynucleotide and is less sensitive than the DNA beacons to the change of salt thus allowing effective detection of nucleic acid targets under various conditions. Though we found stemless DNA beacons improper for diagnostic purposes due to high background fluorescence, we believe that use of these DNA and similar RNA constructs in molecular-biophysical studies may be helpful for analysis of conformational flexibility of single-stranded nucleic acids. With the aid of PNA "openers", molecular beacons were employed for the detection of a chosen target sequence directly in double-stranded DNA (dsDNA). Conditions are found where the stemless PNA beacon strongly discriminates the complementary versus mismatched dsDNA targets. Together with the insensitivity of PNA beacons to the presence of salt and DNA-binding/processing proteins, the latter results demonstrate the potential of these probes as robust tools for recognition of specific sequences within dsDNA without denaturation and deproteinization of duplex DNA.

  4. Quantification of transgene-derived double-stranded RNA in plants using the QuantiGene nucleic acid detection platform.

    PubMed

    Armstrong, Toni A; Chen, Hao; Ziegler, Todd E; Iyadurai, Kelly R; Gao, Ai-Guo; Wang, Yongcheng; Song, Zihong; Tian, Qing; Zhang, Qiang; Ward, Jason M; Segers, Gerrit C; Heck, Gregory R; Staub, Jeffrey M

    2013-12-26

    The expanding use of RNA interference (RNAi) in agricultural biotechnology necessitates tools for characterizing and quantifying double-stranded RNA (dsRNA)-containing transcripts that are expressed in transgenic plants. We sought to detect and quantify such transcripts in transgenic maize lines engineered to control western corn rootworm (Diabrotica virgifera virgifera LeConte) via overexpression of an inverted repeat sequence bearing a portion of the putative corn rootworm orthologue of yeast Snf7 (DvSnf7), an essential component of insect cell receptor sorting. A quantitative assay was developed to detect DvSnf7 sense strand-containing dsRNA transcripts that is based on the QuantiGene Plex 2.0 RNA assay platform from Affymetrix. The QuantiGene assay utilizes cooperative binding of multiple oligonucleotide probes with specificity for the target sequence resulting in exceptionally high assay specificity. Successful implementation of this assay required heat denaturation in the presence of the oligonucleotide probes prior to hybridization, presumably to dissociate primary transcripts carrying the duplex dsRNA structure. The dsRNA assay was validated using a strategy analogous to the rigorous enzyme-linked immunosorbent assay evaluations that are typically performed for foreign proteins expressed in transgenic plants. Validation studies indicated that the assay is sensitive (to 10 pg of dsRNA/g of fresh tissue), highly reproducible, and linear over ∼2.5 logs. The assay was validated using purified RNA from multiple maize tissue types, and studies indicate that the assay is also quantitative in crude tissue lysates. To the best of our knowledge, this is the first report of a non-polymerase chain reaction-based quantitative assay for dsRNA-containing transcripts, based on the use of the QuantiGene technology platform, and will broadly facilitate characterization of dsRNA in biological and environmental samples.

  5. Effects of chemopreventive natural products on non-homologous end-joining DNA double-strand break repair.

    PubMed

    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

  6. Toll-like Receptor 3, RIG-I-like Receptors and the NLRP3 Inflammasome: Key Modulators of Innate Immune Responses to Double-stranded RNA Viruses

    PubMed Central

    Yu, Man; Levine, Stewart J.

    2011-01-01

    Double-stranded RNA (dsRNA), the genetic material for many RNA viruses, induces robust host immune responses via pattern recognition receptors, which include Toll-like receptor 3 (TLR3), retinoic acid-inducible gene-I-like receptors (RLRs) and the multi-protein NLRP3 inflammasome complex. The engagement of dsRNA receptors or inflammasome activation by viral dsRNA initiates complex intracellular signaling cascades that play essential roles in inflammation and innate immune responses, as well as the resultant development of adaptive immunity. This review focuses on signaling pathways mediated by TLR3, RLRs and the NLRP3 inflammasome, as well as the potential use of agonists and antagonists that target these pathways to treat disease. PMID:21466970

  7. Opposing roles of RNF8/RNF168 and deubiquitinating enzymes in ubiquitination-dependent DNA double-strand break response signaling and DNA-repair pathway choice

    PubMed Central

    Nakada, Shinichiro

    2016-01-01

    The E3 ubiquitin ligases ring finger protein (RNF) 8 and RNF168 transduce the DNA double-strand break (DSB) response (DDR) signal by ubiquitinating DSB sites. The depletion of RNF8 or RNF168 suppresses the accumulation of DNA-repair regulating factors such as 53BP1 and RAP80 at DSB sites, suggesting roles for RNF8- and RNF168-mediated ubiquitination in DSB repair. This mini-review provides a brief overview of the RNF8- and RNF168-dependent DDR-signaling and DNA-repair pathways. The choice of DNA-repair pathway when RNF8- and RNF168-mediated ubiquitination-dependent DDR signaling is negatively regulated by deubiquitinating enzymes (DUBs) is reviewed to clarify how the opposing roles of RNF8/RNF168 and DUBs regulate ubiquitination-dependent DDR signaling and the choice of DNA-repair pathway. PMID:26983989

  8. Adsorption of double-stranded DNA to graphene oxide preventing enzymatic digestion

    NASA Astrophysics Data System (ADS)

    Lei, Haozhi; Mi, Lijuan; Zhou, Xuejiao; Chen, Jiajia; Hu, Jun; Guo, Shouwu; Zhang, Yi

    2011-09-01

    Investigation into the interactions between graphene oxide (GO) and biomolecules is very important for broad applications of GO in bioassay and bioanalysis. In this work, we describe the interactions between double-stranded DNA (dsDNA) and GO. We demonstrated that dsDNA can bind to GO forming complexes (dsDNA/GO) in the presence of certain salts, which protects dsDNA from being enzymatically digested. On the other hand, we found that a nonionic surfactant, such as triton X-100, can block the formation of dsDNA/GO complexes, so that the enzymatic digestion of dsDNA is restored. These results lead us to believe that the reason for GO protecting dsDNA from enzymatic digestion is the formation of dsDNA/GO complexes hindering the access of DNA enzymes to dsDNA, rather than direct inactivation of the DNA enzymes.Investigation into the interactions between graphene oxide (GO) and biomolecules is very important for broad applications of GO in bioassay and bioanalysis. In this work, we describe the interactions between double-stranded DNA (dsDNA) and GO. We demonstrated that dsDNA can bind to GO forming complexes (dsDNA/GO) in the presence of certain salts, which protects dsDNA from being enzymatically digested. On the other hand, we found that a nonionic surfactant, such as triton X-100, can block the formation of dsDNA/GO complexes, so that the enzymatic digestion of dsDNA is restored. These results lead us to believe that the reason for GO protecting dsDNA from enzymatic digestion is the formation of dsDNA/GO complexes hindering the access of DNA enzymes to dsDNA, rather than direct inactivation of the DNA enzymes. Electronic supplementary information (ESI) available: Effect of temperature and dsDNA length on the binding and protecting of DNA by GO. See DOI: 10.1039/c1nr10617a

  9. Accumulation of Ku70 at DNA double-strand breaks in living epithelial cells

    SciTech Connect

    Koike, Manabu; Yutoku, Yasutomo; Koike, Aki

    2011-10-15

    Ku70 and Ku80 play an essential role in the DNA double-strand break (DSB) repair pathway, i.e., nonhomologous DNA-end-joining (NHEJ). No accumulation mechanisms of Ku70 at DSBs have been clarified in detail, although the accumulation mechanism of Ku70 at DSBs plays key roles in regulating the NHEJ activity. Here, we show the essential domains for the accumulation and function of Ku70 at DSBs in living lung epithelial cells. Our results showed that EGFP-Ku70 accumulation at DSBs began immediately after irradiation. Our findings demonstrate that three domains of Ku70, i.e., the {alpha}/{beta}, DNA-binding, and Ku80-binding domains, but not the SAP domain, are necessary for the accumulation at or recognition of DSBs in the early stage after irradiation. Moreover, our findings demonstrate that the leucine at amino acid 385 of Ku70 in the Ku80-binding domain, but not the three target amino acids for acetylation in the DNA-binding domain, is involved in the localization and accumulation of Ku70 at DSBs. Furthermore, accumulations of XRCC4 and XLF, but not that of Artemis, at DSBs are dependent on the presence of Ku70. These findings suggest that Artemis can work in not only the Ku-dependent repair process, but also the Ku-independent process at DSBs in living epithelial cells.

  10. Host Double Strand Break Repair Generates HIV-1 Strains Resistant to CRISPR/Cas9.

    PubMed

    Yoder, Kristine E; Bundschuh, Ralf

    2016-01-01

    CRISPR/Cas9 genome editing has been proposed as a therapeutic treatment for HIV-1 infection. CRISPR/Cas9 induced double strand breaks (DSBs) targeted to the integrated viral genome have been shown to decrease production of progeny virus. Unfortunately HIV-1 evolves rapidly and may readily produce CRISPR/Cas9 resistant strains. Here we used next-generation sequencing to characterize HIV-1 strains that developed resistance to six different CRISPR/Cas9 guide RNAs (gRNAs). Reverse transcriptase (RT) derived base substitution mutations were commonly found at sites encoding unpaired bases of RNA stem-loop structures. In addition to RT mutations, insertion and/or deletion (indel) mutations were common. Indels localized to the CRISPR/Cas9 cleavage site were major contributors to CRISPR gRNA resistance. While most indels at non-coding regions were a single base pair, 3 base pair indels were observed when a coding region of HIV-1 was targeted. The DSB repair event may preserve the HIV-1 reading frame, while destroying CRISPR gRNA homology. HIV-1 may be successfully edited by CRISPR/Cas9, but the virus remains competent for replication and resistant to further CRISPR/Cas9 targeting at that site. These observations strongly suggest that host DSB repair at CRISPR/Cas9 cleavage sites is a novel and important pathway that may contribute to HIV-1 therapeutic resistance. PMID:27404981

  11. Genetic and Physical Analysis of Double-Strand Break Repair and Recombination in Saccharomyces Cerevisiae

    PubMed Central

    Rudin, N.; Sugarman, E.; Haber, J. E.

    1989-01-01

    We have investigated HO endonuclease-induced double-strand break (DSB) recombination and repair in a LACZ duplication plasmid in yeast. A 117-bp MATa fragment, embedded in one copy of LACZ, served as a site for initiation of a DSB when HO endonuclease was expressed. The DSB could be repaired using wild-type sequences located on a second, promoterless, copy of LACZ on the same plasmid. In contrast to normal mating-type switching, crossing-over associated with gene conversion occurred at least 50% of the time. The proportion of conversion events accompanied by exchange was greater when the two copies of LACZ were in direct orientation (80%), than when inverted (50%). In addition, the fraction of plasmids lost was significantly greater in the inverted orientation. The kinetics of appearance of intermediates and final products were also monitored. The repair of the DSB is slow, requiring at least an hour from the detection of the HO-cut fragments to completion of repair. Surprisingly, the appearance of the two reciprocal products of crossing over did not occur with the same kinetics. For example, when the two LACZ sequences were in the direct orientation, the HO-induced formation of a large circular deletion product was not accompanied by the appearance of a small circular reciprocal product. We suggest that these differences may reflect two kinetically separable processes, one involving only one cut end and the other resulting from the concerted participation of both ends of the DSB. PMID:2668114

  12. Telomere Dysfunction Triggers Palindrome Formation Independently of Double-Strand Break Repair Mechanisms

    PubMed Central

    Raykov, Vasil; Marvin, Marcus E.; Louis, Edward J.; Maringele, Laura

    2016-01-01

    Inverted chromosome duplications or palindromes are linked with genetic disorders and malignant transformation. They are considered by-products of DNA double-strand break (DSB) repair: the homologous recombination (HR) and the nonhomologous end joining (NHEJ). Palindromes near chromosome ends are often triggered by telomere losses. An important question is to what extent their formation depends upon DSB repair mechanisms. Here we addressed this question using yeast genetics and comparative genomic hybridization. We induced palindrome formation by passaging cells lacking any form of telomere maintenance (telomerase and telomere recombination). Surprisingly, we found that DNA ligase 4, essential for NHEJ, did not make a significant contribution to palindrome formation induced by telomere losses. Moreover RAD51, important for certain HR-derived mechanisms, had little effect. Furthermore RAD52, which is essential for HR in yeast, appeared to decrease the number of palindromes in cells proliferating without telomeres. This study also uncovered an important role for Rev3 and Rev7 (but not for Pol32) subunits of polymerase ζ in the survival of cells undergoing telomere losses and forming palindromes. We propose a model called short-inverted repeat-induced synthesis in which DNA synthesis, rather than DSB repair, drives the inverted duplication triggered by telomere dysfunction. PMID:27334270

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

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

    2016-01-01

    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 lymphocytes in 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

  15. Numerical constraints and feedback control of double-strand breaks in mouse meiosis

    PubMed Central

    Kauppi, Liisa; Barchi, Marco; Lange, Julian; Baudat, Frédéric; Jasin, Maria; Keeney, Scott

    2013-01-01

    Different organisms display widely different numbers of the programmed double-strand breaks (DSBs) that initiate meiotic recombination (e.g., hundreds per meiocyte in mice and humans vs. dozens in nematodes), but little is known about what drives these species-specific DSB set points or the regulatory pathways that control them. Here we examine male mice with a lowered dosage of SPO11, the meiotic DSB catalyst, to gain insight into the effect of reduced DSB numbers on mammalian chromosome dynamics. An approximately twofold DSB reduction was associated with the reduced ability of homologs to synapse along their lengths, provoking prophase arrest and, ultimately, sterility. In many spermatocytes, chromosome subsets displayed a mix of synaptic failure and synapsis with both homologous and nonhomologous partners (“chromosome tangles”). The X chromosome was nearly always involved in tangles, and small autosomes were involved more often than large ones. We conclude that homolog pairing requirements dictate DSB set points during meiosis. Importantly, our results reveal that karyotype is a key factor: Smaller autosomes and heteromorphic sex chromosomes become weak links when DSBs are reduced below a critical threshold. Unexpectedly, unsynapsed chromosome segments trapped in tangles displayed an elevated density of DSB markers later in meiotic prophase. The unsynapsed portion of the X chromosome in wild-type males also showed evidence that DSB numbers increased as prophase progressed. These findings point to the existence of a feedback mechanism that links DSB number and distribution with interhomolog interactions. PMID:23599345

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

    SciTech Connect

    Xiao, Xiao; Gang, Yi; Wang, Honghong; Wang, Jiayin; Zhao, Lina; Xu, Li; Liu, Zhiguo

    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.

  17. Involvement of histone H1.2 in apoptosis induced by DNA double-strand breaks.

    PubMed

    Konishi, Akimitsu; Shimizu, Shigeomi; Hirota, Junko; Takao, Toshifumi; Fan, Yuhong; Matsuoka, Yosuke; Zhang, Lilin; Yoneda, Yoshihiro; Fujii, Yoshitaka; Skoultchi, Arthur I; Tsujimoto, Yoshihide

    2003-09-19

    It is poorly understood how apoptotic signals arising from DNA damage are transmitted to mitochondria, which release apoptogenic factors into the cytoplasm that activate downstream destruction programs. Here, we identify histone H1.2 as a cytochrome c-releasing factor that appears in the cytoplasm after exposure to X-ray irradiation. While all nuclear histone H1 forms are released into the cytoplasm in a p53-dependent manner after irradiation, only H1.2, but not other H1 forms, induced cytochrome c release from isolated mitochondria in a Bak-dependent manner. Reducing H1.2 expression enhanced cellular resistance to apoptosis induced by X-ray irradiation or etoposide, but not that induced by other stimuli including TNF-alpha and UV irradiation. H1.2-deficient mice exhibited increased cellular resistance in thymocytes and the small intestine to X-ray-induced apoptosis. These results indicate that histone H1.2 plays an important role in transmitting apoptotic signals from the nucleus to the mitochondria following DNA double-strand breaks.

  18. Double-strand break-induced mitotic intrachromosomal recombination in the fission yeast Schizosaccharomyces pombe

    SciTech Connect

    Osman, F.; Fortunato, E.A.; Subramani, S.

    1996-02-01

    The Saccharomyces cerevisiae HO gene and MATa cutting site were used to introduce site-specific double-strand breaks (DSBs) within intrachromosomal recombination substrates in Schizosaccharomyces pombe. The recombination substrates consisted of nontandem direct repeats of ade6 heteroalleles. DSB induction stimulated the frequency of recombinants 2000-fold. The spectrum of DSB-induced recombinants depended on whether the DSB was introduced within one of the ade6 repeats or in intervening unique DNA. When the DSB was introduced within unique DNA, over 99.8% of the recombinants lacked the intervening DNA but retained one copy of ade6 that was wild type or either one of the heteroalleles. When the DSB was located in duplicated DNA, 77% of the recombinants were similar to the deletion types described above, but the single ade6 copy was either wild type or exclusively that of the uncut repeat. The remaining 23% of the induced recombinants were gene convertants with two copies of ade6 and the intervening sequences; the ade6 heteroallele in which the DSB was induced was the recipient of genetic information. Half-sectored colonies were isolated, analyzed and interpreted as evidence of heteroduplex DNA formation. The results are discussed in terms of current models for recombination. 81 refs., 9 figs., 3 tabs.

  19. Nanoneedle insertion into the cell nucleus does not induce double-strand breaks in chromosomal DNA.

    PubMed

    Ryu, Seunghwan; Kawamura, Ryuzo; Naka, Ryohei; Silberberg, Yaron R; Nakamura, Noriyuki; Nakamura, Chikashi

    2013-09-01

    An atomic force microscope probe can be formed into an ultra-sharp cylindrical shape (a nanoneedle) using micro-fabrication techniques such as focused ion beam etching. This nanoneedle can be effectively inserted through the plasma membrane of a living cell to not only access the cytosol, but also to penetrate through the nuclear membrane. This technique shows great potential as a tool for performing intranuclear measurements and manipulations. Repeated insertions of a nanoneedle into a live cell were previously shown not to affect cell viability. However, the effect of nanoneedle insertion on the nucleus and nuclear components is still unknown. DNA is the most crucial component of the nucleus for proper cell function and may be physically damaged by a nanoneedle. To investigate the integrity of DNA following nanoneedle insertion, the occurrence of DNA double-strand breaks (DSBs) was assessed. The results showed that there was no chromosomal DNA damage due to nanoneedle insertion into the nucleus, as indicated by the expression level of γ-H2AX, a molecular marker of DSBs.

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

    PubMed

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

    2016-06-28

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

  1. Double strand binding – single strand incision mechanism for human flap endonuclease: implications for the superfamily

    PubMed Central

    Tsutakawa, Susan E.; Tainer, John A.

    2012-01-01

    Detailed structural, mutational, and biochemical analyses of human FEN1/DNA complexes have revealed the mechanism for recognition of 5′ flaps formed during lagging strand replication and DNA repair. FEN1 processes 5′ flaps through a previously unknown, but structurally elegant double-stranded (ds) recognition/single stranded (ss) incision mechanism that both selects for 5′ flaps and selects against ss DNA or RNA, intact dsDNA, and 3′ flaps. Two major DNA binding interfaces, including a K+ bridge between the DNA and the H2TH motif, are spaced one helical turn apart and together select for substrates with dsDNA. A conserved helical gateway and a helical cap protects the two-metal active site and selects for ss flaps with free termini. Structures of substrate and product reveal an unusual step between binding substrate and incision that involves a double base unpairing with incision occurring in the resulting unpaired DNA or RNA. Ordering of the active site requires a disorder-to-order transition induced by binding of an unpaired 3′ flap, which ensures that the product is ligatable. Comparison with FEN superfamily members, including XPG, EXO1, and GEN1, identifies superfamily motifs such as the helical gateway that select for ss-dsDNA junctions and provides key biological insights into nuclease specificity and regulation. PMID:22244820

  2. Tetrameric Ctp1 coordinates DNA binding and bridging in DNA double strand break repair

    PubMed Central

    Andres, Sara N.; Appel, C. Denise; Westmoreland, Jim; Williams, Jessica S.; Nguyen, Yvonne; Robertson, Patrick D.; Resnick, Michael A.; Williams, R. Scott

    2014-01-01

    Ctp1 (aka CtIP or Sae2) collaborates with Mre11–Rad50–Nbs1 to initiate repair of DNA double strand breaks (DSBs), but its function(s) remain enigmatic. We report that tetrameric Schizosaccharomyces pombe Ctp1 harbors multivalent DNA-binding and bridging activities. Through structural and biophysical analyses of the Ctp1 tetramer we define the salient features of Ctp1 architecture: an N-terminal interlocking tetrameric helical dimer-of-dimers (THDD) domain and a central intrinsically disordered region (IDR) linked to C-terminal “RHR” DNA interaction motifs. The THDD, IDR and RHR are required for Ctp1 DNA bridging activity in vitro and both the THDD and RHR are required for efficient DSB repair in S. pombe. Our results establish non-nucleolytic roles for Ctp1 in binding and coordination of DSB repair intermediates and suggest that ablation of human CtIP DNA binding by truncating mutations underlie the CTIP-linked Seckel and Jawad syndromes. PMID:25580577

  3. Probing 3D Collective Cancer Invasion Using Double-Stranded Locked Nucleic Acid Biosensors.

    PubMed

    Dean, Zachary S; Elias, Paul; Jamilpour, Nima; Utzinger, Urs; Wong, Pak Kin

    2016-09-01

    Cancer is a leading cause of death worldwide and metastases are responsible for over 90% of human cancer deaths. There is an urgent need to develop novel therapeutics for suppressing cancer invasion, the initial step of metastasis. Nevertheless, the regulation of cancer invasion is poorly understood due to a paucity of tools for monitoring the invasion process in 3D microenvironments. Here, we report a double-stranded locked nucleic acid (dsLNA) biosensor for investigating 3D collective cancer invasion. By incorporating multiphoton microscopy and the dsLNA biosensor, we perform dynamic single cell gene expression analysis while simultaneously characterizing the biomechanical interaction between the invading sprouts and the extracellular matrix. Gene profiling of invasive leader cells and detached cells suggest distinctive signaling mechanisms involved in collective and individual invasion in the 3D microenvironment. Our results underscore the involvement of Notch signaling in 3D collective cancer invasion, which warrants further investigation toward antimetastasis therapy in the future. PMID:27529634

  4. Double-strand break-induced recombination between ectopic homologous sequences in somatic plant cells.

    PubMed Central

    Puchta, H

    1999-01-01

    Homologous recombination between ectopic sites is rare in higher eukaryotes. To test whether double-strand breaks (DSBs) can induce ectopic recombination, transgenic tobacco plants harboring two unlinked, nonfunctional homologous parts of a kanamycin resistance gene were produced. To induce homologous recombination between the recipient locus (containing an I-SceI site within homologous sequences) and the donor locus, the rare cutting restriction enzyme I-SceI was transiently expressed via Agrobacterium in these plants. Whereas without I-SceI expression no recombination events were detectable, four independent recombinants could be isolated after transient I-SceI expression, corresponding to approximately one event in 10(5) transformations. After regeneration, the F1 generation of all recombinants showed Mendelian segregation of kanamycin resistance. Molecular analysis of the recombinants revealed that the resistance gene was indeed restored via homologous recombination. Three different kinds of reaction products could be identified. In one recombinant a classical gene conversion without exchange of flanking markers occurred. In the three other cases homologous sequences were transferred only to one end of the break. Whereas in three cases the ectopic donor sequence remained unchanged, in one case rearrangements were found in recipient and donor loci. Thus, ectopic homologous recombination, which seems to be a minor repair pathway for DSBs in plants, is described best by recombination models that postulate independent roles for the break ends during the repair process. PMID:10388832

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

    PubMed Central

    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 mapping indicates that the likelihood of GQ formation is substantially diminished in dsDNA, likely due to the competition from the Watson–Crick base pairing. Unlike GQ folding sequence in ssDNA which forms both parallel and antiparallel GQs, dsDNA displays only parallel folding. Additionally, we employed single molecule FRET to obtain a direct quantitation of stably formed-, weakly folded and unfolded GQ conformations. The findings of this study and the method developed here will enable identifying and classifying potential GQ-forming sequences in human genome. PMID:26202971

  6. DNA Double Strand Break Repair Pathway Choice Is Directed by Distinct MRE11 Nuclease Activities

    PubMed Central

    Shibata, Atsushi; Moiani, Davide; Arvai, Andrew S.; Perry, J. Jefferson P.; Harding, Shane M.; Genois, Marie-Michelle; Maity, Ranjan; van Rossum-Fikkert, Sari; Kertokalio, Aryandi; Romoli, Filippo; Ismail, Amani; Ismalaj, Ermal; Petricci, Elena; Matthew, J Neale; Bristow, Robert G; Masson, Jean-Yves; Wyman, Claire; Jeggo, Penny; Tainer, John A.

    2014-01-01

    SUMMARY MRE11 within the MRE11-RAD50-NBS1 (MRN) complex acts in DNA double-strand break repair (DSBR), detection and signaling; yet, how its endo- and exonuclease activities regulate DSB repair by non-homologous end-joining (NHEJ) versus homologous recombination (HR) remains enigmatic. Here we employed structure-based design with a focused chemical library to discover specific MRE11 endo- or exonuclease inhibitors. With these inhibitors we examined repair pathway choice at DSBs generated in G2 following radiation exposure. Whilst endo- or exonuclease inhibition impairs radiation-induced RPA chromatin binding, suggesting diminished resection, the inhibitors surprisingly direct different repair outcomes. Endonuclease inhibition promotes NHEJ in lieu of HR, whilst exonuclease inhibition confers a repair defect. Collectively, the results describe nuclease-specific MRE11 inhibitors, define distinct nuclease roles in DSB repair, and support a mechanism whereby MRE11 endonuclease initiates resection, thereby licensing HR followed by MRE11 exo and EXO1/BLM bidirectional resection towards and away from the DNA end, which commits to HR. PMID:24316220

  7. Host Double Strand Break Repair Generates HIV-1 Strains Resistant to CRISPR/Cas9

    PubMed Central

    Yoder, Kristine E.; Bundschuh, Ralf

    2016-01-01

    CRISPR/Cas9 genome editing has been proposed as a therapeutic treatment for HIV-1 infection. CRISPR/Cas9 induced double strand breaks (DSBs) targeted to the integrated viral genome have been shown to decrease production of progeny virus. Unfortunately HIV-1 evolves rapidly and may readily produce CRISPR/Cas9 resistant strains. Here we used next-generation sequencing to characterize HIV-1 strains that developed resistance to six different CRISPR/Cas9 guide RNAs (gRNAs). Reverse transcriptase (RT) derived base substitution mutations were commonly found at sites encoding unpaired bases of RNA stem-loop structures. In addition to RT mutations, insertion and/or deletion (indel) mutations were common. Indels localized to the CRISPR/Cas9 cleavage site were major contributors to CRISPR gRNA resistance. While most indels at non-coding regions were a single base pair, 3 base pair indels were observed when a coding region of HIV-1 was targeted. The DSB repair event may preserve the HIV-1 reading frame, while destroying CRISPR gRNA homology. HIV-1 may be successfully edited by CRISPR/Cas9, but the virus remains competent for replication and resistant to further CRISPR/Cas9 targeting at that site. These observations strongly suggest that host DSB repair at CRISPR/Cas9 cleavage sites is a novel and important pathway that may contribute to HIV-1 therapeutic resistance. PMID:27404981

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

    PubMed

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

    2011-08-01

    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 apoptotic oocyte death and resulted in the activation of ATM. It appeared that the repair response enabled a minor proportion of oocytes (34.7%) and granulosa cells (12.1%) to survive while the majority succumbed to apoptotic death. Paradoxically, inhibition of ATM by KU-55933 resulted in improved survival, probably via prevention of downstream activation of TAp63α. Furthermore, doxorubicin caused vascular and stromal damage in the human ovary, which might impair ovarian function both pre- and post-menopausally. Chemotherapy-induced premature ovarian aging appears to result from a complex process involving both the germ- and non-germ cell components of the ovary. These effects may have clinical implications in aging both for premenopausal and postmenopausal cancer survivors. PMID:21869459

  9. Cdc14A and Cdc14B Redundantly Regulate DNA Double-Strand Break Repair

    PubMed Central

    Lin, Han; Ha, Kyungsoo; Lu, Guojun; Fang, Xiao; Cheng, Ranran; Zuo, Qiuhong

    2015-01-01

    Cdc14 is a phosphatase that controls mitotic exit and cytokinesis in budding yeast. In mammals, the two Cdc14 homologues, Cdc14A and Cdc14B, have been proposed to regulate DNA damage repair, whereas the mitotic exit and cytokinesis rely on another phosphatase, PP2A-B55α. It is unclear if the two Cdc14s work redundantly in DNA repair and which repair pathways they participate in. More importantly, their target(s) in DNA repair remains elusive. Here we report that Cdc14B knockout (Cdc14B−/−) mouse embryonic fibroblasts (MEFs) showed defects in repairing ionizing radiation (IR)-induced DNA double-strand breaks (DSBs), which occurred only at late passages when Cdc14A levels were low. This repair defect could occur at early passages if Cdc14A levels were also compromised. These results indicate redundancy between Cdc14B and Cdc14A in DSB repair. Further, we found that Cdc14B deficiency impaired both homologous recombination (HR) and nonhomologous end joining (NHEJ), the two major DSB repair pathways. We also provide evidence that Cdh1 is a downstream target of Cdc14B in DSB repair. PMID:26283732

  10. Repairing a double-strand chromosome break by homologous recombination: revisiting Robin Holliday's model.

    PubMed Central

    Haber, James E; Ira, Gregorz; Malkova, Anna; Sugawara, Neal

    2004-01-01

    Since the pioneering model for homologous recombination proposed by Robin Holliday in 1964, there has been great progress in understanding how recombination occurs at a molecular level. In the budding yeast Saccharomyces cerevisiae, one can follow recombination by physically monitoring DNA after the synchronous induction of a double-strand break (DSB) in both wild-type and mutant cells. A particularly well-studied system has been the switching of yeast mating-type (MAT) genes, where a DSB can be induced synchronously by expression of the site-specific HO endonuclease. Similar studies can be performed in meiotic cells, where DSBs are created by the Spo11 nuclease. There appear to be at least two competing mechanisms of homologous recombination: a synthesis-dependent strand annealing pathway leading to noncrossovers and a two-end strand invasion mechanism leading to formation and resolution of Holliday junctions (HJs), leading to crossovers. The establishment of a modified replication fork during DSB repair links gene conversion to another important repair process, break-induced replication. Despite recent revelations, almost 40 years after Holliday's model was published, the essential ideas he proposed of strand invasion and heteroduplex DNA formation, the formation and resolution of HJs, and mismatch repair, remain the basis of our thinking. PMID:15065659

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

    PubMed

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

    2016-03-24

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

  12. Ycs4 is Required for Efficient Double-Strand Break Formation and Homologous Recombination During Meiosis.

    PubMed

    Hong, Soogil; Choi, Eui-Hwan; Kim, Keun Pil

    2015-07-01

    Condensin is not only responsible for chromosome condensation, but is also involved in double-strand break (DSB) processing in the cell cycle. During meiosis, the condensin complex serves as a component of the meiotic chromosome axis, and mediates both proper assembly of the synaptonemal complex and DSB repair, in order to ensure proper homologous chromosome segregation. Here, we used the budding yeast Saccharomyces cerevisiae to show that condensin participates in a variety of chromosome organization processes and exhibits crucial molecular functions that contribute to meiotic recombination during meiotic prophase I. We demonstrate that Ycs4 is required for efficient DSB formation and establishing homolog bias at the early stage of meiotic prophase I, which allows efficient formation of interhomolog recombination products. In the Ycs4 meiosis-specific allele (ycs4S), interhomolog products were formed at substantial levels, but with the same reduction in crossovers and noncrossovers. We further show that, in prophase chromosomal events, ycs4S relieved the defects in the progression of recombination interactions induced as a result of the absence of Rec8. These results suggest that condensin is a crucial coordinator of the recombination process and chromosome organization during meiosis.

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

    SciTech Connect

    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.

  14. Method for colorimetric detection of double-stranded nucleic acid using leuco triphenylmethane dyes.

    PubMed

    Miyamoto, Shigehiko; Sano, Sotaro; Takahashi, Koji; Jikihara, Takaaki

    2015-03-15

    Because loop-mediated isothermal amplification (LAMP) can amplify substantial amounts of DNA under isothermal conditions, its applications for simple genetic testing have attracted considerable attention. A positive LAMP reaction is indicated by the turbidity caused by by-products or by the color change after adding a metallochromic indicator to the reaction solution, but these methods have certain limitations. Leuco crystal violet (LCV), a colorless dye obtained after sodium sulfite treatment of crystal violet (CV), was used as a new colorimetric method for detecting LAMP. LCV is reconverted into CV through contact with double-stranded DNA (dsDNA). Therefore, the positive reaction of LAMP is indicated by color change from colorless to violet. The assay is sensitive enough to detect LAMP products, with a detection limit of 7.1 ng/μl for dsDNA. It is also highly selective to dsDNA, and interference with single-stranded DNA and deoxynucleotide triphosphates (dNTPs) is not observed. LCV facilitates direct colorimetric detection of the main product rather than a by-product of the LAMP reaction; therefore, this method can be used under various reaction conditions such as those with added pyrophosphatase in solution. This colorimetric LAMP detection method using LCV is useful for point-of-care genetic testing given its simplicity. PMID:25575759

  15. DNA double-strand breaks alter the spatial arrangement of homologous loci in plant cells.

    PubMed

    Hirakawa, Takeshi; Katagiri, Yohei; Ando, Tadashi; Matsunaga, Sachihiro

    2015-01-01

    Chromatin dynamics and arrangement are involved in many biological processes in nuclei of eukaryotes including plants. Plants have to respond rapidly to various environmental stimuli to achieve growth and development because they cannot move. It is assumed that the alteration of chromatin dynamics and arrangement support the response to these stimuli; however, there is little information in plants. In this study, we investigated the chromatin dynamics and arrangement with DNA damage in Arabidopsis thaliana by live-cell imaging with the lacO/LacI-EGFP system and simulation analysis. It was revealed that homologous loci kept a constant distance in nuclei of A. thaliana roots in general growth. We also found that DNA double-strand breaks (DSBs) induce the approach of the homologous loci with γ-irradiation. Furthermore, AtRAD54, which performs an important role in the homologous recombination repair pathway, was involved in the pairing of homologous loci with γ-irradiation. These results suggest that homologous loci approach each other to repair DSBs, and AtRAD54 mediates these phenomena.

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

    NASA Astrophysics Data System (ADS)

    Cveticanin, Jelena; Joksic, Gordana; Leskovac, Andreja; Petrovic, Sandra; Valenta Sobot, Ana; Neskovic, Olivera

    2010-01-01

    Carbon nanotubes are unique one-dimensional macromolecules with promising applications in biology and medicine. Since their toxicity is still under debate, here we present a study investigating the genotoxic properties of purified single wall carbon nanotubes (SWCNTs), multiwall carbon nanotubes (MWCNTs), and amide functionalized purified SWCNTs on cultured human lymphocytes employing cytokinesis block micronucleus assay and enumeration of γH2AX foci as a measure of double strand breaks (DSBs) of the DNA in normal human fibroblasts. SWCNTs induce micronuclei (MN) formation in lymphocytes and decrease the proliferation potential (CBPI) of cells. In a fibroblast cell line the same dose of SWCNTs induces γH2AX foci 2.7-fold higher than in a control. Amide functionalized purified SWCNTs behave differently: they do not disturb the cell proliferation potential of harvested lymphocytes, but induce micronuclei to a higher extent than SWCNTs. When applied on fibroblasts, amide functionalized SWCNTs also induce γH2AX foci, 3.18-fold higher than the control. The cellular effects of MWCNTs display the broad spectrum of clastogenic properties seen as the highest incidence of induced lymphocyte micronuclei and anaphase bridges among nuclei in binucleated cells. Surprisingly, the incidence of induced γH2AX foci was not as high as was expected by the micronucleus test, which indicates that MWCNTs act as clastogen and aneugen agents simultaneously. Biological endpoints investigated in this study indicate a close relationship between the electrochemical properties of carbon nanotubes and observed genotoxicity.

  17. DNA double strand break repair pathway choice: a chromatin based decision?

    PubMed

    Clouaire, T; Legube, G

    2015-01-01

    DNA double-strand breaks (DSBs) are highly toxic lesions that can be rapidly repaired by 2 main pathways, namely Homologous Recombination (HR) and Non Homologous End Joining (NHEJ). The choice between these pathways is a critical, yet not completely understood, aspect of DSB repair. We recently found that distinct DSBs induced across the genome are not repaired by the same pathway. Indeed, DSBs induced in active genes, naturally enriched in the trimethyl form of histone H3 lysine 36 (H3K36me3), are channeled to repair by HR, in a manner depending on SETD2, the major H3K36 trimethyltransferase. Here, we propose that these findings may be generalized to other types of histone modifications and repair machineries thus defining a "DSB repair choice histone code". This "decision making" function of preexisting chromatin structure in DSB repair could connect the repair pathway used to the type and function of the damaged region, not only contributing to genome stability but also to its diversity. PMID:25675367

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

    PubMed

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

    2016-01-01

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

  19. Asf1 facilitates dephosphorylation of Rad53 after DNA double-strand break repair.

    PubMed

    Tsabar, Michael; Waterman, David P; Aguilar, Fiona; Katsnelson, Lizabeth; Eapen, Vinay V; Memisoglu, Gonen; Haber, James E

    2016-05-15

    To allow for sufficient time to repair DNA double-stranded breaks (DSBs), eukaryotic cells activate the DNA damage checkpoint. In budding yeast, Rad53 (mammalian Chk2) phosphorylation parallels the persistence of the unrepaired DSB and is extinguished when repair is complete in a process termed recovery or when the cells adapt to the DNA damage checkpoint. A strain containing a slowly repaired DSB does not require the histone chaperone Asf1 to resume cell cycle progression after DSB repair. When a second, rapidly repairable DSB is added to this strain, Asf1 becomes required for recovery. Recovery from two repairable DSBs also depends on the histone acetyltransferase Rtt109 and the cullin subunit Rtt101, both of which modify histone H3 that is associated with Asf1. We show that dissociation of histone H3 from Asf1 is required for efficient recovery and that Asf1 is required for complete dephosphorylation of Rad53 when the upstream DNA damage checkpoint signaling is turned off. Our data suggest that the requirements for recovery from the DNA damage checkpoint become more stringent with increased levels of damage and that Asf1 plays a histone chaperone-independent role in facilitating complete Rad53 dephosphorylation following repair.

  20. Repairing a double-strand chromosome break by homologous recombination: revisiting Robin Holliday's model.

    PubMed

    Haber, James E; Ira, Gregorz; Malkova, Anna; Sugawara, Neal

    2004-01-29

    Since the pioneering model for homologous recombination proposed by Robin Holliday in 1964, there has been great progress in understanding how recombination occurs at a molecular level. In the budding yeast Saccharomyces cerevisiae, one can follow recombination by physically monitoring DNA after the synchronous induction of a double-strand break (DSB) in both wild-type and mutant cells. A particularly well-studied system has been the switching of yeast mating-type (MAT) genes, where a DSB can be induced synchronously by expression of the site-specific HO endonuclease. Similar studies can be performed in meiotic cells, where DSBs are created by the Spo11 nuclease. There appear to be at least two competing mechanisms of homologous recombination: a synthesis-dependent strand annealing pathway leading to noncrossovers and a two-end strand invasion mechanism leading to formation and resolution of Holliday junctions (HJs), leading to crossovers. The establishment of a modified replication fork during DSB repair links gene conversion to another important repair process, break-induced replication. Despite recent revelations, almost 40 years after Holliday's model was published, the essential ideas he proposed of strand invasion and heteroduplex DNA formation, the formation and resolution of HJs, and mismatch repair, remain the basis of our thinking.

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

    PubMed Central

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

    2016-01-01

    Gene targeting is becoming an important tool for precision genome engineering in plants. During gene replacement, a variant of gene targeting, transformed DNA integrates into the genome by homologous recombination (HR) to replace resident sequences. We have analysed gene targeting in barley (Hordeum vulgare) using a model system based on double-strand break (DSB) induction by the meganuclease I-SceI and a transgenic, artificial target locus. In the plants we obtained, the donor construct was inserted at the target locus by homology-directed DNA integration in at least two transformants obtained in a single experiment and was stably inherited as a single Mendelian trait. Both events were produced by one-sided integration. Our data suggest that gene replacement can be achieved in barley with a frequency suitable for routine application. The use of a codon-optimized nuclease and co-transfer of the nuclease gene together with the donor construct are probably the components important for efficient gene targeting. Such an approach, employing the recently developed synthetic nucleases/nickases that allow DSB induction at almost any sequence of a genome of interest, sets the stage for precision genome engineering as a routine tool even for important crops such as barley. PMID:26712824

  2. DNA double strand breaks in rat epidermis following irradiation with electrons

    SciTech Connect

    Shulman, K.

    1986-05-01

    Although radiation induced single strand breaks in rat epidermis are repaired fairly quickly (t-1/2 = 21 minutes), the fate of DNA double strand breaks in the same cells is unclear. Here we have attempted to measure dsb's in rat epidermis by neutral elution. The DNA of 28 day old CD rats was prelabeled with 6 I.P. injections at 2.0 uCi/g body weight of /sup 3/H-TdR. The dorsal skin was irradiated with a 0.8 MeV electron beam. The epidermis was removed by trypsinization at 4/sup 0/C and a single cell suspension was made. The cells were layered onto a polycarbonate filter, lysed, and eluted at pH 9.6. Doses of at least 6000 rads were needed to detect dsb's in vivo. Dsb's were still detectable in the epidermis 3 hours after irradiation. The amount of dsb's had returned to non-irradiated levels 8 hours after irradiation. 77 refs., 2 figs., 1 tab.

  3. A single double-strand break system reveals repair dynamics and mechanisms in heterochromatin and euchromatin.

    PubMed

    Janssen, Aniek; Breuer, Gregory A; Brinkman, Eva K; van der Meulen, Annelot I; Borden, Sean V; van Steensel, Bas; Bindra, Ranjit S; LaRocque, Jeannine R; Karpen, Gary H

    2016-07-15

    Repair of DNA double-strand breaks (DSBs) must be properly orchestrated in diverse chromatin regions to maintain genome stability. The choice between two main DSB repair pathways, nonhomologous end-joining (NHEJ) and homologous recombination (HR), is regulated by the cell cycle as well as chromatin context.Pericentromeric heterochromatin forms a distinct nuclear domain that is enriched for repetitive DNA sequences that pose significant challenges for genome stability. Heterochromatic DSBs display specialized temporal and spatial dynamics that differ from euchromatic DSBs. Although HR is thought to be the main pathway used to repair heterochromatic DSBs, direct tests of this hypothesis are lacking. Here, we developed an in vivo single DSB system for both heterochromatic and euchromatic loci in Drosophila melanogaster Live imaging of single DSBs in larval imaginal discs recapitulates the spatio-temporal dynamics observed for irradiation (IR)-induced breaks in cell culture. Importantly, live imaging and sequence analysis of repair products reveal that DSBs in euchromatin and heterochromatin are repaired with similar kinetics, employ both NHEJ and HR, and can use homologous chromosomes as an HR template. This direct analysis reveals important insights into heterochromatin DSB repair in animal tissues and provides a foundation for further explorations of repair mechanisms in different chromatin domains. PMID:27474442

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

    PubMed Central

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

    2016-01-01

    Abstract 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

  5. The 9-1-1 checkpoint clamp coordinates resection at DNA double strand breaks

    PubMed Central

    Ngo, Greg H.P.; Lydall, David

    2015-01-01

    DNA-end resection, the generation of single-stranded DNA at DNA double strand break (DSB) ends, is critical for controlling the many cellular responses to breaks. Here we show that the conserved DNA damage checkpoint sliding clamp (the 9-1-1 complex) plays two opposing roles coordinating DSB resection in budding yeast. We show that the major effect of 9-1-1 is to inhibit resection by promoting the recruitment of Rad953BP1 near DSBs. However, 9-1-1 also stimulates resection by Exo1- and Dna2-Sgs1-dependent nuclease/helicase activities, and this can be observed in the absence of Rad953BP1. Our new data resolve the controversy in the literature about the effect of the 9-1-1 complex on DSB resection. Interestingly, the inhibitory role of 9-1-1 on resection is not observed near uncapped telomeres because less Rad953BP1 is recruited near uncapped telomeres. Thus, 9-1-1 both stimulates and inhibits resection and the effects of 9-1-1 are modulated by different regions of the genome. Our experiments illustrate the central role of the 9-1-1 checkpoint sliding clamp in the DNA damage response network that coordinates the response to broken DNA ends. Our results have implications in all eukaryotic cells. PMID:25925573

  6. REV7 counteracts DNA double-strand break resection and affects PARP inhibition.

    PubMed

    Xu, Guotai; Chapman, J Ross; Brandsma, Inger; Yuan, Jingsong; Mistrik, Martin; Bouwman, Peter; Bartkova, Jirina; Gogola, Ewa; Warmerdam, Daniël; Barazas, Marco; Jaspers, Janneke E; Watanabe, Kenji; Pieterse, Mark; Kersbergen, Ariena; Sol, Wendy; Celie, Patrick H N; Schouten, Philip C; van den Broek, Bram; Salman, Ahmed; Nieuwland, Marja; de Rink, Iris; de Ronde, Jorma; Jalink, Kees; Boulton, Simon J; Chen, Junjie; van Gent, Dik C; Bartek, Jiri; Jonkers, Jos; Borst, Piet; Rottenberg, Sven

    2015-05-28

    Error-free repair of DNA double-strand breaks (DSBs) is achieved by homologous recombination (HR), and BRCA1 is an important factor for this repair pathway. In the absence of BRCA1-mediated HR, the administration of PARP inhibitors induces synthetic lethality of tumour cells of patients with breast or ovarian cancers. Despite the benefit of this tailored therapy, drug resistance can occur by HR restoration. Genetic reversion of BRCA1-inactivating mutations can be the underlying mechanism of drug resistance, but this does not explain resistance in all cases. In particular, little is known about BRCA1-independent restoration of HR. Here we show that loss of REV7 (also known as MAD2L2) in mouse and human cell lines re-establishes CTIP-dependent end resection of DSBs in BRCA1-deficient cells, leading to HR restoration and PARP inhibitor resistance, which is reversed by ATM kinase inhibition. REV7 is recruited to DSBs in a manner dependent on the H2AX-MDC1-RNF8-RNF168-53BP1 chromatin pathway, and seems to block HR and promote end joining in addition to its regulatory role in DNA damage tolerance. Finally, we establish that REV7 blocks DSB resection to promote non-homologous end-joining during immunoglobulin class switch recombination. Our results reveal an unexpected crucial function of REV7 downstream of 53BP1 in coordinating pathological DSB repair pathway choices in BRCA1-deficient cells.

  7. REV7 counteracts DNA double-strand break resection and impacts PARP inhibition

    PubMed Central

    Xu, Guotai; Yuan, Jingsong; Mistrik, Martin; Bouwman, Peter; Bartkova, Jirina; Gogola, Ewa; Warmerdam, Daniël; Barazas, Marco; Jaspers, Janneke E.; Watanabe, Kenji; Pieterse, Mark; Kersbergen, Ariena; Sol, Wendy; Celie, Patrick H. N.; Schouten, Philip C.; van den Broek, Bram; Salman, Ahmed; Nieuwland, Marja; de Rink, Iris; de Ronde, Jorma; Jalink, Kees; Boulton, Simon J.; Chen, Junjie; van Gent, Dik C.; Bartek, Jiri; Jonkers, Jos; Borst, Piet; Rottenberg, Sven

    2015-01-01

    Summary Error-free repair of DNA double-strand breaks (DSB) is achieved by homologous recombination (HR), and BRCA1 is an important factor for this repair pathway1. In the absence of BRCA1-mediated HR, administration of PARP inhibitors induces synthetic lethality of tumor cells of patients with breast or ovarian cancers2,3. Despite the benefit of this tailored therapy, drug resistance can occur by HR restoration4. Genetic reversion of BRCA1-inactivating mutations can be the underlying mechanism of drug resistance, but this does not explain resistance in all cases5. In particular, little is known about BRCA1-independent restoration of HR. Here, we show that loss of REV7 (also known as MAD2L2) re-establishes CtIP-dependent end resection of DSBs in BRCA1-deficient cells, leading to HR restoration and PARP inhibitor resistance, reversed by ATM kinase inhibition. REV7 is recruited to DSBs in a manner dependent on the H2AX-MDC1-RNF8-RNF168-53BP1 chromatin pathway, and appears to block HR and promote end joining in addition to its regulatory role in DNA damage tolerance6. Finally, we establish that REV7 blocks DSB resection to promote non-homologous end-joining (NHEJ) during immunoglobulin class switch recombination. Our results reveal an unexpected critical function of REV7 downstream of 53BP1 in coordinating pathological DSB repair pathway choices in BRCA1-deficient cells. PMID:25799992

  8. Mechanisms and Consequences of Double-Strand DNA Break Formation in Chromatin.

    PubMed

    Cannan, Wendy J; Pederson, David S

    2016-01-01

    All organisms suffer double-strand breaks (DSBs) in their DNA as a result of exposure to ionizing radiation. DSBs can also form when replication forks encounter DNA lesions or repair intermediates. The processing and repair of DSBs can lead to mutations, loss of heterozygosity, and chromosome rearrangements that result in cell death or cancer. The most common pathway used to repair DSBs in metazoans (non-homologous DNA end joining) is more commonly mutagenic than the alternative pathway (homologous recombination mediated repair). Thus, factors that influence the choice of pathways used DSB repair can affect an individual's mutation burden and risk of cancer. This review describes radiological, chemical, and biological mechanisms that generate DSBs, and discusses the impact of such variables as DSB etiology, cell type, cell cycle, and chromatin structure on the yield, distribution, and processing of DSBs. The final section focuses on nucleosome-specific mechanisms that influence DSB production, and the possible relationship between higher order chromosome coiling and chromosome shattering (chromothripsis). PMID:26040249

  9. Molecular recombination and the repair of DNA double-strand breaks in CHO cells.

    PubMed Central

    Resnick, M A; Moore, P D

    1979-01-01

    Molecular recombination and the repair of DNA double-strand breaks (DSB) have been examined in the G-0 and S phase of the cell cycle using a temperature-sensitive CHO cell line to test i) if there are cell cycle restrictions on the repair of DSB's' ii) the extent to which molecular recombination can be induced between either sister chromatids or homologous chromosomes and iii) whether repair of DSB's involves recombination (3). Mitomycin C (1-2 micrograms/ml) or ionizing radiation (50 krad) followed by incubation resulted in molecular recombination (hybrid DNA) in S phase cells. Approximately 0.03 to 0.10% of the molecules (number average molecular weight: 5.6 x 10(6) Daltons after shearing) had hybrid regions for more than 75% of their length. However, no recombination was detected in G-0 cells. Since the repair of DSB was observed in both stages with more than 50% of the breaks repaired in 5 hours, it appears that DSB repair in G-0 cells does not involve recombination between homologous chromosomes. The possibility is not excluded that repair in G-0 cells involves only small regions (less than 4 x 10(6) Daltons). PMID:493136

  10. DNA double-strand breaks alter the spatial arrangement of homologous loci in plant cells

    PubMed Central

    Hirakawa, Takeshi; Katagiri, Yohei; Ando, Tadashi; Matsunaga, Sachihiro

    2015-01-01

    Chromatin dynamics and arrangement are involved in many biological processes in nuclei of eukaryotes including plants. Plants have to respond rapidly to various environmental stimuli to achieve growth and development because they cannot move. It is assumed that the alteration of chromatin dynamics and arrangement support the response to these stimuli; however, there is little information in plants. In this study, we investigated the chromatin dynamics and arrangement with DNA damage in Arabidopsis thaliana by live-cell imaging with the lacO/LacI-EGFP system and simulation analysis. It was revealed that homologous loci kept a constant distance in nuclei of A. thaliana roots in general growth. We also found that DNA double-strand breaks (DSBs) induce the approach of the homologous loci with γ-irradiation. Furthermore, AtRAD54, which performs an important role in the homologous recombination repair pathway, was involved in the pairing of homologous loci with γ-irradiation. These results suggest that homologous loci approach each other to repair DSBs, and AtRAD54 mediates these phenomena. PMID:26046331

  11. Thermodynamically modulated partially double-stranded linear DNA probe design for homogeneous real-time PCR.

    PubMed

    Huang, Shihai; Salituro, John; Tang, Ning; Luk, Ka-Cheung; Hackett, John; Swanson, Priscilla; Cloherty, Gavin; Mak, Wai-Bing; Robinson, John; Abravaya, Klara

    2007-01-01

    Real-time PCR assays have recently been developed for diagnostic and research purposes. Signal generation in real-time PCR is achieved with probe designs that usually depend on exonuclease activity of DNA polymerase (e.g. TaqMan probe) or oligonucleotide hybridization (e.g. molecular beacon). Probe design often needs to be specifically tailored either to tolerate or to differentiate between sequence variations. The conventional probe technologies offer limited flexibility to meet these diverse requirements. Here, we introduce a novel partially double-stranded linear DNA probe design. It consists of a hybridization probe 5'-labeled with a fluorophore and a shorter quencher oligo of complementary sequence 3'-labeled with a quencher. Fluorescent signal is generated when the hybridization probe preferentially binds to amplified targets during PCR. This novel class of probe can be thermodynamically modulated by adjusting (i) the length of hybridization probe, (ii) the length of quencher oligo, (iii) the molar ratio between the two strands and (iv) signal detection temperature. As a result, pre-amplification signal, signal gain and the extent of mismatch discrimination can be reliably controlled and optimized. The applicability of this design strategy was demonstrated in the Abbott RealTime HIV-1 assay.

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

    PubMed

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

    1994-08-11

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

  13. Different repair kinetics for short and long DNA double-strand gaps in Saccharomyces cervisiae.

    PubMed

    Glasunov, A V; Frankenberg-Schwager, M; Frankenberg, D

    1995-10-01

    The kinetics of recombinational repair of plasmid DNA double-strand breaks (dsb) and gaps (dsg) of different sizes and ends were studied. For this purpose we used the mutant rad54-3 of the yeast Saccharomyces cerevisiae, which is temperature dependent with respect to genetic recombination and rejoining of dsb/dsg, allowing us to stop these processes by shifting cells to the restrictive temperature. We found that the kinetics of repair of cohesive-ended dsb and small gaps (up to 400 bp) are similar and characterized by two phases separated by a plateau. In contrast, large gap (1.4 kbp) repair proceeds with different kinetics exhibiting only the second phase. We also investigated the repair kinetics of 400 bp gaps introduced into plasmid DNA with and without homology to chromosomal DNA allowing recombinational repair and non-recombinational repair (ligation), respectively. We found that gaps introduced in plasmid sequences homologous to chromosomal DNA are rapidly repaired by recombination. In contrast, recircularization of the gapped plasmid by ligation is as slow and inefficient as ligation of a cohesive-ended dsb. The kinetics of repair of gapped plasmids may be explained by assuming a constitutive level of enzymes responsible for the first phase of recombinational repair, while inducible enzymes, which become available at the end of the plateau, carry out the second phase of repair. PMID:7594968

  14. The Mechanism of Double-Strand DNA Break Repair by the Nonhomologous DNA End Joining Pathway

    PubMed Central

    Lieber, Michael R.

    2011-01-01

    Double-strand DNA breaks are common events in eukaryotic cells, and there are two major pathways for repairing them: homologous recombination and nonhomologous DNA end joining (NHEJ). The diverse causes of DSBs result in a diverse chemistry of DNA ends that must be repaired. Across NHEJ evolution, the enzymes of the NHEJ pathway exhibit a remarkable degree of structural tolerance in the range of DNA end substrate configurations upon which they can act. In vertebrate cells, the nuclease, polymerases and ligase of NHEJ are the most mechanistically flexible and multifunctional enzymes in each of their classes. Unlike repair pathways for more defined lesions, NHEJ repair enzymes act iteratively, act in any order, and can function independently of one another at each of the two DNA ends being joined. NHEJ is critical not only for the repair of pathologic DSBs as in chromosomal translocations, but also for the repair of physiologic DSBs created during V(D)J recombination and class switch recombination. Therefore, patients lacking normal NHEJ are not only sensitive to ionizing radiation, but also severely immunodeficient. PMID:20192759

  15. Chromatin dynamics during repair of chromosomal DNA double-strand breaks

    PubMed Central

    Sinha, Manisha; Peterson, Craig L

    2010-01-01

    The integrity of a eukaryotic genome is often challenged by DNA double-strand breaks (DSBs). Even a single, unrepaired DSB can be a lethal event, or such unrepaired damage can result in chromosomal instability and loss of genetic information. Furthermore, defects in the pathways that respond to and repair DSBs can lead to the onset of several human pathologic disorders with pleiotropic clinical features, including age-related diseases and cancer. For decades, studies have focused on elucidating the enzymatic mechanisms involved in recognizing, signaling and repairing DSBs within eukaryotic cells. The majority of biochemical and genetic studies have used simple, DNA substrates, whereas only recently efforts have been geared towards understanding how the repair machinery deals with DSBs within chromatin fibers, the nucleoprotein complex that packages DNA within the eukaryotic nucleus. The aim of this review is to discuss our recent understanding of the relationship between chromatin structure and the repair of DSBs by homologous recombination. In particular, we discuss recent studies implicating specialized roles for several, distinct ATP-dependent chromatin remodeling enzymes in facilitating multiple steps within the homologous recombination process. PMID:20495614

  16. Short 5'-phosphorylated double-stranded RNAs induce RNA interference in Drosophila.

    PubMed

    Boutla, A; Delidakis, C; Livadaras, I; Tsagris, M; Tabler, M

    2001-11-13

    Double-stranded (ds) RNA causes the specific degradation of homologous RNAs in a process called "RNA interference (RNAi)"[1-4]; this process is called "posttranscriptional gene silencing (PTGS)" in plants [5-7]. Both classes of gene silencing have been reviewed extensively [8-13]. The duplex RNA becomes processed by Dicer [14] or another RNase III-like enzyme to short dsRNA fragments of about 21-23 nucleotides (nt) [15], which are incorporated in the RNA-induced silencing complex (RISC)[16] that directs target-specific RNA degradation [17, 18]. Here, we show that different synthetic dsRNA cassettes, consisting of two 5'-phosphorylated RNA strands of 22 nt each, can initiate RNAi in Drosophila embryos. The cassettes were active at similar quantities required to initiate RNAi by conventional dsRNA. Their sequence specificity was confirmed using synthetic dsRNA cassettes for two different genes, Notch and hedgehog; each time, only the relevant embryonic phenotype was observed. Introduction of point mutations had only a moderate effect on the silencing potential, indicating that the silencing machinery does not require perfect sequence identity. 5'-phosphorylated synthetic RNA was more active than its hydroxylated form. Substitution of either RNA strand by DNA strongly reduced activity. Synthetic cassettes of siRNA will provide a new tool to induce mutant phenotypes of genes with unknown function.

  17. Homing endonuclease I-TevIII: dimerization as a means to a double-strand break

    PubMed Central

    Robbins, Justin B.; Stapleton, Michelle; Stanger, Matthew J.; Smith, Dorie; Dansereau, John T.; Derbyshire, Victoria; Belfort, Marlene

    2007-01-01

    Homing endonucleases are unusual enzymes, capable of recognizing lengthy DNA sequences and cleaving site-specifically within genomes. Many homing endonucleases are encoded within group I introns, and such enzymes promote the mobility reactions of these introns. Phage T4 has three group I introns, within the td, nrdB and nrdD genes. The td and nrdD introns are mobile, whereas the nrdB intron is not. Phage RB3 is a close relative of T4 and has a lengthier nrdB intron. Here, we describe I-TevIII, the H–N–H endonuclease encoded by the RB3 nrdB intron. In contrast to previous reports, we demonstrate that this intron is mobile, and that this mobility is dependent on I-TevIII, which generates 2-nt 3′ extensions. The enzyme has a distinct catalytic domain, which contains the H–N–H motif, and DNA-binding domain, which contains two zinc fingers required for interaction with the DNA substrate. Most importantly, I-TevIII, unlike the H–N–H endonucleases described so far, makes a double-strand break on the DNA homing site by acting as a dimer. Through deletion analysis, the dimerization interface was mapped to the DNA-binding domain. The unusual propensity of I-TevIII to dimerize to achieve cleavage of both DNA strands underscores the versatility of the H–N–H enzyme family. PMID:17289754

  18. Host Double Strand Break Repair Generates HIV-1 Strains Resistant to CRISPR/Cas9.

    PubMed

    Yoder, Kristine E; Bundschuh, Ralf

    2016-07-12

    CRISPR/Cas9 genome editing has been proposed as a therapeutic treatment for HIV-1 infection. CRISPR/Cas9 induced double strand breaks (DSBs) targeted to the integrated viral genome have been shown to decrease production of progeny virus. Unfortunately HIV-1 evolves rapidly and may readily produce CRISPR/Cas9 resistant strains. Here we used next-generation sequencing to characterize HIV-1 strains that developed resistance to six different CRISPR/Cas9 guide RNAs (gRNAs). Reverse transcriptase (RT) derived base substitution mutations were commonly found at sites encoding unpaired bases of RNA stem-loop structures. In addition to RT mutations, insertion and/or deletion (indel) mutations were common. Indels localized to the CRISPR/Cas9 cleavage site were major contributors to CRISPR gRNA resistance. While most indels at non-coding regions were a single base pair, 3 base pair indels were observed when a coding region of HIV-1 was targeted. The DSB repair event may preserve the HIV-1 reading frame, while destroying CRISPR gRNA homology. HIV-1 may be successfully edited by CRISPR/Cas9, but the virus remains competent for replication and resistant to further CRISPR/Cas9 targeting at that site. These observations strongly suggest that host DSB repair at CRISPR/Cas9 cleavage sites is a novel and important pathway that may contribute to HIV-1 therapeutic resistance.

  19. Dynamic distributions of long double-stranded RNA in Tetrahymena during nuclear development and genome rearrangements.

    PubMed

    Woo, Tai-Ting; Chao, Ju-Lan; Yao, Meng-Chao

    2016-03-01

    Bi-directional non-coding transcripts and their ∼29-nt small RNA products are known to guide DNA deletion in Tetrahymena, leading to the removal of one-third of the genome from developing somatic nuclei. Using an antibody specific for long double-stranded RNAs (dsRNAs), we determined the dynamic subcellular distributions of these RNAs. Conjugation-specific dsRNAs were found and show sequential appearances in parental germline, parental somatic nuclei and finally in new somatic nuclei of progeny. The dsRNAs in germline nuclei and new somatic nuclei are likely transcribed from the sequences destined for deletion; however, the dsRNAs in parental somatic nuclei are unexpected, and PCR analyses suggested that they were transcribed in this nucleus. Deficiency in the RNA interference (RNAi) pathway led to abnormal aggregations of dsRNA in both the parental and new somatic nuclei, whereas accumulation of dsRNAs in the germline nuclei was only seen in the Dicer-like gene mutant. In addition, RNAi mutants displayed an early loss of dsRNAs from developing somatic nuclei. Thus, long dsRNAs are made in multiple nuclear compartments and some are linked to small RNA production whereas others might participate in their regulations. PMID:26769902

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

    NASA Astrophysics Data System (ADS)

    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.

  1. Asf1 facilitates dephosphorylation of Rad53 after DNA double-strand break repair.

    PubMed

    Tsabar, Michael; Waterman, David P; Aguilar, Fiona; Katsnelson, Lizabeth; Eapen, Vinay V; Memisoglu, Gonen; Haber, James E

    2016-05-15

    To allow for sufficient time to repair DNA double-stranded breaks (DSBs), eukaryotic cells activate the DNA damage checkpoint. In budding yeast, Rad53 (mammalian Chk2) phosphorylation parallels the persistence of the unrepaired DSB and is extinguished when repair is complete in a process termed recovery or when the cells adapt to the DNA damage checkpoint. A strain containing a slowly repaired DSB does not require the histone chaperone Asf1 to resume cell cycle progression after DSB repair. When a second, rapidly repairable DSB is added to this strain, Asf1 becomes required for recovery. Recovery from two repairable DSBs also depends on the histone acetyltransferase Rtt109 and the cullin subunit Rtt101, both of which modify histone H3 that is associated with Asf1. We show that dissociation of histone H3 from Asf1 is required for efficient recovery and that Asf1 is required for complete dephosphorylation of Rad53 when the upstream DNA damage checkpoint signaling is turned off. Our data suggest that the requirements for recovery from the DNA damage checkpoint become more stringent with increased levels of damage and that Asf1 plays a histone chaperone-independent role in facilitating complete Rad53 dephosphorylation following repair. PMID:27222517

  2. Cohesin Protects Genes against γH2AX Induced by DNA Double-Strand Breaks

    PubMed Central

    Caron, Pierre; Aymard, Francois; Iacovoni, Jason S.; Briois, Sébastien; Canitrot, Yvan; Bugler, Beatrix; Massip, Laurent; Losada, Ana; Legube, Gaëlle

    2012-01-01

    Chromatin undergoes major remodeling around DNA double-strand breaks (DSB) to promote repair and DNA damage response (DDR) activation. We recently reported a high-resolution map of γH2AX around multiple breaks on the human genome, using a new cell-based DSB inducible system. In an attempt to further characterize the chromatin landscape induced around DSBs, we now report the profile of SMC3, a subunit of the cohesin complex, previously characterized as required for repair by homologous recombination. We found that recruitment of cohesin is moderate and restricted to the immediate vicinity of DSBs in human cells. In addition, we show that cohesin controls γH2AX distribution within domains. Indeed, as we reported previously for transcription, cohesin binding antagonizes γH2AX spreading. Remarkably, depletion of cohesin leads to an increase of γH2AX at cohesin-bound genes, associated with a decrease in their expression level after DSB induction. We propose that, in agreement with their function in chromosome architecture, cohesin could also help to isolate active genes from some chromatin remodelling and modifications such as the ones that occur when a DSB is detected on the genome. PMID:22275873

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

    PubMed Central

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

    2009-01-01

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

  4. Sequence conservation of the rad21 Schizosaccharomyces pombe DNA double-strand break repair gene in human and mouse

    SciTech Connect

    McKay, M.J.; Troelstra, C.; Kanaar, R.

    1996-09-01

    The rad21 gene of Schizosaccharomyces pombe is involved in the repair of ionizing radiation-induced DNA double-strand breaks. The isolation of mouse and human putative homologs of rad21 is reported here. Alignment of the predicted amino acid sequence of Rad21 with the mammalian proteins showed that the similarity was distributed across the length of the proteins, with more highly conserved regions at both termini. The mHR21{sup sp} (mouse homolog of Rad21, S. pombe) and hHR21{sup sp} (human homolog 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 mHR21{sup sp} mRNA was abundant in all adult mouse tissues examined, with highest expression in testis and thymus. In addition to a 3.1-kb constitutive mRNA transcript, a 2.2-kb transcript was present at a high level in postmeiotic spermatids, while expression of the 3.1-kb mRNA in testis was confined to the meiotic compartment. hHR21{sup sp} mRNA was cell-cycle regulated in human cells, increasing in late S phase to a peak in G2 phase. The level of hHR21{sup sp} transcripts was not altered by exposure of normal diploid fibroblasts to 10 Gy ionizing radiation. In situ hybridization showed that mHR21{sup sp} resided on chromosome 15D3, whereas hHR21{sup sp} localized to the syntenic 8q24 region. Elevated expression of mHR21{sup sp} in testis and thymus supports a possible role for the rad21 mammalian homologs in V(D)J and meiotic recombination, respectively. Cell cycle regulation of rad21, retained from S. pombe to human, is consistent with a conservation of function between S. pombe and human rad21 genes. 62 refs., 8 figs., 1 tab.

  5. Cisplatin enhances the formation of DNA single- and double-strand breaks by hydrated electrons and hydroxyl radicals.

    PubMed

    Rezaee, Mohammad; Sanche, Léon; Hunting, Darel J

    2013-03-01

    The synergistic interaction of cisplatin with ionizing radiation is the clinical rationale for the treatment of several cancers including head and neck, cervical and lung cancer. The underlying molecular mechanism of the synergy has not yet been identified, although both DNA damage and repair processes are likely involved. Here, we investigate the indirect effect of γ rays on strand break formation in a supercoiled plasmid DNA (pGEM-3Zf-) covalently modified by cisplatin. The yields of single- and double-strand breaks were determined by irradiation of DNA and cisplatin/DNA samples with (60)Co γ rays under four different scavenging conditions to examine the involvement of hydrated electrons and hydroxyl radicals in inducing the DNA damage. At 5 mM tris in an N2 atmosphere, the presence of an average of two cisplatins per plasmid increased the yields of single- and double-strand breaks by factors of 1.9 and 2.2, respectively, relative to the irradiated unmodified DNA samples. Given that each plasmid of 3,200 base pairs contained an average of two cisplatins, this represents an increase in radiosensitivity of 3,200-fold on a per base pair basis. When hydrated electrons were scavenged by saturating the samples with N2O, these enhancement factors decreased to 1.5 and 1.2, respectively, for single- and double-strand breaks. When hydroxyl radicals were scavenged using 200 mM tris, the respective enhancement factors were 1.2 and 1.6 for single- and double-strand breaks, respectively. Furthermore, no enhancement in DNA damage by cisplatin was observed after scavenging both hydroxyl radicals and hydrated electrons. These findings show that hydrated electrons can induce both single- and double-strand breaks in the platinated DNA, but not in unmodified DNA. In addition, cisplatin modification is clearly an extremely efficient means of increasing the formation of both single- and double-strand breaks by the hydrated electrons and hydroxyl radicals created by ionizing

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

    PubMed Central

    Karmakar, Saswata; Madsen, Andreas S.; Guenther, Dale C.; Gibbons, Bradley C.; Hrdlicka, Patrick J.

    2014-01-01

    Despite advances with triplex-forming oligonucleotides, peptide nucleic acids, polyamides and - more recently - engineered proteins, there remains an urgent need for synthetic ligands that enable specific recognition of double-stranded (ds) DNA to accelerate studies aiming at detecting, regulating and modifying genes. Invaders, i.e., energetically activated DNA duplexes with interstrand zipper arrangements of intercalator-functionalized nucleotides, are emerging as an attractive approach toward this goal. Here, we characterize and compare Invaders based on 1-, 2- and 4-pyrenyl-functionalized O2′-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 (ΔTm/modification up to +14.0 °C), provides the driving force for dsDNA recognition. In contrast, Z-modified Invaders show much lower dsDNA recognition efficiency. Thus, even very conservative changes in the chemical makeup of the intercalator-functionalized nucleotides used to activate Invader duplexes, affects dsDNA-recognition efficiency of the probes, which highlights the importance of systematic structure-property studies. The insight from this study will guide future design of Invaders for applications in molecular biology and nucleic acid diagnostics. PMID:25144705

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

    NASA Technical Reports Server (NTRS)

    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.

  8. Tetrameric structure of the restriction DNA glycosylase R.PabI in complex with nonspecific double-stranded DNA

    PubMed Central

    Wang, Delong; Miyazono, Ken-ichi; Tanokura, Masaru

    2016-01-01

    R.PabI is a type II restriction enzyme that recognizes the 5′-GTAC-3′ sequence and belongs to the HALFPIPE superfamily. Although most restriction enzymes cleave phosphodiester bonds at specific sites by hydrolysis, R.PabI flips the guanine and adenine bases of the recognition sequence out of the DNA helix and hydrolyzes the N-glycosidic bond of the flipped adenine in a similar manner to DNA glycosylases. In this study, we determined the structure of R.PabI in complex with double-stranded DNA without the R.PabI recognition sequence by X-ray crystallography. The 1.9 Å resolution structure of the complex showed that R.PabI forms a tetrameric structure to sandwich the double-stranded DNA and the tetrameric structure is stabilized by four salt bridges. DNA binding and DNA glycosylase assays of the R.PabI mutants showed that the residues that form the salt bridges (R70 and D71) are essential for R.PabI to find the recognition sequence from the sea of nonspecific sequences. R.PabI is predicted to utilize the tetrameric structure to bind nonspecific double-stranded DNA weakly and slide along it to find the recognition sequence. PMID:27731370

  9. Homology Requirements for Double-Strand Break-Mediated Recombination in a Phage λ-Td Intron Model System

    PubMed Central

    Parker, M. M.; Court, D. A.; Preiter, K.; Belfort, M.

    1996-01-01

    Many group I introns encode endonucleases that promote intron homing by initiating a double-strand break-mediated homologous recombination event. A td intron-phage λ model system was developed to analyze exon homology effects on intron homing and determine the role of the λ 5'-3' exonuclease complex (Redαβ) in the repair event. Efficient intron homing depended on exon lengths in the 35- to 50-bp range, although homing levels remained significantly elevated above nonbreak-mediated recombination with as little as 10 bp of flanking homology. Although precise intron insertion was demonstrated with extremely limiting exon homology, the complete absence of one exon produced illegitimate events on the side of heterology. Interestingly, intron inheritance was unaffected by the presence of extensive heterology at the double-strand break in wild-type λ, provided that sufficient homology between donor and recipient was present distal to the heterologous sequences. However, these events involving heterologous ends were absolutely dependent on an intact Red exonuclease system. Together these results indicate that heterologous sequences can participate in double-strand break-mediated repair and imply that intron transposition to heteroallelic sites might occur at break sites within regions of limited or no homology. PMID:8807281

  10. Label-free photoelectrochemical detection of double-stranded HIV DNA by means of a metallointercalator-functionalized electrogenerated polymer.

    PubMed

    Haddache, Fatima; Le Goff, Alan; Reuillard, Bertrand; Gorgy, Karine; Gondran, Chantal; Spinelli, Nicolas; Defrancq, Eric; Cosnier, Serge

    2014-11-17

    The design of photoactive functionalized electrodes for the sensitive transduction of double-stranded DNA hybridization is reported. Multifunctional complex [Ru(bpy-pyrrole)2 (dppn)](2+) (bpy-pyrrole=4-methyl-4'-butylpyrrole-2,2'-bipyridine, dppn=benzo[i]dipyrido[3,2-a:2',3'-c]phenazine) exhibiting photosensitive, DNA-intercalating, and electropolymerizable properties was synthesized and characterized. The pyrrole groups undergo oxidative electropolymerization on planar electrodes forming a metallopolymer layer on the electrode. Thanks to the photoelectrochemical and intercalating properties of the immobilized Ru(II) complex, the binding of a double-stranded HIV DNA target was photoelectrochemically detected on planar electrodes. Photocurrent generation through visible irradiation was correlated to the interaction between double-stranded DNA and the metallointercalator polymer. These interactions were well fitted by using a Langmuir isotherm, which allowed a dissociation constant of 2×10(6)  L mol(-1) to be estimated. The low detection limit of 1 fmol L(-1) and sensitivity of 0.01 units per decade demonstrate excellent suitability of these modified electrodes for detection of duplex DNA.

  11. RecBCD is required to Complete Chromosomal Replication: Implications for Double-Strand Break Frequencies and Repair Mechanisms

    PubMed Central

    Courcelle, Justin; Wendel, Brian M.; Livingstone, Dena D.; Courcelle, Charmain T.

    2015-01-01

    Several aspects of the mechanism of homologous double strand break repair remain unclear. Although intensive efforts have focused on how recombination reactions initiate, far less is known about the molecular events that follow. Based upon biochemical studies, current models propose that RecBCD processes double strand ends and loads RecA to initiate recombinational repair. However, recent studies have shown that RecBCD plays a critical role in completing replication events on the chromosome through a mechanism that does not involve RecA or recombination. Here, we examine several studies, both early and recent, that suggest RecBCD also operates late in the recombination process- after initiation, strand invasion, and crossover resolution have occurred. Similar to its role in completing replication, we propose a model in which RecBCD is required to resect and resolve the DNA synthesis associated with homologous recombination at the point where the missing sequences on the broken molecule have been restored. We explain how the impaired ability to complete chromosome replication in recBC and recD mutants is likely to account for the loss of viability and genome instability in these mutants, and conclude that spontaneous double strand breaks and replication fork collapse occur far less frequently than previously speculated. PMID:26003632

  12. Fine resolution mapping of double-strand break sites for human ribosomal DNA units.

    PubMed

    Pope, Bernard J; Mahmood, Khalid; Jung, Chol-Hee; Park, Daniel J

    2016-12-01

    DNA breakage arises during a variety of biological processes, including transcription, replication and genome rearrangements. In the context of disease, extensive fragmentation of DNA has been described in cancer cells and during early stages of neurodegeneration (Stephens et al., 2011 Stephens et al. (2011) [5]; Blondet et al., 2001 Blondet et al. (2001) [1]). Stults et al. (2009) Stults et al. (2009) [6] reported that human rDNA gene clusters are hotspots for recombination and that rDNA restructuring is among the most common chromosomal alterations in adult solid tumours. As such, analysis of rDNA regions is likely to have significant prognostic and predictive value, clinically. Tchurikov et al. (2015a, 2016) Tchurikov et al. (2015a, 2016) [7], [9] have made major advances in this direction, reporting that sites of human genome double-strand breaks (DSBs) occur frequently at sites in rDNA that are tightly linked with active transcription - the authors used a RAFT (rapid amplification of forum termini) protocol that selects for blunt-ended sites. They reported the relative frequency of these rDNA DSBs within defined co-ordinate 'windows' of varying size and made these data (as well as the relevant 'raw' sequencing information) available to the public (Tchurikov et al., 2015b). Assay designs targeting rDNA DSB hotspots will benefit greatly from the publication of break sites at greater resolution. Here, we re-analyse public RAFT data and make available rDNA DSB co-ordinates to the single-nucleotide level. PMID:27656414

  13. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage

    PubMed Central

    Komor, Alexis C.; Kim, Yongjoo B.; Packer, Michael S.; Zuris, John A.; Liu, David R.

    2016-01-01

    Current genome-editing technologies introduce double-stranded (ds) DNA breaks at a target locus as the first step to gene correction.1,2 Although most genetic diseases arise from point mutations, current approaches to point mutation correction are inefficient and typically induce an abundance of random insertions and deletions (indels) at the target locus from the cellular response to dsDNA breaks.1,2 Here we report the development of base editing, a new approach to genome editing that enables the direct, irreversible conversion of one target DNA base into another in a programmable manner, without requiring dsDNA backbone cleavage or a donor template. We engineered fusions of CRISPR/Cas9 and a cytidine deaminase enzyme that retain the ability to be programmed with a guide RNA, do not induce dsDNA breaks, and mediate the direct conversion of cytidine to uridine, thereby effecting a C→T (or G→A) substitution. The resulting “base editors” convert cytidines within a window of approximately five nucleotides (nt), and can efficiently correct a variety of point mutations relevant to human disease. In four transformed human and murine cell lines, second- and third-generation base editors that fuse uracil glycosylase inhibitor (UGI), and that use a Cas9 nickase targeting the non-edited strand, manipulate the cellular DNA repair response to favor desired base-editing outcomes, resulting in permanent correction of ∼15-75% of total cellular DNA with minimal (typically ≤ 1%) indel formation. Base editing expands the scope and efficiency of genome editing of point mutations. PMID:27096365

  14. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage.

    PubMed

    Komor, Alexis C; Kim, Yongjoo B; Packer, Michael S; Zuris, John A; Liu, David R

    2016-05-19

    Current genome-editing technologies introduce double-stranded (ds) DNA breaks at a target locus as the first step to gene correction. Although most genetic diseases arise from point mutations, current approaches to point mutation correction are inefficient and typically induce an abundance of random insertions and deletions (indels) at the target locus resulting from the cellular response to dsDNA breaks. Here we report the development of 'base editing', a new approach to genome editing that enables the direct, irreversible conversion of one target DNA base into another in a programmable manner, without requiring dsDNA backbone cleavage or a donor template. We engineered fusions of CRISPR/Cas9 and a cytidine deaminase enzyme that retain the ability to be programmed with a guide RNA, do not induce dsDNA breaks, and mediate the direct conversion of cytidine to uridine, thereby effecting a C→T (or G→A) substitution. The resulting 'base editors' convert cytidines within a window of approximately five nucleotides, and can efficiently correct a variety of point mutations relevant to human disease. In four transformed human and murine cell lines, second- and third-generation base editors that fuse uracil glycosylase inhibitor, and that use a Cas9 nickase targeting the non-edited strand, manipulate the cellular DNA repair response to favour desired base-editing outcomes, resulting in permanent correction of ~15-75% of total cellular DNA with minimal (typically ≤1%) indel formation. Base editing expands the scope and efficiency of genome editing of point mutations. PMID:27096365

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

    PubMed

    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 Wi_{c}, 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. PMID:27176342

  16. Preferential Repair of DNA Double-strand Break at the Active Gene in Vivo*

    PubMed Central

    Chaurasia, Priyasri; Sen, Rwik; Pandita, Tej K.; Bhaumik, Sukesh R.

    2012-01-01

    Previous studies have demonstrated transcription-coupled nucleotide/base excision repair. We report here for the first time that DNA double-strand break (DSB) repair is also coupled to transcription. We generated a yeast strain by introducing a homing (Ho) endonuclease cut site followed by a nucleotide sequence for multiple Myc epitopes at the 3′ end of the coding sequence of a highly active gene, ADH1. This yeast strain also contains the Ho cut site at the nearly silent or poorly active mating type α (MATα) locus and expresses Ho endonuclease under the galactose-inducible GAL1 promoter. Using this strain, DSBs were generated at the ADH1 and MATα loci in galactose-containing growth medium that induced HO expression. Subsequently, yeast cells were transferred to dextrose-containing growth medium to stop HO expression, and the DSB repair was monitored at the ADH1 and MATα loci by PCR, using the primer pairs flanking the Ho cut sites. Our results revealed a faster DSB repair at the highly active ADH1 than that at the nearly silent MATα locus, hence implicating a transcription-coupled DSB repair at the active gene in vivo. Subsequently, we extended this study to another gene, PHO5 (carrying the Ho cut site at its coding sequence), under transcriptionally active and inactive growth conditions. We found a fast DSB repair at the active PHO5 gene in comparison to its inactive state. Collectively, our results demonstrate a preferential DSB repair at the active gene, thus supporting transcription-coupled DSB repair in living cells. PMID:22910905

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

    NASA Astrophysics Data System (ADS)

    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. Accumulation of DNA Double-Strand Breaks in Normal Tissues After Fractionated Irradiation

    SciTech Connect

    Ruebe, Claudia E.

    2010-03-15

    Purpose: There is increasing evidence that genetic factors regulating the recognition and/or repair of DNA double-strand breaks (DSBs) are responsible for differences in radiosensitivity among patients. Genetically defined DSB repair capacities are supposed to determine patients' individual susceptibility to develop adverse normal tissue reactions after radiotherapy. In a preclinical murine model, we analyzed the impact of different DSB repair capacities on the cumulative DNA damage in normal tissues during the course of fractionated irradiation. Material and Methods: Different strains of mice with defined genetic backgrounds (SCID{sup -/-} homozygous, ATM{sup -/-} homozygous, ATM{sup +/-}heterozygous, and ATM{sup +/+}wild-type mice) were subjected to single (2 Gy) or fractionated irradiation (5 x 2 Gy). By enumerating gammaH2AX foci, the formation and rejoining of DSBs were analyzed in organs representative of both early-responding (small intestine) and late-responding tissues (lung, kidney, and heart). Results: In repair-deficient SCID{sup -/-} and ATM{sup -/-}homozygous mice, large proportions of radiation-induced DSBs remained unrepaired after each fraction, leading to the pronounced accumulation of residual DNA damage after fractionated irradiation, similarly visible in early- and late-responding tissues. The slight DSB repair impairment of ATM{sup +/-}heterozygous mice was not detectable after single-dose irradiation but resulted in a significant increase in unrepaired DSBs during the fractionated irradiation scheme. Conclusions: Radiation-induced DSBs accumulate similarly in acute- and late-responding tissues during fractionated irradiation, whereas the whole extent of residual DNA damage depends decisively on the underlying genetically defined DSB repair capacity. Moreover, our data indicate that even minor impairments in DSB repair lead to exceeding DNA damage accumulation during fractionated irradiation and thus may have a significant impact on normal

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

    PubMed Central

    Haber, James E.

    2016-01-01

    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. PMID:27074148

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

    NASA Technical Reports Server (NTRS)

    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.